*4.1.1 Advantages*

**79**

**Figure 2.**

*Theranostics working on tumor cell.*

*Theranostics: New Era in Nuclear Medicine and Radiopharmaceuticals*

• Nuclear medicine procedure less costly and painless.

• Myelosuppression may occur before chemotherapy.

• Nuclear medicine procedures are safe with no side effects [24].

• Prolonged in convenience and discomfort can be experienced by patients due

• Nuclear medicine tests cannot use for pregnant women because of potential

Theranostics is a combination of two words **Thera**peutics and diag**nostics**. This is an emerging field of medicine where drugs and/or techniques are used in combination for treatment as well as treatment. It's a game changer as it provides diagnosis as well as therapy in single combination. It is economical as well as less time consuming. It uses PET scan to target tumor receptors which are present in tumor cell. If it is found in the cells radioactive drug is used to treat it. There are not much clinical trials found related to use of application of theranostics in prostate cancer by Australian Medicines

The Nuclear Medicine Imaging approach is revolutionized by use theranostics [targeted therapeutic (Rx) + companion diagnostic (DX)] to establish tools for specific molecular targeting. It provides personalized treatment plan for the patient by targeting specific targets. Various department of nuclear science can utilize

Regulator—The Therapeutic Goods Administration (TGA) [25] (**Figure 2**).

*DOI: http://dx.doi.org/10.5772/intechopen.91868*

• Some allergic reactions can be seen.

to administration of multiple fraction.

**5.1 Theranostics: Nuclear Medicine Imaging**

• Risk of radiation is associated.

risk to unborn babies.

**5. Theranostics**

theranostic agents [26].

*4.1.2 Disadvantages*

*Theranostics: New Era in Nuclear Medicine and Radiopharmaceuticals DOI: http://dx.doi.org/10.5772/intechopen.91868*


Thermal Protective Performance of Turnout Gear at High Flux Environment

*Rumeel Ahmad Bhutta and Sengkwan Choi*

## **Abstract**

Thermal protective performance (TPP) tests are conducted at a heat flux of 84 kW*=*m2 to evaluate a firefighter's turnout gear performance. The test criterion used is based on a study conducted by Behnke in 1984. However, an average heat flux of 200 kW*=*m2 has been documented in the literature over the past decade. Henceforth, experiments are conducted on the turnout gear at a higher heat flux level of 126 kW/ m2 , which represents a typical heat flux during the initial phase of a fully developed fire. The analytical analysis provides insights into air gaps, the effect of smoke layers on radiative attenuation and the thermal decomposition of the fabric layers. Numerical techniques were employed to introduce a survival curve to effectively gauge the degree of spatial burn at variable levels of heat fluxes, moisture effects and superficial burns. The sustainability of high resistance fabrics in terms of toxicity of flame retardants and recycling of these textiles have been discussed. Bio-composites as flame retardants are being introduced to replace traditional flame-retardant chemicals.

**Keywords:** firefighter, high heat flux, survival curve, thermal protective clothing, turnout gear

#### **1. Introduction**

Turnout gear is worn to safeguard individuals employed in hazardous fields like firefighting, high-speed sports, the oil and gas industry and the military. The protective gear can be either thermal protective clothing for firefighting, chemical resistant clothing for the oil and gas industry or puncture- and cut-resilient clothing for the military [1]. Thermal protective clothing is crucial for firefighters because fire is a common occurrence and poses a significant threat to people's safety. Firefighter protective clothing can be divided into two types: station wear, which is a single-layer garment worn while at the fire station, and turnout gear, which is a multiple-layer garment worn while responding to a fire incident [2]. The turnout gear is a combination of three fabric layers: an outer shell (OS), a moisture barrier (MB) and a thermal liner (TL).

The outer shell is the primary layer in a multi-layered clothing system and is part of a family of fabrics that are naturally resistant to fire [3, 4]. It has a highly organized structure of aromatic groups attached to a backbone chain, which prevents it from

breaking down into smaller molecules and producing less smoke when exposed to fire [5, 6]. When this aramid fabric group is combined with other fire-resistant materials using lamination, coating or bonding, it creates a moisture barrier. This porosity of the membrane is a determining factor in moisture control, that is, it is permeable to sweat vapor and acts as a barrier for water droplets. To make a thermal liner, a batting fabric made from fire-resistant fibers is sewn or quilted onto a face fabric, which is also made using fire-resistant fibers. Its main function is to reduce heat dissipation, provide comfort and release metabolic heat away from the wearer's body.

The maximum evaluation criterion for thermal protective clothing was proposed by Behnke [7]. He exposed swatches of the protective fabric made of Nomex®IIIA to several incident fluxes. The amount of incident flux that replicated the appearance from damaged clothing was estimated to be 84 kW*=*m2 ≈2 cal*=*cm2 ð Þs for 8 seconds of exposure. This estimated value was proposed to be a representation of flashover.

The conditions of a fire, whether it's inside a building or in a forest, can vary from one incident to another due to the unpredictable nature of the materials that are burning [8]. That is why firefighters' protective clothing is designed to be effective under a range of heat levels that are representative of several types of fires, including flashovers, backdrafts, fully developed fires, and forest fires. These garments are classified into routine, hazardous and emergency categories [9, 10]. Recent experiments [11–19] reported an average of 200 kW*=*m2 heat flux under extreme conditions in buildings and other enclosed spaces. These are higher levels of fire severity than previously measured. Henceforth, a new upper limit of 126 kW*=*m2 is recommended, and the performance of turnout gear is tested at this level.

It corelates with the building data collected by the end of the past decade which emphasize modern construction trends in the housing sector [20, 21]. According to this data, the home area has increased by approximately 232*:*3 m2 in 2008, as compared to 144 m2 in 1973. The 26% of homes built in 2008 were bigger than 278*:*7 m2; additionally, a growth of 56% in the construction of 2-storey homes (1-level above ground floor, US standard) was reported. Also, the current housing incorporates taller ceilings of 4.3–6.1 m, than the traditional 2.4 m. All these factors contribute to increased area and volume. As a result, the smoke spread during a fire incident remains above the ongoing fire; hence, more oxygen is available, causing the fire to grow rapidly and violently. This fire spread is also linked to the decorations and contents in the accommodation that have transitioned from natural to synthetic materials, such as cotton being replaced by polyurethane foam [22].

The effectiveness of a multi-layered protective garment can be evaluated using either a full-scale manikin test or a laboratory bench-scale test using the TPP rating system [23–25]. A full-scale manikin test assesses the performance of the entire garment against a convective/radiative thermal load, while a bench-scale test is performed on small patches of the garment. To capture thermal load on the skin, fullscale manikin testing facilities have been developed using a network of over 100 sensors distributed across the manikin's surface [26–30]. However, due to the high cost of full-scale tests, bench-scale tests are preferred as they are more affordable and can still replicate real-world room fires. Standards for bench-scale tests have been developed for diverse types of thermal loads, such as convective, radiant or a mix of both. The assessment criteria such as ISO 9151 [31], ISO 6942 [32], ASTM F2702 [33], ISO 17492 [24], ASTM F2703 [34] and NFPA 1971 [35] vary depending on the heat source and are representative of several types of fire incidents. The data from these experiments are then input into analytical models to predict skin burns.

#### **1.1 Skin burns**

The skin layers of interest in burn degree evaluation are the epidermis, dermis and subcutaneous. The second layer, dermis, is of the most significance as its temperature profile corelates to second-degree burns. The human body maintains a skin temperature of 37°C during daily activities. Pain is induced when the body temperature increases by 8°C, which occurs at a temperature of 44°C. A first-degree (superficial) burn occurs at 48°C, and a second-degree burn occurs with continued exposure to heat at 55°C. At 72°C, the skin tissues cannot sustain thermal equilibrium and are destroyed. Two widely accepted criteria are applied to assess this damage, Stoll's and Henrique's.

Stoll's criterion is applied to evaluate protection against second-degree burns [36]. It involves recording temperature data from a benchtop test, and the intersection of the experimental curve with Stoll's documented experimental curve predicts tolerance time. This criterion assumes a rectangular heat pulse, and its validity depends on this assumption.

In contrast, Henrique's is based on a chemical rate process and uses temperature and time to determine skin damage [37]. Skin-simulant sensors are used in modern thermal manikin experiments to predict third-degree burns using Henrique's burn integral, Ω, as shown in Eq. 1. The values of physical constant have been presented in **Table 1**. While Stoll's criterion is easy to use, Henrique integral has shown more accurate predictions for incident heat flux range not covered experimentally by Stoll. Henceforth, it is a preferred choice when assessing superficial burns at heat fluxes above 41 kW*=*m2.

$$\Omega = \int\_0^t \text{Pexp}\left(-\frac{\Delta \mathcal{E}}{\text{RT}}\right) \,\text{d}t \tag{1}$$

Ω, Henrique integral, second-degree burn occurs when *Ω* is unity

ΔE, the activation energy (J/mol) P, a pre-exponential factor

T, time-dependent absolute temperature of the basal layer.

#### **Table 1.**

*Henrique burn model physical constant values [8, 38].*

## **2. Experimental methodology**

To conduct novel tests on the thermal protective garment subjected to a fire load of 126 kW*=*m2, a purpose-built experimental apparatus was designed by cooperation between the Korean Conformity Laboratories (KCL) and Ulster University (UU), as shown in **Figure 1**. It comprises a radiant panel, a specimen assembly and a trolley. Two-layered halogen quartz tubes can output a consistent radiant flux of 126 kW*=*m2 for more than 60 seconds. More details on the development, configuration and limitations of the test apparatus can be found in the preliminary study of KCL [39].

The experimental setup consists of an outer shell (OS), a moisture barrier (MB), a thermal liner (TL) and a substrate. The addition of a substrate represents closed-back configuration with variations in the air gap between the skin and the innermost layer [40, 41]. The temperature measurement at the back surface of the thermal liner is vital. Therefore, four type-K thermocouples were prepared to record the thermal liner back surface temperature. Constant contact is needed between the thermocouples and thermal liner back surface, for which sewing of the thermocouple was first implemented, as suggested by the National Institute of Standard and Technology (NIST) [42]. However, it was difficult to keep the bead in contact with fabric; plus, sewing made it demanding to retain the stripped wire away from the fabric surface. As four thermocouples are required to be placed in proper contact with the back surface, sewing proved to be impractical. Additionally, the application of heat-resistant tape is not recommended [42]. To overcome this, a new mechanism was developed based on pressure contact.

