systems   biology is a discipline that examines the organizational relationships between biological structures in an organism , and thus it covers a very broad scope of biology from the macroscopic to the microscopic biology of organisms , ranging from mankind and other animals , plants , and microorganisms to their organs , tissues , cells , and subcellular organelles and structures through to molecular structures and the interactions between different molecular structures and systems .
this broad scope of systems biology demands many different types of instruments for different aspects of imaging and signal detection .
instruments and devices such as cameras and x - ray imaging systems are used for whole organism imaging in man and larger animals [ 1 , 2 ] , ivis imaging system has been developed for small animal imaging in vivo 
 , ultrasonic devices are applied for the organ and tissue imaging , the ultraview living cell imager has been developed for cells and subcellular imaging , and various types of biochip have been designed for the analysis of the tissue , cells , and molecular structures . except for ultrasonic devices ,
all of the above detection - analysis systems require excellent optical design for the best performance of their particular objectives
. one important type of optical system useful for the analysis of subcellular and molecular structures is the confocal chip scanner with a low 
 background noise .
advanced biochip analysis platforms [ 7 , 8 ] analyze the content of a particular microarray slide chip , including gene chips , protein chips , cell chips , tissue chips , and 
 others .
many researchers work with small molecule biochips , used for the detection of dna variations by dna 
 hybridization , of proteins by the immunoreaction of proteins , and other more specialist analyses such as for dna sequencing , ligase chain reaction ( lcr ) , and others .
some important biochip detection systems [ 9 , 10 ] have been developed , such as the fluorescence microscope imaging system and the laser confocal scanner .
in all these detection systems , optical design has played a keyrole in obtaining high - clarity images of microscopic objects , and the optical objective is crucial to the detection sensitivity , the 
 resolving power , and the working distance of the detection system . usually , the bigger the numerical aperture of 
 the objective , the higher its resolving power , and the higher the power of collecting the fluorescence signals 
 bound on the tested object , however to achieve this structure , the working distance of the objective must also be shorter .
for example , when the conventional microscope objective has an na  >  0.6 , then its working distance 
 wd is usually smaller than 1  mm . 

for example , the zeiss plan objective 440050 , magnification 40 ,   na=0.65 , wd=0.6  mm , and the nikon plan objective model 40 ,    
 na=0.65 , and wd=0.57  mm . in this paper ,
a novel confocal optical system design and a dual laser confocal scanner are described in which   the materials and lenses employed are as small as practicable ; the optical design and the potential pitfalls of an objective with a long working distance and a high numerical aperture have been considered and resolved for application to biochips .
the collecting fluorescence has very high efficiency , and 

 generally the instrument has excellent resolving power and an excellent signal - to - noise ratio .
the design of a confocal optical system with high - performance detection of biological sample slides and biochips centers around the image quality , including the consideration of the resolution , zoom , aberration , and optical transfer 
 functions , each of which can be optimized by using rays tracing calculations . in the process of optical design ,
all parameters of the optical system structure , including the surface curvature radius of each lens , the thickness of the lenses , the transmitted materials used , the separation distances , and the surface apertures can be varied to obtain an excellent optical specificity and image quality .
the functional relationships between the specificity , image quality , and structural parameters of the system can be described as follows : 
 ( 1)1(1,  ,i)=1k(1,  ,i)=k , 
 where i and k are two natural numbers , 1k are various aberrations of the optical specificity 
 and the image quality , 
 1k are functions of the specificity , the image quality , and the structural parameters of system , 
 1i correspond to all the structural parameters of system . based on the polynomial expansion and the minimum binary iterative method ,
when all structural parameters 
 1i are modified repeatedly , an approximate minimum aberration will be obtained in 
 ( 1 ) . applying the above optical design method , an optimum confocal optical system structure for the detection of 
 biochips
the objective system is a combination of seven lenses including two doublets and uses only three glass materials , zk7 , zk11 , and zf2 .
