the same piece of membrane in different solutions is the distinct advantage of the outside-out patch relative to the cell-attached method. on the other hand, it is more difficult to accomplish. the longer formation process involves more steps that could fail and results in a lower frequency of usable patches. perforated patch perforated patch technique this variation of the patch clamp method is very similar to the whole-cell configuration. the main difference lies in the fact that when the experimenter forms the gigaohm seal, suction is not used to rupture the patch membrane. instead, the electrode solution contains small amounts of an antifungal or antibiotic agent, such as amphothericin-b, nystatin, or gramicidin, which diffuses into the membrane patch and forms small pores in the membrane, providing electrical access to the cell interior. when comparing the whole-cell and perforated patch methods, one can think of the whole-cell patch as an open door, in which there is complete exchange between molecules in the pipette solution and the cytoplasm. the perforated patch can be likened to a screen door that only allows the exchange of certain molecules from the pipette solution to the cytoplasm of the cell. advantages of the perforated patch method, relative to whole-cell recordings, include the properties of the antibiotic pores, that allow equilibration only of small monovalent ions between the patch pipette and the cytosol, but not of larger molecules that cannot permeate through the pores. this property maintains endogenous levels of divalent ions such as ca2+ and signaling molecules such as camp. consequently, one can have recordings of the entire cell, as in whole-cell patch clamping, while retaining most intracellular signaling mechanisms, as in cell-attached recordings. as a result, there is reduced current rundown, and stable perforated patch recordings can last longer than one hour. disadvantages include a higher access resistance, relative to whole-cell, due to the partial membrane occupying the tip of the electrode. this may decrease current resolution and increase recording noise. it can also take a significant amount of time for the antibiotic to perforate the membrane (about 15 minutes for amphothericin-b, and even longer for gramicidin and nystatin). the membrane under the electrode tip is weakened by the perforations formed by the antibiotic and can rupture. if the patch ruptures, the recording is then in whole-cell mode, with antibiotic contaminating the inside of the cell. loose patch loose patch clamp technique a loose patch clamp is different from the other techniques discussed here in that it employs a loose seal (low electrical resistance) rather than the tight gigaseal used in the conventional technique. this technique was used as early as the year 1961, as described in a paper by strickholm on the impedance of a muscle cell's surface, but received little attention until being brought up again and given a name by almers, stanfield, and stühmer in 1982, after patch clamp had been established as a major tool of electrophysiology. to achieve a loose patch clamp on a cell membrane, the pipette is moved slowly towards the cell, until the electrical resistance of the contact between the cell and the pipette increases to a few times greater resistance than that of the electrode alone. the closer the pipette gets to the membrane, the greater the resistance of the pipette tip becomes, but if too close a seal is formed, and it could become difficult to remove the pipette without damaging the cell. for the loose patch technique, the pipette does not get close enough to the membrane to form a gigaseal or a permanent connection, nor to pierce the cell membrane. the cell membrane stays intact, and the lack of a tight seal creates a small gap through which ions can pass outside the cell without entering the pipette. a significant advantage of the loose seal is that the pipette that is used can be repeatedly removed from the membrane after recording, and the membrane will remain intact. this allows repeated measurements in a variety of locations on the same cell without destroying the integrity of the membrane. this flexibility has been especially useful to researchers for studying muscle cells as they contract under real physiological conditions, obtaining recordings quickly, and doing so without resorting to drastic measures to stop the muscle fibers from contracting. a major disadvantage is that the resistance between the pipette and the membrane is greatly reduced, allowing current to leak through the seal, and significantly reducing the resolution of small currents. this leakage can be partially corrected for, however, which offers the opportunity to compare and contrast recordings made from different areas on the cell of interest. given this, it has been estimated that the loose patch technique can resolve currents smaller than 1 ma/cm2. patch-seq a combination of cellular imaging, rna sequencing and patch clamp this method is used to fully characterize neurons across multiple modalities. as neural tissues are one of the most transcriptomically diverse populations of cells, classifying neurons into cell types in order to understand the circuits they form is a major challenge for neuroscientists. combining classical classification methods with single cell rna-sequencing post-hoc has proved to be difficult and slow. by combining multiple data modalities such as electrophysiology, sequencing and microscopy, patch-seq allows for neurons to be characterized in multiple ways simultaneously. it currently suffers from low throughput relative to other sequencing methods mainly due to the manual labor involved in achieving a successful patch-clamp recording on a neuron. investigations are currently underway to automate patch-clamp technology which will improve the throughput of patch-seq as well. automatic patch clamping automated patch clamp systems have been developed in order to collect large amounts of data inexpensively in a shorter period of time. such systems typically include a single-use microfluidic device, either an injection molded or a polydimethylsiloxane (pdms) cast chip, to capture a cell or cells, and an integrated electrode. in one form of such an automated system, a pressure differential is used to force the cells being studied to be drawn towards the pipette opening until they form a gigaseal. then, by briefly exposing the pipette tip to the atmosphere, the portion of the membrane protruding from the pipette bursts, and the membrane is now in the inside-out conformation, at the tip of the pipette. in a completely automated system, the pipette and the membrane patch can then be rapidly moved through a series of different test solutions, allowing different test compounds to be applied to the intracellular side of the membrane during recording.