Thermocouple beads were soldered to a copper sheet of circular cut with a thickness of 0.35 mm. Thermocouple wires ran across the slits on a rod made of high thermal-resistant plastic. It was then put through circular holes in the substrate. The distance between the copper sheet and the circular rod was kept to greater than 6 mm to ensure proper air circulation. In this manner, four probes were prepared and attached at four distinct points of the fabric back surface. This method of pressure contact can record the spatial variations of the temperature, and it has been validated [43].

**Figure 1.** *Bench-scale apparatus.*

*Thermal Protective Performance of Turnout Gear at High Flux Environment DOI: http://dx.doi.org/10.5772/intechopen.114293*

**Figure 2.** *Turnout gear layup for experimental studies.*

Three types of fabric configurations, Type A, B and C as described in **Figure 2**, were examined at two different exposure levels of 84 and 126 kW*=*m2, representative of flashover and post-flashover. Specimens were exposed to the incident radiant flux for three different exposure times, of 10, 20 and 25 seconds. Type-A configuration is of a conventional layup. It is commercially available in the market and, therefore, is treated as a benchmark to access the performance of Type B and C. A 2 mm (2A) air spacing is ensured between the fabric layers and 6.5 mm between TL and substrate as per research standards [29, 41, 42, 44].


**2. Preventive awareness and control care for oral cancers**

Preventive medicine acts as a unique control for oral cancers and also charges at all-time low costs to states across the world [2, 15]. In this way, many actions can be taken by health organizations, especially by the World Health Organization in the risky geographic regions of the world [28]. Prevention of oral cancer is mainly a management of those diseases, so public awareness should be the first priority for health workers (e.g., by using media to promote fast outcomes) [3, 14]. For awareness about encountering precancerous lesions [1, 22, 28], Jeihooni et al. [9] reported that 30.4% of patients had preventable oral cancer, while 50% did not, and the remaining 19.6% of patients were unclear as to whether or not oral cancer is preventable.

To prevent oral cancers, the etiological factors should be known, and measures must be taken according to those causes. Etiological factors are tobacco use, especially in individuals older than 40 years of age, alcohol consumption, betel chewing, HPV infection or carriage, dietary intakes such as salted meat consumption, poor oral hygiene, genetic predisposition, Epstein-Barr virus (EBV) infection, long-term exposure to sunlight, and immune system disease such as organ transplant recipients and HIV-infected patients, especially as it causes lip cancer [1, 3, 4, 9, 13, 14, 29, 30].

HPV-related oral cancers, especially oropharyngeal cancers, differ from other cause-related cancers as HPV-related cancers have been seen in young, non- or former tobacco users, nonalcoholic patients, and also patients with small T tumors but with nodal involvement (fortunately, their prognosis is more satisfactory than non-related HPV tumors). In addition, HPV-related tumors are well-defined borderline, more exophytic with smaller sizes and lymph node metastasis with dominant cystic features [19]. To prevent HPV-related oral cancers, vaccination against HPV should be performed to decrease the incidence of oropharyngeal SCC [31], whereas Owosho et al. [31] reported that HPV-related oropharyngeal cancer reached its highest rate in the year 2006 and has since started to decline but not at a statistically significant rate. In the literature, vaccination programs have primarily focused on HPV infection related to cervical cancer [1]. Thus, Center for Disease Control and Prevention (CDC) is recommending that the vaccination program for HPV-related oropharyngeal cancers should be carried out for children at ages 11–12 in two doses by health providers [31].

multidisciplinary approach, including oral and maxillofacial surgeons, oral and maxillofacial radiologists, ENT specialists, medical and radiological oncologists, prosthodontists, dentists, general surgeons, primary care clinicians (such as family medicine), physical therapy and rehabilitation specialists, dietitians, speech therapists, supportive care experts, and also

After radical, surgical, and other treatment modalities such as radiotherapy and chemotherapy especially in stages III and IV patients—improving a patient's quality of life is very important [7, 14, 24, 26, 27]. Besides these, if patients have more advanced surgical (and other) treatment modality, the important thing is, of course, survival. Also, the second issue is improving quality of life. In this aspect, prosthodontics, speech therapy, dental implant-supported restoration with oral and maxillofacial surgeons, and other supportive specialties such as providers like physical therapists, mental health professionals, and dietitians and applications improve

Preventive medicine acts as a unique control for oral cancers and also charges at all-time low costs to states across the world [2, 15]. In this way, many actions can be taken by health organizations, especially by the World Health Organization in the risky geographic regions of the world [28]. Prevention of oral cancer is mainly a management of those diseases, so public awareness should be the first priority for health workers (e.g., by using media to promote fast outcomes) [3, 14]. For awareness about encountering precancerous lesions [1, 22, 28], Jeihooni et al. [9] reported that 30.4% of patients had preventable oral cancer, while 50% did not, and the remaining 19.6% of patients were unclear as to whether or not oral cancer is preventable. To prevent oral cancers, the etiological factors should be known, and measures must be taken according to those causes. Etiological factors are tobacco use, especially in individuals older than 40 years of age, alcohol consumption, betel chewing, HPV infection or carriage, dietary intakes such as salted meat consumption, poor oral hygiene, genetic predisposition, Epstein-Barr virus (EBV) infection, long-term exposure to sunlight, and immune system disease such as organ transplant recipients and HIV-infected patients, especially as it causes lip cancer [1,

HPV-related oral cancers, especially oropharyngeal cancers, differ from other cause-related cancers as HPV-related cancers have been seen in young, non- or former tobacco users, nonalcoholic patients, and also patients with small T tumors but with nodal involvement (fortunately, their prognosis is more satisfactory than non-related HPV tumors). In addition, HPV-related tumors are well-defined borderline, more exophytic with smaller sizes and lymph node metastasis with dominant cystic features [19]. To prevent HPV-related oral cancers, vaccination against HPV should be performed to decrease the incidence of oropharyngeal SCC [31], whereas Owosho et al. [31] reported that HPV-related oropharyngeal cancer reached its highest rate in the year 2006 and has since started to decline but not at a statistically significant rate. In the literature, vaccination programs have primarily focused on HPV infection related

pathologists or, if possible, oral and maxillofacial pathologists [2, 24, 25].

**2. Preventive awareness and control care for oral cancers**

a patient's quality of life [2, 24].

54 Prevention, Detection and Management of Oral Cancer

3, 4, 9, 13, 14, 29, 30].

Other efforts to prevent oral cancer should be done by controlling tobacco use or reduction programs [3, 6, 7, 11, 14, 29]. Alcohol consumption should be reduced or cut off as well [3, 7, 14]. If tobacco and alcohol are used together by the same individuals, that increases the oral cancer rate and may be a dramatically worse prognosis than HPV-related cancers [3, 5, 7, 11]. Unfortunately, alcohol is related to a very risky factor for cancer occurring even in nonsmoking individuals [3, 5, 7].

On the other hand, improving especially rich vegetable and fruit diet intake awareness programs, physical activity-increasing programs in daily life, regular sexual education for adolescents, and oral hygiene education activities may be helpful for reducing oral cancer rates in the future [1, 14, 32]. Awareness programs and activities should be conducted by healthcare providers or governments worldwide, especially in more risky regions such as Southeast Asia. For example, images of early oral cancer can be placed on cigarette packages [4].

In the risky region, or those patients that have high risk, they should be encouraged to carry out self-inspection of their oral mucosa, and if they see any mouth abnormality, they are obligated to take professional advice or treatment [4]. In addition, oral hygiene education and encouragement to improve it in individuals are key factors in preventing or early detection of oral cancer [9]. Moreover, Rahmati-Najarkolaei et al. [32] reported that awareness activities with students about risky factors decreased fear of oral cancer and also changed their attitude about knowing whether they have oral cancer. Lack of public awareness about cancer-causing factors and predictors produces diagnosis of oral cancer at stages as late as III or IV [4]. Therefore, awareness may provide early diagnosing of lesions; it reduces the cost of treatment and also reduces treatment morbidity [16, 22].

To date, many strategies have experimented with community awareness in the literature, such as mass media campaigns, which have gained some success from increased public awareness [1, 3, 8]. Unfortunately, these improvements are still not enough in terms of public awareness. Nowadays, Internet-based or online connections such as social media are very popular communication tools across the whole world. For example, the online-supported education or activities for tobacco cessation counseling revealed limited success in a randomized experiment [1, 8]. Internet-based education also applied for alcohol counseling with some success gained [1]. Billboards have also not been successful in delivering improved awareness to risky populations [1]. Prevention is the main management of oral cancers, and public awareness is a key part of prevention: where, when, and how it should be done and how it is possible is not important.

#### **2.1. Look and see: precancerous mouth lesions**

Many types of oral lesions have malignant transformation potential, aptly named premalignant or precancerous lesions. Premalignant lesions are described by using clinical, pathological, histochemical, and also many other screening techniques for which lesions such as leukoplakia, lichen planus with oral and cutaneous form, erythroplakia, stomatitis nicotina, and submucous fibrosis are named or classified [3, 10, 15, 18, 22, 33–35]. In addition, there is less malign transformation potential in lesions such as discoid lupus erythematosus and also a lesser likelihood of malignant transformation in hereditary entities such as epidermolysis bullosa and dyskeratosis congenita [34]. For the lip cancer, there are precancerous lesions like xeroderma pigmentosum, radiodermatitis, and chronic cheilitis [18]. However, histological investigation has only produced knowledge about malignant transformation potential named dysplasia—and has been indicated or used as a prediction of malignant changes [34]. According to the WHO's (2005) statement, carcinoma in situ has the highest degree of dysplasia and is also defined as a premalignant lesion [3, 10, 34]. Premalignant lesions are usually clinically screened for mouth mucosa such as white, red, or red-white mixed patches and are also called leukoplakia or erythroplakia [34, 35].

and malignant cells have higher nucleic acid content; hence, it is stained with dyes that can be identified under the microscope due to nucleic acid and have been used since 1980 [34]. Toluidine blue stain, ViziLite, and VEL scope sensitivity and specificity in oral dysplasia patients are presented by Awan et al. [37] to be 84.1 and 77.3%, 15.3 and 27.8%, and 65.8 and

Early Detection and Multidisciplinary Approach to Oral Cancer Patients

http://dx.doi.org/10.5772/intechopen.81126

57

Exfoliative cytology procures cells from a wide surface area of the affected tissue with fewer invasive effects on the tissue than biopsy. It also involves a lower contamination risk prior to DNA obtaining than surgical intervention [5]. Exfoliative cytology is obtained by scrapping the mucosa lesion using a blade as tongue blade. The obtained material is spread on a dry, clean glass lam and fixed with 100% ethanol. The lam is then sent to an experienced pathologist for examination under a light microscope for dysplastic evidences in the cells [35]. In addition, serum and saliva are the most commonly used as less invasive, easily accessible, cost-effective, and convenient samples for cancer detection [15]. The sensitivity and specificity of exfoliative cytology for oral cancer detection were revealed as 93.5 and 50.6%, respectively [15]. Although exfoliative cytology is less invasive, it is highly subjective and dependent upon the expertise of examiners. Moreover, exfoliative cytology related to the DNA aneuploidy and quantitative cytomorphometry has low specificity due to the collection of disassembled cells [15]. If the exfoliative cytology is used on heavy smoker and alcohol-using patients with negative malignancy findings, a biopsy procedure should be carried out as an additional supportive test [22]. OralCDx or Orcellex® brush cytology is a bit advanced for complementary forms of the exfoliative cytology, due to it including representative cells of all layers of epithelial tissue [34, 38]. Moreover, brush cytology has provided diagnostic accuracy because of computer assistance screening [34, 38]. Studies demonstrated that OralCDx or Orcellex® cytology has a potential value as an adjunct to oral diagnosing or screening in identifying premalignant pathologies at early stages that provides surgical or curative treatment that is most effective [39]. In the future, developing automatic, cytometric, or cytomorphometric techniques combined with genetic and related features may enhance screening strategies [34]. Affecting tissue with any pathology sample technique is still recommended if there is a strong suspicion of any lesion

with malignancy regardless of the oral brush cytology result [34].