the objective system has a high numerical 
 aperture of 0.72 for collecting the signal from the object , a focal length of 13.06  mm , and a front focal length of 3.22  mm to provide a working distance of approximately 3.0  mm .
there is a parallel ray path between the objective and the magnifying lenses , to which it is convenient to also add the filters and the dichroic mirror for the incident laser when building the confocal scanning system .
the optical structure parameters of confocal scanning system were optimized in figure 
 1 , where there are an objective and a magnifying lenses , the objective consists of seven lenses , the magnifying lenses consists of five lenses , there is a parallel beam between the objective and magnifying lenses .
a configuration of the objective collecting fluorescence is shown in figure 
 2 , where the distance from the focal plane to the front surface of first lens of objective is z , 
 r is the radius of effective aperture of the objective , 
  is the half of aperture angle .
the emission fluorescence ( em - fluor ) of molecule bound on biochips is ideally a spherical wave , and the fluorescence bound on the biochip collected by the objective as shown in figure 2 is described approximately by the formula 
 ( 2)i(z)k02z2r2 , 
 where k0 is a constant , z is the distance from the center of source in the focal plane to the first surface of objective , r is the radius of effective aperture of the objective .
when biochips are placed at the focal plane of the objective , and the objective has a numerical apture 
 na = nsin , where n is the refractive index , then the formula ( 2 ) can be 
 simplified to formula 
 ( 3)i(z)1n2k02na2(1+na2+na4+ ) . 

formula ( 3 ) indicates that the intensity of the collecting fluorescence of molecules on biochips has a direct ratio to the square of the higher power of the na .
by developing a structure form using formula ( 3 ) , we created a confocal optical system with a numerical aperture of 0.72 , and an efficiency of collected fluorescence of more than 2-fold better than that of other commercial confocal biochip scanners [ 9 , 10 ] whose objective has a smaller numerical aperture than 0.5 . in a confocal scanning system
the smaller the laser scanning spot of optical system , the higher the scanning resolution power of the confocal 
 system .
the scanning spot diagrams are referenced to the real chief ray as shown in figure 
 3 .
this option allows selection of two other reference points , the centroid and the middle .
the middle is defined so that the maximum ray errors are equal in both the x- and the y - directions .
when a laser with a real beam diameter of about 1  mm is imaged by the objective shown above in figure 1 , then the spot diagram on the focal plane is as shown in figure 3 , where the scale is 0.4  m , the spot diameter on the focal plane is smaller than 0.4  m , which is an ideal scanning probe beam with a more high resolving power of < 0.5  m . the optical speciality and the aberration of the imaging system of our novel optical structure can be analyzed 
 using the optical design software zemax - ee .
for the spot diagram shown in figure 3 , the root mean square ( rms ) radius on the focal plane is 0.105  m , and the geometric radius on the focal plane is 0.183  m , which corresponds to a confocal optical system with a scanning resolution power smaller than 0.4  m . the optical path difference ( opd ) is a scalar quantity and it is identical to those for ray aberration fans at the tangential and sagittal directions px and py , respectively .
the data plotted in figure 4(a ) is the optical path difference of the system in figure 1 , which is the difference between the optical path length of the ray and the optical path length of the chief ray .
the vertical axis scale of graph is one wave , while the opd maximum of system in 
 figure 1 is smaller than 5 waves . in figure 4(a ) , there is a small optical path difference among 3 wavelengths 570  nm , 620  nm , and 670  nm , but the maximum optical path difference is smaller than 3 waves , which is lower than a normal visible light imaging system of 5 waves and can be used for confocal scanning system very well .
the encircled energy diagram is the percentage of total energy enclosed as a function of distance from either the chief ray or the centroid at the image of a point object , while the diffraction limit curve is for the aberration - free encircled 
 energy computed on - axis .
the encircled energy diagram of the system is shown in figure 
 4(b ) , where the horizontal coordinate is the radius , and the vertical coordinate is the normalized fraction of the enclosed energy .