Biological, chemical, or reactional molecular agents named cell markers and biomarkers mean that signs of living organisms and the obviousness of their availability as tumor necrosis factor-alpha (TNF-α), epithelial growth factor (EGFR), vascular endothelial growth factor (VEGF), IL-8 and IL-8 mRNA, and interleukin 6 (IL-6) [10, 15]. Biomarker investigation of abnormalities of oral tissues as normal, tumorous, and inflammatory keratinocyte proteomes is likely to find new biomarker agents for oral cancer diagnosis, treatment, follow-up, and the development of personalized therapies for oral cancer and other tumorous regions [16].

The most visible oral premalignant or precancerous lesion is oral leukoplakia (OL) that has been studied for its establishment of a biomarker that signals the malign transformation of OL [22]. For instance, OL has low prevalence in western countries, so the development of a new biomarker is challenging due to the low rate of malignant transformation, and it requires long follow-up periods to achieve a new biomarker [22]. Besides these, loss of heterozygosity (LOH) was described as the strongest and most valuable biomarker by Mao et al. at the end

56.8%, respectively.


**1. Introduction**

Infective endocarditis (IE) risk is strongly associated with underlying cardiac disease. This chapter will review the pathology, mechanisms and estimated risks according to lesionspecific groups. Echocardiographic predictors of IE will be discussed along with the increasingly reported occurrence of IE in 'normal valves'.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


*2.7. Heterotrophy*

Heterotrophic carbon can become a significant energy source for some coral species when phototrophic carbon is unavailable, such as during a bleaching event (Table 1) [118]. Some studies have shown how heterotrophy replenished energy reserves in corals exposed to high temperatures [119] and during the recovery phase [120]. Similarly, Borell and Bischof [121] showed higher photochemical efficiency in fed corals compared to unfed corals after a mild thermal stress experiment. Also, Borell et al. [122] demonstrated how heterotrophy sustained photosynthetic activity and energy reserves in thermally stressed corals.

In a study which developed an energy-budget model linking coral bleaching and mortality risk, authors concluded that the time between the start of severe bleaching and the beginning of mortality is influenced by the amount of lipid stores corals have before the bleaching event and their capacity to acquire energy through heterotrophy [123]. With a stable isotope 13C pulse-chase labelling experiment, Hughes et al. [124] demonstrated that, after exposure to high temperatures, coral hosts incorporated heterotrophic labelled carbon for storage and to stimulate endosymbiont recovery. Even after recovery from bleaching, 75% of carbon in newly acquired lipids was sourced heterotrophically [125], and corals continued assimilating heterotrophic carbon for up to 11 months after the bleaching experiment [126].

Nonetheless, the capacity for heterotrophic plasticity is compromised after two consecutive bleaching events [127]. Researchers experimentally bleached corals for 2.5 weeks, transferred corals to the field for recovery, and then repeated the bleaching experiment after one year. After the first thermal stress experiment, zooplankton and dissolved organic carbon (DOC) allowed the metabolic demand of bleached corals to be met; however, neither form of heterotrophic carbon was able to contribute to the energy budget of both species after the second bleaching experiment, suggesting that the capacity for heterotrophic plasticity is compromised under annual bleaching events [127] and corals need to depend on their energy reserves and/or symbiont association to survive repeated bleaching [128].

#### **3. Perspectives for the Future**

Because the loss of corals around the world would be a devastating consequence of human influence on earth, strategies to mitigate the damage and improve coral's thermal tolerance are currently being taken into consideration. For example, assisted colonization, migration and/or gene flow contemplate the movement of colonies or larvae of the same species living at different latitudes. 'Warm-adapted' corals can be transplanted to high latitude areas, where conspecifics living in colder environments, are vulnerable to thermal stress [129–131]. Assisted evolution has the potential to increase thermal stress tolerance in corals through various approaches: preconditioning acclimatisation (see Section: 'Thermal stress acclimatisation') and trans-generational acclimatisation, changes in microbial communities [132], selective breeding [133], mutagenesis [134], and the use of "CRISPR/Cas9" genome editing technology [135]. The use of 'strong corals' naturally adapted to high temperature extremes, such as corals originating from the Persian Gulf or Red Sea, as possible seedlings to repopulate areas where corals have disappeared [117,136–138] is also being considered. Unfortunately, none of

these measures seem to be able to keep pace with the current rate of climate change, with the time between recurrent bleaching events becoming increasingly too short to allow complete recovery of coral reef ecosystems [139]. Despite recent advances in research methods and technology, such as transcriptomics [140], financial and logistical limitations to implement these actions remain [141], especially at large scales [130], and it takes many years to safely deploy new technology after social and political scrutiny [142].

Other conservation measures under consideration include designing better marine protected areas (MPAs) [143] or networks of MPAs [144–146], taking into consideration larval dispersal, connectivity and distribution patterns in areas with thermally tolerant corals [147] and including 'refugia' in areas where coral reefs have proven to be resilient to climate change [21,43,148,149]. This might help avoid the "protection paradox" in MPAs, in which vulnerable species are protected from local pressures, like fishing; yet while these species recover, they might be more sensitive to global pressures, like bleaching events [144]. Nevertheless, well-protected reefs within MPAs are not shielded from thermal stress [150,151]. After the last bleaching event, this was confirmed for MPAs [152], and for remote and isolated reefs with almost no direct human pressures [23,24,27,153–155].

The integration of assisted evolution [131,134] into coral reef restoration programs [156,157] to increase the resilience of already degraded ecosystems [41] is one strategy that has proven to be successful. Morikawa and Palumbi [158] used naturally thermal-tolerant corals from American Samoa to show that resilient corals can survive multiple bleaching events, providing the first proof that ecosystem engineering for conservation might be a resilience restoration tool of great importance in our climate changed future [158].

Evidence from reciprocally transplanted coral clones between sites with different thermal histories shows how individual coral colonies can shift their thermal threshold and thermal tolerance [93,159,160]. It is clear that many coral species are acclimatising and adapting to rapid changes in climate and their mechanisms differ among species and localities [67,82,83,89,90,113,115,161]. However, under current greenhouse gas emission projections, coral reefs worldwide are likely to change into new configurations with new assemblages of species [19,149,162–165]. These changes are happening fast, the GBR being the best example. After the 2014–2017 mass bleaching event, even the most 'pristine' areas in the northern GBR saw high mortality regardless of reefs' individual management status, proving that current management toolsets are insufficient to protect coral reef ecosystems from climate change [20,152]. The Paris Agreement was a first step to tackle the climate crisis, but no major industrialized country is meeting its pledges to control and reduce their greenhouse gas emissions [166]. It is imperative that societies completely change our dependence on fossil fuels, therefore addressing the root causes of climate change.

**Author Contributions:** Conceptualization, R.C.-B. and C.A.C.; methodology, R.C.-B.; software, R.C.-B.; validation, R.C.-B. and D.S.; formal analysis, R.C.-B.; investigation, R.C.-B.; resources, C.A.C.; data curation, R.C.-B.; writing—original draft preparation, R.C.-B. and D.S.; writing—review and editing, R.C.-B., D.S. and C.A.C.; visualization, R.C.-B.; supervision, C.A.C.; project administration, C.A.C.; funding acquisition, C.A.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Academia Sinica for Life Science Research (no. 4010) and Ministry of Science and Technology, Taiwan (MOST 101-2621-B-001-005-MY3) to C.A.C.

**Acknowledgments:** Many thanks to LK Chou and D Huang for their invitation to contribute to the special issue of "Coral Reef Resilience". We also thank three anonymous reviewers for helping improve this manuscript. R.C.-B. and D.S. are the receipts of PhD fellowship from Taiwan International Graduate Program (TIGP)-Biodiversity in the Academia Sinica. This review is supported by funds from Academia Sinica and Ministry of Science and Technology, Taiwan to C.A.C.

**Conflicts of Interest:** The authors declare no conflict of interest and the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.


**3. High average power wavelength conversion of picosecond solid-state lasers**

The extreme ultraviolet lithography is now in an introductory phase in semiconductor industry. The EUVL has been developed in the field of various component technologies such as Mo/Si high reflectivity mirror at 13.5 nm wavelength, new types of resist of higher sensitivity at this wavelength, and plasma-based 100 W class stable EUV sources. Further increase in average power is expected for large-scale manufacturing to kW level and shorter wavelength to 6.7 nm where a higher reflectivity mirror seems available. The present source architecture is the laser-produced plasma (LPP) and is recently considered in its practical scaling limitation in average power in the range of kW. Free electron laser has been emerging as the new shortwavelength source in the EUV to X-ray region in the past decade. The present generation is based on lower repetition rate operation for scientific applications, but the next generation is aiming at high repetition rate for high average power. Several research papers are discussing on the possibility of high repetition rate FEL by superconducting RF cavity technology for the generation of more than kW average power at 13.5 nm wavelength [28, 29]. The present FEL is operated in the SASE mode, in which the pulses are generated in undulator and composed of many short-pulse length spikes. The typical pulse parameters are 0.1 mJ pulse energy, 100 fs pulse length, and the beam diameter is 1 mm. The beam fluence is higher than the ablation threshold of a resist [30], and the high spatial coherence results in much higher localized peak fluence on the resist. The interaction mechanism is now in a basic study to overcome these effects compared to the present LPP-generated 100 kHz, mJ EUV pulses with no coherence and longer pulse length as 10 ns.

The scaling of the FEL technology to kW average power level requires the photocathode operation in higher repetition rate in industrial environment together with optical technology to optimize the FEL beam for lithography application and scaling to the 6.7 nm wavelength region.

The first consideration is the industrial operation of photocathode at >MHz repetition rate. The bunch charge is typically 1 nC. Metal photocathode is robust, but the quantum efficiency (QE) is lower for higher charge generation. Several semiconductor cathodes were studied for higher efficiency to reduce the requirement for the driver laser average power in the repetition rate mode. The Advanced Photo-Injector (APEX) experiment in Lawrence Berkley National Laboratory is working to realize a high repetition rate at MHz, high-brightness photocathode. The photocathode is a normal conducting, 187 MHz RF cavity in the CW mode, and designed for short bunches as 1–10 ps of 750 keV energy up to 1 nC charges. Several semiconductor cathode materials are tested for better beam emittance for various operational conditions. CsK2Sb is irradiated by SHG of Yb fiber pulses, and Cs2Te is irradiated by 4HG. Both semi‐ conductor cathodes have nearly 1% quantum efficiency. The laser pulse energy is 0.5 μJ with the MHz repetition rate, and the average power is 0.5 W [31]. Cu and Mg photocathodes were studied for use in an RF photocathode. The gun was manufactured by a technique of hot isostatic pressing with diamond polishing and tested under a peak electric field of 57 MV/m. The quantum efficiency of the Cu cathode was 10−4, while Mg cathode achieved a high QE of up to 10−3 under 262 nm laser-light illumination. The QE of the Mg cathode under 349 nm laserlight illumination was measured to be 2.2 × 10−5. The experimental setup and the results of the photocathode QE measurement are shown in **Figure 9** [32].

**3. High average power wavelength conversion of picosecond solid-state**

The extreme ultraviolet lithography is now in an introductory phase in semiconductor industry. The EUVL has been developed in the field of various component technologies such as Mo/Si high reflectivity mirror at 13.5 nm wavelength, new types of resist of higher sensitivity at this wavelength, and plasma-based 100 W class stable EUV sources. Further increase in average power is expected for large-scale manufacturing to kW level and shorter wavelength to 6.7 nm where a higher reflectivity mirror seems available. The present source architecture is the laser-produced plasma (LPP) and is recently considered in its practical scaling limitation in average power in the range of kW. Free electron laser has been emerging as the new shortwavelength source in the EUV to X-ray region in the past decade. The present generation is based on lower repetition rate operation for scientific applications, but the next generation is aiming at high repetition rate for high average power. Several research papers are discussing on the possibility of high repetition rate FEL by superconducting RF cavity technology for the generation of more than kW average power at 13.5 nm wavelength [28, 29]. The present FEL is operated in the SASE mode, in which the pulses are generated in undulator and composed of many short-pulse length spikes. The typical pulse parameters are 0.1 mJ pulse energy, 100 fs pulse length, and the beam diameter is 1 mm. The beam fluence is higher than the ablation threshold of a resist [30], and the high spatial coherence results in much higher localized peak fluence on the resist. The interaction mechanism is now in a basic study to overcome these effects compared to the present LPP-generated 100 kHz, mJ EUV pulses with no coherence

The scaling of the FEL technology to kW average power level requires the photocathode operation in higher repetition rate in industrial environment together with optical technology to optimize the FEL beam for lithography application and scaling to the 6.7 nm wavelength

The first consideration is the industrial operation of photocathode at >MHz repetition rate. The bunch charge is typically 1 nC. Metal photocathode is robust, but the quantum efficiency (QE) is lower for higher charge generation. Several semiconductor cathodes were studied for higher efficiency to reduce the requirement for the driver laser average power in the repetition rate mode. The Advanced Photo-Injector (APEX) experiment in Lawrence Berkley National Laboratory is working to realize a high repetition rate at MHz, high-brightness photocathode. The photocathode is a normal conducting, 187 MHz RF cavity in the CW mode, and designed for short bunches as 1–10 ps of 750 keV energy up to 1 nC charges. Several semiconductor cathode materials are tested for better beam emittance for various operational conditions. CsK2Sb is irradiated by SHG of Yb fiber pulses, and Cs2Te is irradiated by 4HG. Both semi‐ conductor cathodes have nearly 1% quantum efficiency. The laser pulse energy is 0.5 μJ with the MHz repetition rate, and the average power is 0.5 W [31]. Cu and Mg photocathodes were studied for use in an RF photocathode. The gun was manufactured by a technique of hot isostatic pressing with diamond polishing and tested under a peak electric field of 57 MV/m. The quantum efficiency of the Cu cathode was 10−4, while Mg cathode achieved a high QE of

**lasers**

112 High Energy and Short Pulse Lasers

and longer pulse length as 10 ns.

region.

**Figure 9.** Left: Experimental setup of photocathode QE measurement. Right: Electron charge vs. input laser energy (266 nm) from Mg photocathode, • is for before laser cleaning.

It is concluded that 5 μJ, 266 nm, picosecond pulse is enough for Mg photocathode operation and the required average power at the MHz repetition rate is 5 W. Cu photocathode is proven to be robust material and usable by a 50 W 4HG picosecond laser. The progress of the laser technology is now making the metal photocathode again usable for the emerging requirement for long-life industrial application.

The other consideration is the reduction of the temporal microspikes in the SASE FEL pulses. Coherence is characterized in a report on the FLASH operation at 8.0 nm wavelength [33]. The single FEL femtosecond beam is passed through double pinholes for diffraction pattern, and the measured transverse coherence length is 6.2 ± 0.9 μm in the horizontal and 8.7 ± 1.0 μm in the vertical directions. The mutual coherence function K is given as 0.42, and a measurement of K by a laser plasma source is 3.2 × 10−9. It is concluded from these measurements that a beam spatial homogenization is required at EUV wavelength by using total reflection. Temporal coherence was also reported by using a split and delay unit. The coherence time of the pulses produced in the same operation conditions of FLASH was measured to be 1.75 fs. The measured coherence time has a value, which corresponds to about 65.5 ± 0.5 wave cycles (*cτ*/*λ*). It is well known that the SASE FEL pulses are composed of many small spikes and random spectrum due to SASE process. It is reported that the averaged spectrum has a 1.4% bandwidth typically, which is favorable for the Mo/Si EUV multilayer mirror at 13.5 nm (bandwidth 2%). It is necessary to smooth the temporal spikes to avoid random EUV flux change in the resist absorption process. The requirement is similar to most FEL applications, and we must consider efficient seed technology for MHz repetition rate operation.

It is desirable to increase the brightness and pointing/energy stability compared to SASE mode. An efficient seeding method was established by using a UV wavelength laser, in which the seed laser modulates the electron beam into coherent bunching at the harmonics of the seed laser wavelength. The bunching is intensified in another undulator for coherent FEL action, and the method is called as high-gain higher harmonic generation (HGHG). FERMI is the leading institute in this specific technology, and it is reported on the double-stage-seeded FEL with the fresh bunch injection technique [13]. The main limitation for the direct extension of the HGHG to shorter wavelength is the required small electron beam energy spread and higher average power seed laser source. The fresh bunch scheme is the solution for this problem, in which the FEL radiation is initially produced in an earlier stage undulator and used as the seeder for shorter wavelength generation (**Table 1**).

**Table 1.** Comparison of wavelengths for HGHG operation in FERMI FEL-2 and EUV FEL.

The external seed laser was the third harmonic of a Ti:Sapphire laser with a duration of ∼180 fs (FWHM) and up to 20 μJ energy per pulse. Its transverse size in the modulator was made larger than the electron beam size to ensure as uniform as possible electron beam energy modulation. Once the same laser energy is required for MHz EUV FEL, 20 W average power is required for 324 nm with 180 fs at MHz repetition rate. There are two approaches to generate such laser pulses, the first is based on the MHz repetition rate Ti:Sapphire laser with 100 μJ level pulses, and the second one is based on OPCPA.

The short-pulse, short-wavelength laser technology is now advancing to realize the specifica‐ tion described here in a compact box, due to the new suitable laser configuration as thin-disc laser and an efficient wavelength conversion method.

An ultrafast thin-disc multipass laser amplifier demonstrated the advantage recently by delivering 1.4 kW of average output power with 4.7 mJ pulse energy and duration of 8 ps at a repetition rate of 300 kHz [18]. The beam quality factor was better than M2 = 1.4. The experi‐ ments show that the thin-disc multipass amplifier can scale pulse energy and average output power independently for the investigated repetition rates between 300 and 800 kHz. Frequency doubling by means of an LBO crystal generated 820 W SHG average power at the wavelength of 515 nm with 1170 W of incident IR power, which corresponds to a conversion efficiency of 70% and an SHG pulse energy of 2.7 mJ. By sum-frequency generation between the beams at 1030 and 515 nm in a second LBO crystal, an average UV power of 234 W (780 μJ of pulse energy) was generated at the wavelength of 343 nm THG with a conversion efficiency of 32%.