the encircled energy diagram shows a diffused intensity spot in focal plane of system , the smaller the radius of encircled energy diagram is , the more the fluorescence energy is 
 collected by the pinhole in the focal plane . in figure 4(b ) , the radius of encircled 
 energy diagram is smaller than 10  m , where the efficiency of fluorescence 
 collected is near to 100% , when a pinhole with a radius of 10  m is set at the focal plane of the magnifying lenses , then the efficiency of collection of the 
 fluorescence of an object is near to the diffraction limit .
geometric image analysis is used to model extended sources , to analyze useful resolution , to represent 
 the appearance of imaged objects , and to provide intuition as to image rotation .
the diffraction image analysis accounts for the finite pass band and other diffraction - related effects of real optical systems based upon fourier optics .
the geometric image analysis of the optical system is shown in figure 5(a ) , while the diffraction image analysis of the optical system in the object area illuminated by the scanning spot is shown in 
 figure 5(b ) , both of which have a high efficiency of 100% . in figure 5(a ) , the geometric image analysis shows a nice roundness of model extended sources , there 
 is good image rotation invariability of system . 
 in figure 5(b )
, the diffraction image analysis shows a nice uniformity and clear outline of the letter f as an object , there is good image quality of system to the object area illuminated . applying the above optical design parameters
, we constructed a new confocal optical scanning system which has been further developed into the advanced confocal scanner specially for biochip application , as illustrated in 
 figure 6 , where the objective and magnifying lenses are designed in 
 figure 1 , two filters for the dye cy3 and the dye cy5 are bought from chroma corporation , pmt ( photomultiplier tube ) is bought from hamamatsu corporation , laser 1 is a solid laser with wavelength 532  nm and power 25  mw , laser 2 is a semiconductor laser with wavelength 635  nm and power 25  mw , splitter is a dichroic mirror from chroma   corporation ,   the mirror is machined into an elliptical mirror with a small hole 1 mm in center ,   xy scanning platform is designed with 2  m moving control precision .
pinhole is a small hole with diameter 20  m , a / d electronic card is designed with precision 16  bit and frequency 1  mhz .
computer is chosen with piii cpu or higher cpu speed , biochip is developed by capitalbio corporation .
it is characterized by an objective with a large numeral aperture of na=0.72 , a long working distance of 3.0  mm , and a sensitivity of fluorescence detection of about 0.1 fluors/m .
compared to other similar commercial scanners , it has a higher - resolution power and an excellent signal - to - noise ratio . in figure 6 ,
when the laser beam irradiates the biochip on the xy scanning platform from the laser 1 with wavelength 532  nm , or from the laser 2 with wavelength 635  nm , the fluorescence of biological sample on the biochip is induced and collected by pmt .
after a / d ( analogue / digital ) transfer is complete , the fluorescence signal from the biochip is loaded into the computer 
 for digital image processing . when pairs of identical gradient signal biochips from full moon ( full moon biosystems , sunnyvale , calif , usa ) were labeled with either cy3-tagged or cy5-tagged probes , and dual color fluorescence was detected by using our newly developed confocal scanner and by another commercial confocal scanner ( us popular brand s ) , the features of the scanning image analyses are shown in figures 7(a)7(d ) , where the right small spot array image is a local area magnifying view for left scanning images .
our newly developed confocal scanner shows a high scanning resolution , an excellent contrast , and a signal - to - noise ratio seen in figures 7(a ) and 7(c ) .
the detection sensitivity of the new confocal scanner was further illustrated in figure 7(e ) , where a full moon ( full moon biosystems , sunnyvale , calif , usa ) normal molecular density biochip was 
 used and the unit of density is the molecule number per the square micron , and the signal is the relative intensity in the range of 0 to 65  535 . in figure 7(e ) , a molecule with a density of 0.071 fluors/m was detected with a signal of 547 , where snr ( signal - to - noise ratio ) is greater than 2 , which 
 indicates that the new confocal scanner is with a sensitivity of fluorescence about 0.1 fluors/m .