A wavelength conversion experiment was performed in the HiLASE project to evaluate the high average power generation of picosecond harmonics, namely, SHG (515 nm) and FHG (257.5 nm), in LBO and BBO/CLBO crystals, respectively [34]. The pumping of the crystals was performed by the PERLA C Yb:YAG thin-disc laser operating at 100 kHz and 60 W average power with 4 ps pulse duration. The average output power of 6 W DUV was achieved in CLBO at a spectral bandwidth of 0.2 nm and the FHG/fundamental conversion efficiency was 10%. The basic optical configuration is shown in **Figure 10** together with a photo. The input beam (upper left) is reflected by two motorized mirrors controlled by a beam stabilizer ensuring pointing stability better than 20 μrad (RMS). The following half-wave plate and polarizer is used for energy tuning. The beam is frequency doubled in an LBO at 50°C and 10 mm long, cut for the critically phase-matched generation at *θ* = 90° and *ϕ* = 12.8°, and antireflection coated for 1030 and 515 nm. The second harmonic beam passes two dichroic mirrors and is injected into an argon-filled box with a BBO or CLBO crystal. To ensure a stable long-term functioning of the crystals the temperature was kept at 150°C. The experimental results are shown in **Figure 11**. It is visible that the 4HG/SHG conversion in the CLBO crystal has 30% higher efficiency than in the BBO crystal. The next step of the experiment is to increase the pumping power to 500 W to confirm the linearity of the conversion efficiency for 50 W FHG output power.

laser wavelength. The bunching is intensified in another undulator for coherent FEL action, and the method is called as high-gain higher harmonic generation (HGHG). FERMI is the leading institute in this specific technology, and it is reported on the double-stage-seeded FEL with the fresh bunch injection technique [13]. The main limitation for the direct extension of the HGHG to shorter wavelength is the required small electron beam energy spread and higher average power seed laser source. The fresh bunch scheme is the solution for this problem, in which the FEL radiation is initially produced in an earlier stage undulator and used as the

The external seed laser was the third harmonic of a Ti:Sapphire laser with a duration of ∼180 fs (FWHM) and up to 20 μJ energy per pulse. Its transverse size in the modulator was made larger than the electron beam size to ensure as uniform as possible electron beam energy modulation. Once the same laser energy is required for MHz EUV FEL, 20 W average power is required for 324 nm with 180 fs at MHz repetition rate. There are two approaches to generate such laser pulses, the first is based on the MHz repetition rate Ti:Sapphire laser with 100 μJ

The short-pulse, short-wavelength laser technology is now advancing to realize the specifica‐ tion described here in a compact box, due to the new suitable laser configuration as thin-disc

An ultrafast thin-disc multipass laser amplifier demonstrated the advantage recently by delivering 1.4 kW of average output power with 4.7 mJ pulse energy and duration of 8 ps at a repetition rate of 300 kHz [18]. The beam quality factor was better than M2 = 1.4. The experi‐ ments show that the thin-disc multipass amplifier can scale pulse energy and average output power independently for the investigated repetition rates between 300 and 800 kHz. Frequency doubling by means of an LBO crystal generated 820 W SHG average power at the wavelength of 515 nm with 1170 W of incident IR power, which corresponds to a conversion efficiency of 70% and an SHG pulse energy of 2.7 mJ. By sum-frequency generation between the beams at 1030 and 515 nm in a second LBO crystal, an average UV power of 234 W (780 μJ of pulse energy) was generated at the wavelength of 343 nm THG with a conversion efficiency of 32%.

A wavelength conversion experiment was performed in the HiLASE project to evaluate the high average power generation of picosecond harmonics, namely, SHG (515 nm) and FHG (257.5 nm), in LBO and BBO/CLBO crystals, respectively [34]. The pumping of the crystals was performed by the PERLA C Yb:YAG thin-disc laser operating at 100 kHz and 60 W average power with 4 ps pulse duration. The average output power of 6 W DUV was achieved in CLBO

**Fermi FEL-2 EUV FEL**

seeder for shorter wavelength generation (**Table 1**).

114 High Energy and Short Pulse Lasers

level pulses, and the second one is based on OPCPA.

laser and an efficient wavelength conversion method.

Seed wavelength (nm) 260 324 1nd FEL (nm) 32 40.5 2nd FEL (nm) 10.8 13.5

**Table 1.** Comparison of wavelengths for HGHG operation in FERMI FEL-2 and EUV FEL.

**Figure 10.** Optical configuration of the SHG and FHG, and SHG light is introduced into a box filled with argon.

**Figure 11.** Left: Fourth harmonic output power dependence on the second harmonic in BBO (AR coated) and CLBO (uncoated) crystals. Right: Relevant FHG spectra from CLBO.

A small part of the beam is absorbed in the crystal and converted into heat that leads to temperature gradients in the crystal in the high average power wavelength conversion. This causes partial phase mismatch and reduces the conversion efficiency. It is estimated that the fundamental power absorption at the 60 W input is <20 mW in the 10 mm long LBO crystal. The total absorbed power may be higher due to the fact that a green laser beam has higher absorption than the fundamental beam [35]. The absorption in the antireflective coating increases the temperature, which causes the mismatch more than the bulk absorption [36].

A tunable, 112 W optical parametric chirped-pulse amplifier (OPCPA) was demonstrated for FEL seeding in a burst mode with center frequencies ranging from 720 to 900 nm, pulse energies up to 1.12 mJ, and a pulse duration of 30 fs at a repetition rate of 100 kHz [14]. The results demonstrated the feasibility of 112 W femtosecond OPCPA in a burst mode with a duty cycle of 8 × 10−3, where no heating effects were observed. It was indicated from the measurements of absorption coefficients of BBO and LBO and calculations, the feasibility of much higher powers up to 1 kW in continuous mode was expected. Absorption causes a spatially and temporally varying temperature distribution in the sample. This leads to local changes of the refractive index and results in the development of a thermal lens. Especially in anisotropic crystals, this has consequences on increased phase mismatch in optical parametric processes with a conversion efficiency decrease. In the case of anisotropic crystal in electro-optical devices such as Pockels cells, the thermally induced depolarization reduces the contrast ratio. Though the absorption of the anisotropic crystal in these applications is usually very low, the related effects can be significant with input powers at the kilowatt level. In order to estimate the influence of thermal effects and taking it into account in the optical system design, the comprehensive knowledge of material absorption at the operation wavelength is unavoidable. An extended photothermal method was demonstrated for the quantitative determination of laser-induced wavefront deformations, which enables the separation of bulk and surface contributions to absorption in the more complex case of optically anisotropic crystalline media [36]. Experimental setup is shown in **Figure 12**. The wavefront deformations of the test beam (light source) are measured and used for absorption evaluations. The results show that the absorption is highest at the AR-coated KTP surface of input side (**Figure 13**, left), while it is higher at the surface of the output side of noncoated KTP (**Figure 13**, right). This photothermal method is usable in the real OPCPA for a better cooling system installation.

**Figure 12.** Setup of the photothermal method by crossed beam measurement. Wavefront measurement is performed by a Hartmann-Shack sensor.

High-Brightness Solid-State Lasers for Compact Short-Wavelength Sources http://dx.doi.org/10.5772/64147 117

**Figure 13.** Wavefront deformation measurement results for AR-coated (left) and noncoated KTP samples. Blue indi‐ cates the largest wavefront deformation. The heating laser beam comes into the crystal horizontally from the left, while the probe beam passes vertically to the readers.

#### **4. Cryogenic laser technology for high pulse energy picosecond amplifier**

The basic principle of the laser Compton short-wavelength source is similar to an undulator emission, and high-intensity laser field is used as the modulating electromagnetic field. Basic principle of the laser Compton X-ray source is well studied, and a single-shot imaging is critical for many practical applications. The required specification is explained as the laser pulse must exceed some threshold parameters. It is known that the highest peak brightness is obtained in the case of counterpropagating laser pulse and electron beam bunch, in the minimum focusing area before nonlinear threshold. **Figure 14** describes the schematic of the laser Compton interaction between the electron beam and the laser.

**Figure 14.** Schematic of the laser Compton scattering process.

A small part of the beam is absorbed in the crystal and converted into heat that leads to temperature gradients in the crystal in the high average power wavelength conversion. This causes partial phase mismatch and reduces the conversion efficiency. It is estimated that the fundamental power absorption at the 60 W input is <20 mW in the 10 mm long LBO crystal. The total absorbed power may be higher due to the fact that a green laser beam has higher absorption than the fundamental beam [35]. The absorption in the antireflective coating increases the temperature, which causes the mismatch more than the bulk absorption [36].

A tunable, 112 W optical parametric chirped-pulse amplifier (OPCPA) was demonstrated for FEL seeding in a burst mode with center frequencies ranging from 720 to 900 nm, pulse energies up to 1.12 mJ, and a pulse duration of 30 fs at a repetition rate of 100 kHz [14]. The results demonstrated the feasibility of 112 W femtosecond OPCPA in a burst mode with a duty cycle of 8 × 10−3, where no heating effects were observed. It was indicated from the measurements of absorption coefficients of BBO and LBO and calculations, the feasibility of much higher powers up to 1 kW in continuous mode was expected. Absorption causes a spatially and temporally varying temperature distribution in the sample. This leads to local changes of the refractive index and results in the development of a thermal lens. Especially in anisotropic crystals, this has consequences on increased phase mismatch in optical parametric processes with a conversion efficiency decrease. In the case of anisotropic crystal in electro-optical devices such as Pockels cells, the thermally induced depolarization reduces the contrast ratio. Though the absorption of the anisotropic crystal in these applications is usually very low, the related effects can be significant with input powers at the kilowatt level. In order to estimate the influence of thermal effects and taking it into account in the optical system design, the comprehensive knowledge of material absorption at the operation wavelength is unavoidable. An extended photothermal method was demonstrated for the quantitative determination of laser-induced wavefront deformations, which enables the separation of bulk and surface contributions to absorption in the more complex case of optically anisotropic crystalline media [36]. Experimental setup is shown in **Figure 12**. The wavefront deformations of the test beam (light source) are measured and used for absorption evaluations. The results show that the absorption is highest at the AR-coated KTP surface of input side (**Figure 13**, left), while it is higher at the surface of the output side of noncoated KTP (**Figure 13**, right). This photothermal

method is usable in the real OPCPA for a better cooling system installation.

by a Hartmann-Shack sensor.

116 High Energy and Short Pulse Lasers

**Figure 12.** Setup of the photothermal method by crossed beam measurement. Wavefront measurement is performed

The general formula of obtainable X-ray photon flux *N*<sup>0</sup> is calculated in the counter collision by the following expression:

$$\mathrm{N}\_{0} \propto (\sigma\_{\mathrm{c}} \,\mathrm{Ne} \,\mathrm{Np}) / (4\pi \mathrm{r}^{2})$$

where *σ*<sup>c</sup> is the Compton cross section (6.7 × 10−25 cm<sup>2</sup> ), Ne is the total electron number, Np is the total laser photon number, and *r* is the interaction area radius. It is predicted that an increase in Ne and Np, and the reduction in *r* results in the increase in the photon flux *N*0. The practical limitation of these operations are the instrumental condition of electron beam emittance in higher charge, M2 of laser beam at higher pulse energy, and optimization for reduced focusing diameter *r*. It is possible to assume these parameters as 1 nC charge with 3 ps pulse duration to be focused down to 10 μm at 38 MeV voltage. Another limitation is the maximum of singlepulse laser intensity to reach the nonlinear threshold of the higher harmonics generation in the X ray region. The nonlinear Compton threshold is characterized by the laser field strength

#### a0 eE/m LC = w

where parameters *E*, *ω*L, and *C* correspond to the amplitude of the laser electric field, laser frequency, and the speed of light, respectively. The laserfield strength is a function of the laser wavelength. The nonlinear threshold *a*<sup>0</sup> is given around 0.6 which corresponds to 1 J pulse energy in 1 ps pulse duration at 10 μm focusing intensity in the solid-state laser wavelength. The threshold laser energy for a single-shot imaging is similarto this critical laser pulse energy in the expected tight focus condition. The laser technology was not matured to realize such parameters simultaneously in the past, and usual approach was to increase the repetition rate of the event to increase the effective obtainable X-ray photon average flux in the affordable imaging time period such as <millisecond for bioimaging. The first approach is the pulsed laser storage in an optical enhancement cavity for laser Compton X-ray sources [37]. It is descri‐ bed in the experimental report that "the enhancement factor *P* inside the optical cavity was 600 (circulating laser power was 42 kW), in which the Finess was more than 2000, and the laser beam waist of 30 μm (2σ) was stably achieved using a 1 μm wavelength Nd:Vanadium modelocked laser with repetition rate 357 MHz, pulse width 7 ps, and average power 7 W." The second approach is the multipass optical cavity, in which the laser Compton generation focus exists inside the multipass cavity. The minimum focusing diameter is limited due to the requirement of the cavity design. SHG picosecond pulse of 0.2 J pulse energy is circulated 32 times to collide electron bunches [38].

An approach is undertaken by the thin-disc laser technology to generate 1 J, picosecond high beam quality pulses at 100 Hz repetition rate in the Max Born Institute, Berlin, Germany. The development is based on a ring cavity concept combined with chirped pulse amplification (CPA) [39]. The regenerative amplifier produced more than 300 mJ energy when pumped with the maximum available pump power of 1.7 kW. The regenerative amplifier is followed by a large aperture ring amplifier that increases the pulse energy further to 600 mJ. This ring

amplifier consists of a Pockels cell and a set of polarizers for the in- and out-coupling, two amplifier heads and a spatial filter in between. The amplifier heads are equipped with 750 μm thick Yb:YAG (7%) discs of 25 mm diameter. Each disc is pumped by 1 ms long pulses of 4 × 1.5 kW. Booster amplifierfor 1 J pulse is based on the large aperture ring amplifier design withoutinternal Pockels cell. The amplifier discs were pumped by diode modules that deliver 6 kW peak power out of a 2 mm fiber. Each amplifier is equipped with two of these pump modules, which together provide about twice the pump power compared to the large aperture ring amplifier. The booster amplifieris changed from former multipass configuration to a large aperture ring amplifier. The result is a multiple amplifier stage configuration with many thindisc laser modules.

The general formula of obtainable X-ray photon flux *N*<sup>0</sup> is calculated in the counter collision

<sup>2</sup> N ( Ne Np) / (4 r ) <sup>0</sup> ¥

the total laser photon number, and *r* is the interaction area radius. It is predicted that an increase in Ne and Np, and the reduction in *r* results in the increase in the photon flux *N*0. The practical limitation of these operations are the instrumental condition of electron beam emittance in higher charge, M2 of laser beam at higher pulse energy, and optimization for reduced focusing diameter *r*. It is possible to assume these parameters as 1 nC charge with 3 ps pulse duration to be focused down to 10 μm at 38 MeV voltage. Another limitation is the maximum of singlepulse laser intensity to reach the nonlinear threshold of the higher harmonics generation in the X ray region. The nonlinear Compton threshold is characterized by the laser field strength

> a0 eE/m LC = w

where parameters *E*, *ω*L, and *C* correspond to the amplitude of the laser electric field, laser frequency, and the speed of light, respectively. The laserfield strength is a function of the laser wavelength. The nonlinear threshold *a*<sup>0</sup> is given around 0.6 which corresponds to 1 J pulse energy in 1 ps pulse duration at 10 μm focusing intensity in the solid-state laser wavelength. The threshold laser energy for a single-shot imaging is similarto this critical laser pulse energy in the expected tight focus condition. The laser technology was not matured to realize such parameters simultaneously in the past, and usual approach was to increase the repetition rate of the event to increase the effective obtainable X-ray photon average flux in the affordable imaging time period such as <millisecond for bioimaging. The first approach is the pulsed laser storage in an optical enhancement cavity for laser Compton X-ray sources [37]. It is descri‐ bed in the experimental report that "the enhancement factor *P* inside the optical cavity was 600 (circulating laser power was 42 kW), in which the Finess was more than 2000, and the laser beam waist of 30 μm (2σ) was stably achieved using a 1 μm wavelength Nd:Vanadium modelocked laser with repetition rate 357 MHz, pulse width 7 ps, and average power 7 W." The second approach is the multipass optical cavity, in which the laser Compton generation focus exists inside the multipass cavity. The minimum focusing diameter is limited due to the requirement of the cavity design. SHG picosecond pulse of 0.2 J pulse energy is circulated 32

An approach is undertaken by the thin-disc laser technology to generate 1 J, picosecond high beam quality pulses at 100 Hz repetition rate in the Max Born Institute, Berlin, Germany. The development is based on a ring cavity concept combined with chirped pulse amplification (CPA) [39]. The regenerative amplifier produced more than 300 mJ energy when pumped with the maximum available pump power of 1.7 kW. The regenerative amplifier is followed by a large aperture ring amplifier that increases the pulse energy further to 600 mJ. This ring

*c*

 p

), Ne is the total electron number, Np is

s

by the following expression:

118 High Energy and Short Pulse Lasers

where *σ*<sup>c</sup> is the Compton cross section (6.7 × 10−25 cm<sup>2</sup>

times to collide electron bunches [38].

Cryogenic laser technology is suitable for the generation of large-pulse energy in a laser configuration of lower stages. A cryogenic thick-disc Yb:YAG laser was reported as 1 J was generated at 100 Hz repetition rate [3]. The picosecond CPA laser was developed for driving high average power soft X-ray lasers. This is one of the greatest breakthroughs in the history of high-energy solid-state laser, and it is described in the report on the configuration and operation as "Seed pulses of 100 mJ energy were produced by the laser frontend and ampli‐ fied to 1.5 Joules pulse energy by the five-pass power amplifier which consists of two Yb:YAG disks mounted in vacuum on a single cryo-cooling head. The Yb:YAG disks are bonded on all lateral sides with a Cr:YAG cladding to eliminate feedback of spontaneous emission into the active region to prevent amplified spontaneous emission (ASE) losses and transverse parasit‐ ic lasing. Cryogenic cooling of Yb:YAG to liquid nitrogen temperature increases the heat conductivity and reduces the saturation fluence, allowing for efficient high energy pulse generation at high repetition rates. High capacity cooling was accomplished by flowing cryogenic liquid coolant through the laser head. Each disk was pumped with 1.5 ms dura‐ tion, 4 kW pulses from a *λ* = 940 nm laser diode array. At the maximum pump power, 1.5 J laser pulses were obtained. These pulses were compressed by a dielectric grating pair producing 1 J, 5 ps FWHM duration pulses at 100 Hz repetition rate." The repetition rate is recently increased to 500 Hz and the picosecond pulse energy is 1 J, and the resulting aver‐ age power is 500 W. Temporally pulse-shaped laser pulses were focused into a ∼5 mm long, 30 μm FWHM wide line on a solid target using cylindrical optics. The beam quality is indicated by the focusing specification. The resulting plasma was in the Ni-like stage, and strong collisional excitation leads to a large transient population inversion on the 4d1S0 → 4p1P1 transition of Ni-like ions at wavelengths ranging from 10.9 to 18.9 nm.

Cryogenic solid-state laser is preferred for power scalability with better beam quality, especially in higher pulse energy mode, and improvement of efficiency at the cost of longer pulse length [40]. Yb:YAG is the most tested material due to its low quantum defect and still broadband absorption in low temperatures. Various thermal optical properties are reported for base materials as YAG (ceramic and single crystal), GGG, GdVO4, and Y2O3 on the thermal conductivity, thermal expansion, refractive index, absorption cross section, emission cross section, and fluorescence lifetime in the cryogenic condition.

One of the key features of the cryogenic laser is its better beam quality. A quantitative evaluation is important for a practical laser design for dedicated applications and a measure‐

ment was performed on the wavefront distortion caused by the thermal origin in a cryogen‐ ic Yb:YAG crystal in the temperature range 250–130 K in nonlasing condition [41]. The wavefront aberration was evaluated by a wavefront sensor. The measurement results showed a significant reduction of the wavefront aberration in lower temperature. The thermal defocus was concluded as originated to the thermal lensing effect together with electronically induced change of the refractive index by the excitation of ion activators (electronic lensing). The dominant reason of the aberration was found as the thermal lensing in the experimental condition as 6.3 kW/cm2 pumping intensity and pumping repetition rate of 100 Hz. The Strehl ratio was observed to be improved in the lower temperature even the absorbed energy was increased. The experiment showed the advantage of the cryogenic technology in terms of efficiency and beam quality.

**Figure 15.** LEFT: Experimental configuration of the aberration measurement. FLD1, fiber-coupled pump diode at 936.6 nm; FLD2, fiber-coupled probe beam laser diode at 1065 nm; GT, Galilean telescope; W, windows; M, turning mirrors; DM, dichroic mirrors; A1, 2, achromatic doublets with focal lengths of 100 and 250 mm, respectively, L1, 2, lenses with a focal length of 250 mm; ND, neutral density filters; LPF, longpass filter with cutoff wavelength at 1050 nm. Right: Multipass amplification configuration. WS, wavefront sensor; PM, power meter.