the gene expression analysis is an important method to study the different gene functions of organisms , 
  which is usually performed on high - density biochips with several tens of thousands of probes in a small 20  mm    60  mm area , where dual fluorescent color labels with the dyes 
 cy3 and cy5 can be used to produce a gene expression spectrum . in order to analyze the gene expression of biochips ,
it is important for the scanning system to possess high scanning resolution , an excellent contrast , and signal - to - noise ratio , over a wide range of signal intensities .
the newly developed confocal optical scanning system can be readily used for the gene expression analysis of biochips . the gene expression spectrum for a high - yield variety of cotton as shown in 
 figure 8 was obtained by using the developed confocal scanner . in figure 8
, the dual fluorescent color - labeled biochips were hybridized with 15 reference probes at the beginning of first row and with 687 gene reporter probes representing expressed genes 
 from the two cottons .
messanger rna from common cotton was labeled with cy3 , and mrna from a high - yield cotton was labeled with cy5 .
the dual fluorescent color - labeled biochips were scanned twice using the new confocal scanner , where one scan was illuminated by the green laser with a wavelength 532  nm to induce fluorescence of cy3 , and the second scan was illuminated by the red laser with a wavelength 635  nm to induce the fluorescence of cy5 .
figure 8(a ) is the image of the gene expression spectrum of the common cotton labeled with cy3 , and figure 8(b ) is the image of the gene expression spectrum of the high - yield cotton labeled with cy5 .
figure 8(c ) is the combined images of the gene expression spectrums from both the high - yield cotton and the common cotton , where there is an obvious color change if there is a difference between the level of gene expression of mrnas labeled by cy3 and by cy5 .
figure 
 8(d ) is the scatter plot of gene expression to analyze the differences between the gene message labeled with cy3 to message labeled with cy5 , where the identical level of expression of the same genes in the two plant varieties distributes along the line direction of 45 degrees with increasing relative intensity of signals .
the 
 larger the difference of gene expression between the two varieties , the farther the position of gene in the scatter plot is away from the 45-degree line . in figure 8(d ) , except for 15-reference probes at row 1 , 
 there are 36 genes with a 2-fold signal difference between the common cotton and the high - yield cotton , distributed over a broad range of relative signal intensities , which shows that there are 36 important genes to 
 improve the yield of cotton .
the new confocal scanner is also useful for fluorescence detection of the immunoreactions of proteins on chips .
we describe here a protein microarray chip for the parallel detection of autoantibodies in the serum of patients with 
 autoimmune diseases , including systemic lupus erythmatosus ( sle ) , mixed connective tissue disease ( mctd ) , sjgren 's syndrome ( ss ) , sjgren 's syndrome a ( ssa ) , sjgren 's syndrome b(ssb ) , smith ( sm ) , ribonucleoprotein ( rnp ) , scleroderma ( scl ) , systemic sclerosis ( ssc ) , dermotomyositis ( dm ) , double - stranded dna(dsdna ) , phosphate - buffered saline tween-20(pbst ) , and polymyositis ( pm ) .
purified autoantigens ( ssa-52 , ssa-60 , ssb , sm , rnp-68 , scl-70 , jo-1 , dsdna , centromere b , ribosomal p0 , and extracts of hep-2 cells ) are immobilized on the gel chip as shown in figure 
 9(a ) , where qc  quality control , bc  blank control , rc  reaction control , nc  negative control , 1-jo-1 , 2-sm , 3-scl-70 , 4-cenp - b , 5-dsdna , 6-ssb , 7-ssa-52 , 8-extracts of hep-2 cells , 9-ssa-60 , 10-ribosomal p0 , 11-rnp-68 .
the protein microarray chip was incubated with 30  l of a five diseases - mixed serum ( diluted 1 : 100 with pbst ) for 30 minutes at room temperature . after being rinsed and washed one time for 5 minutes with pbst ,   the chip was incubated with 30  l of cy3-labeled goat antihuman igg antibody for 30 minutes at room temperature .