The experimental setup used for the measurement of the wavefront aberrations in a cryogen‐ ically cooled Yb:YAG slab is shown in **Figure 15** (left). The right side figure shows the configuration of multipass amplification from the 100 mJ level input. A Yb:YAG crystal is mounted in a copper holderin a closed-loop pulse tube cryostat (Q Drive). The cooling capacity is 12 W at 100 K. The crystal was supplied from Crytur, Czech Republic, and the specifica‐ tion was thickness 2 mm, diameter 10 mm, and doping concentration 3 at%. A fiber-coupled laser diode (DILAS) pumped the crystal from one side. The peak wavelength was 936.9 nm, and the peak intensity was 6.3 kW/cm2 . Two achromatic doublet lens of focal length 100 and 250 mm imaged the 1 mm core of the fiber with NA 0.22 to the Yb:YAG surface. The result‐ ing pump spot size was 2.5 mm (1/e2) in super-Gaussian intensity distribution. **Table 2(a)** summarizes the absorbed energy per pump pulse at each temperature. The absorbed energy increases by about 19 % if the temperature decreases by 120 K from initial 250 K. The absor‐ bed power and thus generated heat is higher with decreasing temperature, and the aberra‐ tions are lower because of higher thermal conductivity, lower d*n*/d*T*, and lower expansion coefficient. Theoretical thermal decay time constants were calculated according to the formula *t*T = *r*<sup>p</sup> 2 /4*κ*, where *r*p is the radius of the pump, and *κ* is the thermal diffusion coefficient, which is defined as *k*/(*ρ*cp) where *k* is the thermal conductivity, *ρ* is the mass density, and cp is the specific heat. The estimated values are shown in **Table 2(b)** for the thermal relaxation time constants for different temperature conditions in 3 at% doped Yb:YAG as an estimation from data for 2 and 4 at% doped crystals. The thermal decay time is around 31 ms at 150 K, which is three times longer than the time interval between pumping at 100 Hz. This value is 93 ms at 250 K.

ment was performed on the wavefront distortion caused by the thermal origin in a cryogen‐ ic Yb:YAG crystal in the temperature range 250–130 K in nonlasing condition [41]. The wavefront aberration was evaluated by a wavefront sensor. The measurement results showed a significant reduction of the wavefront aberration in lower temperature. The thermal defocus was concluded as originated to the thermal lensing effect together with electronically induced change of the refractive index by the excitation of ion activators (electronic lensing). The dominant reason of the aberration was found as the thermal lensing in the experimental condition as 6.3 kW/cm2 pumping intensity and pumping repetition rate of 100 Hz. The Strehl ratio was observed to be improved in the lower temperature even the absorbed energy was increased. The experiment showed the advantage of the cryogenic technology in terms of

**Figure 15.** LEFT: Experimental configuration of the aberration measurement. FLD1, fiber-coupled pump diode at 936.6 nm; FLD2, fiber-coupled probe beam laser diode at 1065 nm; GT, Galilean telescope; W, windows; M, turning mirrors; DM, dichroic mirrors; A1, 2, achromatic doublets with focal lengths of 100 and 250 mm, respectively, L1, 2, lenses with a focal length of 250 mm; ND, neutral density filters; LPF, longpass filter with cutoff wavelength at 1050 nm. Right:

The experimental setup used for the measurement of the wavefront aberrations in a cryogen‐ ically cooled Yb:YAG slab is shown in **Figure 15** (left). The right side figure shows the configuration of multipass amplification from the 100 mJ level input. A Yb:YAG crystal is mounted in a copper holderin a closed-loop pulse tube cryostat (Q Drive). The cooling capacity is 12 W at 100 K. The crystal was supplied from Crytur, Czech Republic, and the specifica‐ tion was thickness 2 mm, diameter 10 mm, and doping concentration 3 at%. A fiber-coupled laser diode (DILAS) pumped the crystal from one side. The peak wavelength was 936.9 nm,

250 mm imaged the 1 mm core of the fiber with NA 0.22 to the Yb:YAG surface. The result‐ ing pump spot size was 2.5 mm (1/e2) in super-Gaussian intensity distribution. **Table 2(a)** summarizes the absorbed energy per pump pulse at each temperature. The absorbed energy increases by about 19 % if the temperature decreases by 120 K from initial 250 K. The absor‐ bed power and thus generated heat is higher with decreasing temperature, and the aberra‐ tions are lower because of higher thermal conductivity, lower d*n*/d*T*, and lower expansion

. Two achromatic doublet lens of focal length 100 and

Multipass amplification configuration. WS, wavefront sensor; PM, power meter.

and the peak intensity was 6.3 kW/cm2

efficiency and beam quality.

120 High Energy and Short Pulse Lasers

**Table 2.** (a) Absorbed energy per pump pulse by the 2 mm thick, 3 at% doped Yb:YAG slab pumped by energy of 310 mJ at a wavelength of 936.6 nm for different temperatures of the cooling finger. (b) Calculated thermal decay time for 3 at% Yb:YAG crystal.

Cryogenic cooling is usually applied in booster amplifiers with more than one pass of the seed beam through the active medium in order to efficiently extract the stored energy. Therefore, it is assumed to evaluate four beam passes, and the measured wavefront with subtracted tilt and defocus was four times multiplied to calculate the real Strehl ratio in multipass amplifi‐ cation. The calculated Strehl ratio was 0.96 at 130 K and decreased to 0.93 at 250 K as shown in **Figure 16**. The practical Strehl ratio to obtain the same pulse energy decreases at higher temperature to obtain the same pulse energy in lower gain. The measurement indicates the linearity of the Strehl ratio to the temperature decrease, and it is expected further increase in the beam quality is possible in lower temperature of about less than 130 K.

In the last part of this chapter, a large aperture cryogenic laser is evaluated. The perform‐ ance of a gas-cooled multislab laser is recently reported from the DiPOLE project within the Central Laser Facility (CLF RAL STFC), UK. The development is aiming at an efficient high pulse energy diode-pumped solid-state laser (DPSSL) architecture based on cryogenic gascooled, multislab ceramic Yb:YAG amplifier technology. A prototype amplifier is delivering up to 10.8 J pulse energy at 1030 nm wavelength with 10 Hz repetition rate. The optical-optical conversion efficiency is 22.5% [42]. The long-term energy stability was observed as 0.85% RMS with 7 J pulse energy for 48 h operation (2 million shots). An extension of the cryogenic technology is now under test in the DiPOLE 100 to confirm the cryogenic concept at 100 J, 10 Hz region (kW average power). The present laser system is built for the HiLASE project and will deliver 100 J temporally shaped ns pulses at 10 Hz with a fully integrated control system. A second system is also under development for the high-energy density (HED) beamline of the European XFEL project.

**Figure 16.** Strehl ratio for the four passes of the probe beam through the Yb:YAG slab and absorbed pump power per single pass as a function of temperature. Lines represent a linear fit with slopes of –6 × 10−4/K and −0.064%/K, respec‐ tively.

The 10 J amplifier architecture is based on the multislab approach. The gain medium is composed of four circular Yb:YAG slabs with two different Yb doping levels as 1.1 and 2.0 at % to confirm a uniform temperature distribution among each slab. The diameter of the circular slab is 45 mm with a 5 mm thickness, and the pump area is square of 23 × 23 mm2 . The pump beam is supplied from 939 nm diodes in stack with a pumping time duration of 700 μs at 10 Hz. The Yb:YAG circular slab is cladded with a 5 mm wide Cr:YAG absorber with 6 cm−1 absorption coefficient. This is effective to prevent amplified spontaneous emission (ASE) and parasitic oscillations. A cryogenic He flow cools the slab and keeps the temperature as 150– 170 K. The pressure of the He flow is 10 bar. **Figure 17** shows the optical arrangement for amplification. A seed beam is injected into the amplifier through a dichroic mirror and then image relayed by a spatial filter (*f* = 1 m) to a back reflector and reflected back to the amplifi‐ er module. One spatial filter locates on each side of the amplifier head. Each pass is com‐ posed by a set of separate mirrors. A deformable mirror is placed in the amplifier after the third pass for the aberration compensation. After seven passes, the beam is extracted from the amplifier with pulse energy increased to 9 J with a size of 21 mm × 21 mm2 .

Numerical modeling of the multislab amplifier is conducted in the HiLASE project to ensure the scaling of the cryogenic technology forfurtherincreased parameterregion in pulse energy, repetition rate, and better beam quality. Comsol Multiphysics software was chosen to model the thermal and stress effects in the amplifiers [43]. The sources of heat were calculated in the ASE code [44]. The axial surfaces of the slabs are assumed to be cooled only by flowing helium gas at 160 K. The slab was assumed to have no thermal contact with its 2 cm thick Invar holder; and all heat is removed by convection through the faces. From the temperature and stress maps of the slab, the optical path difference (OPD) and birefringence depolarization losses were calculated for a single slab according to a prior approach. The gradual decrease of cooling efficiency in the direction of gas flow, caused by He heating, results in the loss of left-right symmetry of the temperature, stress, depolarization, and OPD maps.

**Figure 17.** Schematic of the 10 J cryogenic multislab amplifier. It consists of Yb:YAG ceramic slabs in the laser head, dichroic beam splitters (DBSs), lens arrays (LAs), vacuum spatial filters (VSFs), and homogenized pump diode laser modules (PDs).

It is planned to increase the repetition rate of the 10 J amplifier to 100 Hz by keeping the basic performance. Once the operation is as expected, the cryogenic laser offers a technological stage for a single module to generate picosecond multipulses of a few joules of energies, for a singleshot imaging Compton source.


**3. Parasite factors associated with infection duration**

After inoculation of sporozoites and the subsequent release of merozoites from the liver, there is a period of time when parasites are present in the blood at concentrations undetectable by conventional diagnostics. In many individuals, parasites then multiply to reach detectable densities, however in other individuals, parasites may remain at low densities that are undetectable. Human challenge studies on non-immune individuals in which parasites are monitored both by molecular methods and by microscopy have estimated that infections are detectable by PCR an average of 3.7 days (range 2–4 days) [35] or 3.1 days (range 0–4) [36] before being detectable by microscopy. Controlled human infections also suggest that the parasite stages that precede blood invasion might influence asexual blood stage dynamics. For example, Churcher and colleagues [37] observed that the inoculum size (the estimated number of sporozoites injected by infected *Anopheles* mosquitoes) influences the time it takes for infections to become patent: individuals receiving five bites from mosquitoes with more than 1000 sporozoites have detectable parasitaemia at least 2 days earlier than those volunteers infected by mosquitoes with 11–101 sporozoites. Quantification of sporozoite counts in wild-caught mosquitoes is necessary to confirm the relevance of this finding in natural settings. In Papua New Guinea, it was estimated that infected malaria vectors had on average (geometric mean) 4000 sporozoites [38], which is of the same order of magnitude as sporozoite counts in mosquitoes used in controlled infections.

gametocyte densities. Data from epidemiological studies confirm that most asymptomatic infections with patent or sub-patent asexual stage parasite levels have sub-patent gametocytaemia [42], only detectable by RNA-based molecular methods. However, a few asymptomatic individuals with low-density infections have relatively high gametocyte densities, which could be related to symptomless fluctuations in parasitaemia that result in higher gametocytaemia a few days later. The rate of commitment of asexually replicating parasites to sexual development is another factor that influences gametocyte levels in malaria infections. Adults, who on average carry lower asexual stage parasite densities, have a higher sexual to asexual density ratio [43]. This could be related to an unequal increase in clearance rates of asexual and sexual parasites with age, or potentially to changes in commitment to gametocytogenesis [43]. Consistent with the latter, parasite investment in transmission stages has been shown to vary in areas with different transmission levels, being higher in settings with lower endemicity. Recent data suggest that parasite variations in commitment to gametocytes are epigeneti-

Understanding the Importance of Asymptomatic and Low-Density Infections…

http://dx.doi.org/10.5772/intechopen.77293

135

As articulated above, the importance of asymptomatic infections for malaria transmission does not lie in their average sexual stage parasite densities but in the durations of gametocyte carriage and infectiousness over time. A mathematical model [45] fitted to both asexual parasite and gametocyte malariotherapy data estimated infectivity over the course of an infection based on gametocyte density data. This analysis concluded that the majority of infectivity was usually concentrated early in infection, although some patients were significantly infectious later on. However, in this model, it was assumed relatively low infectivity of low gametocyte densities compared with other analyses [46]. While these data are extremely detailed, it is not known whether these dynamics are similar to those in naturally infected individuals who have immunity. Furthermore, specific *P. falciparum* strains were selected for malariotherapy because they were 'benign' and may not exhibit the same behaviour in terms of parasite multiplication rates and gametocyte commitment as parasites in

Although asymptomatic infections do not prompt treatment-seeking behaviour, during community mass treatment campaigns that involve treatment regardless of symptomatology (e.g., mass drug administration (MDA) or mass screening and treatment), these infections are cleared with antimalarials. In a meta-analysis of trials with gametocyte density data [47], the combinations artesunate-mefloquine and artemether-lumefantrine were more effective in preventing the appearance of gametocytes and in clearing existing sexual stage parasites compared to dihydroartemisinin-piperaquine. The choice of drugs to be used during control interventions thus may be important to limit residual transmission from these infections. In Section 4, we discuss the impact of different interventions that target asymptomatic and

Two variables linked to blood sampling for parasite detection can influence prevalence and density estimates: volume and timing. Even sensitive molecular assays will not detect low parasite densities in samples if nucleic acids are isolated from small blood volumes. Highvolume PCR has been used in epidemiological studies in Southeast Asia to circumvent

cally imprinted and higher in parasites in lower endemicity settings [44].

endemic areas.

symptomatic infections.

**3.2. Underestimations of parasitaemia linked to sampling**

Microscopy has limited sensitivity to quantify low parasite densities and this will affect its utility for studying any chronicity in infection dynamics. Histidine rich protein 2 (HRP-2), a protein the parasite secretes in the plasma, is considered to be a more accurate measure of total falciparum parasite burden [39], however, this measure does not distinguish between monoclonal and multiclonal infections. Molecular tools are more sensitive and allow discrimination of different parasite genotypes. They have been used to assess the effects of super-infection and exposure to different parasite clones on clinical malaria risk. A study that involved daily blood sampling of children with initially asymptomatic infections [40] suggests that development of symptoms is often associated with appearance of a new parasite strain in the blood and increases in parasite levels. Correspondingly, recent data from Papua New Guinea [16] showed that incidence of infections by new clones correlates with clinical malaria risk. This indicates that clinical malaria is often associated with new infection, presumably by a parasite clone with a previously unencountered antigenic profile.

Consistent with this, genetic analysis of malaria parasite populations in Zambia [41] found that in some settings individuals with symptomatic infections had different parasite strains compared to asymptomatic individuals. One hypothesis for this is that symptomatic infections originated from imported or recently introduced strains and that immunity to these strains is insufficient. This indicates that the rate of importation may play a role in the proportion of infections that are asymptomatic and symptomatic, especially in areas approaching elimination. An infection with a clone to which immunity has been acquired might lead to infections with shorter duration (e.g., **Figure 1**, yellow line).

#### **3.1.  Data from epidemiological studies confirm that most asymptomatic infections with patent or sub-patent asexual stage parasite levels have sub-patent gametocytaemia [42], only detectable by RNA-based molecular methods. However, a few asymptomatic individuals with low-density infections have relatively high gametocyte densities, which could be related to symptomless fluctuations in parasitaemia that result in higher gametocytaemia a few days later. The rate of commitment of asexually replicating parasites to sexual development is another factor that influences gametocyte levels in malaria infections. Adults, who on average carry lower asexual stage parasite densities, have a higher sexual to asexual density ratio [43]. This could be related to an unequal increase in clearance rates of asexual and sexual parasites with age, or potentially to changes in commitment to gametocytogenesis [43]. Consistent with the latter, parasite investment in transmission stages has been shown to vary in areas with different transmission levels, being higher in settings with lower endemicity. Recent data suggest that parasite variations in commitment to gametocytes are epigenetically imprinted and higher in parasites in lower endemicity settings [44].

**3. Parasite factors associated with infection duration**

134 Towards Malaria Elimination - A Leap Forward

After inoculation of sporozoites and the subsequent release of merozoites from the liver, there is a period of time when parasites are present in the blood at concentrations undetectable by conventional diagnostics. In many individuals, parasites then multiply to reach detectable densities, however in other individuals, parasites may remain at low densities that are undetectable. Human challenge studies on non-immune individuals in which parasites are monitored both by molecular methods and by microscopy have estimated that infections are detectable by PCR an average of 3.7 days (range 2–4 days) [35] or 3.1 days (range 0–4) [36] before being detectable by microscopy. Controlled human infections also suggest that the parasite stages that precede blood invasion might influence asexual blood stage dynamics. For example, Churcher and colleagues [37] observed that the inoculum size (the estimated number of sporozoites injected by infected *Anopheles* mosquitoes) influences the time it takes for infections to become patent: individuals receiving five bites from mosquitoes with more than 1000 sporozoites have detectable parasitaemia at least 2 days earlier than those volunteers infected by mosquitoes with 11–101 sporozoites. Quantification of sporozoite counts in wild-caught mosquitoes is necessary to confirm the relevance of this finding in natural settings. In Papua New Guinea, it was estimated that infected malaria vectors had on average (geometric mean) 4000 sporozoites [38], which is of the same order of magnitude as sporozoite counts in mosquitoes used in controlled infections. Microscopy has limited sensitivity to quantify low parasite densities and this will affect its utility for studying any chronicity in infection dynamics. Histidine rich protein 2 (HRP-2), a protein the parasite secretes in the plasma, is considered to be a more accurate measure of total falciparum parasite burden [39], however, this measure does not distinguish between monoclonal and multiclonal infections. Molecular tools are more sensitive and allow discrimination of different parasite genotypes. They have been used to assess the effects of super-infection and exposure to different parasite clones on clinical malaria risk. A study that involved daily blood sampling of children with initially asymptomatic infections [40] suggests that development of symptoms is often associated with appearance of a new parasite strain in the blood and increases in parasite levels. Correspondingly, recent data from Papua New Guinea [16] showed that incidence of infections by new clones correlates with clinical malaria risk. This indicates that clinical malaria is often associated with new infection, presumably by a parasite clone with a previously unencountered antigenic profile.

Consistent with this, genetic analysis of malaria parasite populations in Zambia [41] found that in some settings individuals with symptomatic infections had different parasite strains compared to asymptomatic individuals. One hypothesis for this is that symptomatic infections originated from imported or recently introduced strains and that immunity to these strains is insufficient. This indicates that the rate of importation may play a role in the proportion of infections that are asymptomatic and symptomatic, especially in areas approaching elimination. An infection with a clone to which immunity has been acquired might lead to

Gametocytes derive from a small percentage of asexual parasites that commit to sexual development; therefore, asymptomatic infections with low asexual levels may also have low

infections with shorter duration (e.g., **Figure 1**, yellow line).

**3.1. Gametocytes in asymptomatic infections**

As articulated above, the importance of asymptomatic infections for malaria transmission does not lie in their average sexual stage parasite densities but in the durations of gametocyte carriage and infectiousness over time. A mathematical model [45] fitted to both asexual parasite and gametocyte malariotherapy data estimated infectivity over the course of an infection based on gametocyte density data. This analysis concluded that the majority of infectivity was usually concentrated early in infection, although some patients were significantly infectious later on. However, in this model, it was assumed relatively low infectivity of low gametocyte densities compared with other analyses [46]. While these data are extremely detailed, it is not known whether these dynamics are similar to those in naturally infected individuals who have immunity. Furthermore, specific *P. falciparum* strains were selected for malariotherapy because they were 'benign' and may not exhibit the same behaviour in terms of parasite multiplication rates and gametocyte commitment as parasites in endemic areas.

Although asymptomatic infections do not prompt treatment-seeking behaviour, during community mass treatment campaigns that involve treatment regardless of symptomatology (e.g., mass drug administration (MDA) or mass screening and treatment), these infections are cleared with antimalarials. In a meta-analysis of trials with gametocyte density data [47], the combinations artesunate-mefloquine and artemether-lumefantrine were more effective in preventing the appearance of gametocytes and in clearing existing sexual stage parasites compared to dihydroartemisinin-piperaquine. The choice of drugs to be used during control interventions thus may be important to limit residual transmission from these infections. In Section 4, we discuss the impact of different interventions that target asymptomatic and symptomatic infections.

#### **3.2. Underestimations of parasitaemia linked to sampling**

Two variables linked to blood sampling for parasite detection can influence prevalence and density estimates: volume and timing. Even sensitive molecular assays will not detect low parasite densities in samples if nucleic acids are isolated from small blood volumes. Highvolume PCR has been used in epidemiological studies in Southeast Asia to circumvent this problem and less than 30% of all falciparum infections are estimated to be missed by this method [48]. The timing of blood sampling in parasitological surveys might also affect parasite detection and quantification because asexual falciparum parasites do not circulate continuously; sequestration of falciparum schizonts starts 12-18 hours after merozoite invasion and during this period they might not be detectable. An intensive longitudinal study in Tanzania showed that periodic changes in parasite densities are common. The periodicity of clone-specific detectability indicates that in natural infections, synchronised sequestration of clonal parasite populations occurs [49]. A study [50] that collected samples on two consecutive days found a prevalence disparity of approximately 25% between the two samples. Periodic changes in parasite levels could have a direct impact on the selection of diagnostics, for example by favouring assays that detect more persistent markers, such as HRP-2.

The detection of either asexual or sexual stage parasites is sufficient to establish the diagnosis of infection. Although gametocytes are not known to periodically sequester, there is evidence of periodic variation in gametocyte levels [51] in peripheral blood. For several decades now [52], accumulation of mature gametocytes in the skin [53] has been hypothesised as a possible mechanism of transmission enhancement. If confirmed, this would imply that subpatent gametocytaemias in peripheral blood might be associated with higher-than-expected infectivity.