after another rinse and 5-minutes pbst wash , the chip was briefly centrifuged to dry it . the mixed serum of sle , ss , ssc , mctd , dm , and pm positive sera patient was tested on the protein microarray chip , which was then scanned using our confocal scanner .
the result of scanning image in figure 9(b ) clearly showed that this mixed sera contained anti - jo-1 at row 2 from column 1 to column 3 , anti - sm at row 2 from column 4 to column 6 , anti - scl-70 at row 2 from column 7 to column 9 , anticentromere b at row 3 from column 1 to column 3 , anti - dsdna at row 3 from column 4 to column 6 , anti - ssb at row 3 from column 7 to column 9 , anti - ssa-52 at row 4 from column 1 to column 3 , antinuclear antibodies at row 4 from column 7 to column 9 , anti - ssa-60 at row 5 from column 1 to column 3 , anti - ribosomal p0 at row 5 from column 4 to column 6 , and anti - rnp-68 at row 5 from column 7 to column 9 .
this developed confocal scanner is good for some applications of biochips , such as dna hybridization and immuno - reaction of proteins .
the long working distance of the newly developed confocal scanner has a particular advantage for work with biochips enclosed with a hybridization gasket , or conventional microscope slides with thick covers , or even with an uncovered liquid surface .
this feature is necessary for observation of real - time ( rt ) events on the chip surfaces , such as monitoring rt - fluorescence pcr .
the high scanning resolution power available with a 
 scanning beam of 0.4  m and with a sensitivity of detected fluorescence of 0.1 fluors/m are both important for obtaining an excellent images of small objects with quite clear definition , high contrast , and with a high signal - to - noise ratio .
this improved clarity of the images could be seen when comparing scans of a full moon normal molecular density biochip by using our developed confocal scanner with that of using another commercial confocal scanner ( see figure 7 ) . when compared to other common microscope objective designs where the objective of a typical microscope has a numerical aperture of na=0.65 and a working distance smaller than 1  mm ( such as the zeiss plan objective 440050 , magnification 40 ,    
 na=0.65 , wd=0.6 , wd=0.6  mm , and the nikon plan objective model 
 40 ,    
 na=0.65 , wd=0.6 , wd=0.57  mm ) , it is obvious that the optical design of the newly developed confocal scanning system is highly advanced , with an optimum combination of lenses , including a high numerical aperture of 0.72 , a long working distance of 3.0  mm .
the use of only 7 lenses and only 3 different kinds of optical glasses of zk7 , zk11 , and zf2 is also beneficial .
the limited number of glasses and lenses reduces the compound aberration in the use of 
 large numbers of lenses and multiple glasses .
in addition , these glass materials favor manufacture with small material error and small machining error .
furthermore , the developed confocal scanner can be used for scanning cells and tissues slide , and because the 
 high numerical aperture optical design has a very short focal depth 0 . 
7 m , it is also good for the tomography imaging of a cubic object .
 a novel confocal optical system design and a dual laser confocal scanner have been developed to meet the requirements of highly sensitive detection of biomolecules on microarray chips , which is characterized by a long working distance ( wd>3.0  mm ) , high numerical aperture ( na=0.72 ) , and only   3 materials and 7 lenses used .   
 this confocal optical system has a high scanning resolution , an excellent contrast and signal - to - noise ratio , and an efficiency of collected fluorescence of more than 2-fold better than that of other commercial confocal biochip scanners .   
 the scanner is as equally good for the molecular imaging detection of enclosed biochips as for the detection of   biological samples on a slide surface covered with a cover - slip glass .   
 some applications of gene and protein imagings using the dual laser confocal scanner are described . 
1. INTRODUCTION
2. CONFOCAL OPTICAL SYSTEM DESIGN  AND 
ANALYSIS
3. THE APPLICATION TO BIOCHIPS
4. DISCUSSION AND CONCLUSION