Introduction and Principle

Visual patching and imaging chamber is a temperature control device employed to maintain the specimen temperature, thereby preserving it in optimal conditions for a longer duration during imaging or electrophysiological experiments. Maintaining the specimen/slice at its physiological temperature during imaging or other experiments is requisite in some cases, and visual imaging and patching chamber plays a significant role. 

The visual patching and imaging chamber comprises a heat exchange system to maintain accurate temperature and feedback control to ensure bath temperature stability. The device also contains a separate temperature probe for in situ measurements of bath conditions. These temperature control instruments are usually calibrated with highly sensitive feedback sensors that facilitate greater temperature resolution, i.e., up to two decimal places. 

In the experiments, the visual patching and imaging chamber are usually mounted on the stage of an upright top-down microscope. The bath chamber is fed with the perfusate, and a constant flow of perfusate is maintained into and across the bath by the suction needle outflow. It moves waste perfusate to the waste vessel and keeps the chamber volume constant. The perfusion tubes are positioned properly, and a small amount of silicone is applied to avoid capillary action backflow and stabilize the tubing. 

Apparatus and Equipment

A thin heating element heat the aluminum heat exchange plate. The heat exchange system provides concentric heat to the chamber and the incoming perfusate, ensuring uniform thermal distribution. A thermistor embedded in the plate gives temperature feedback to the control system. The annular grooves present in the upper surface of the plate can carry up to four perfusate tubes. These perfusate tubes are designed to carry perfusate to the central chamber at a very low level, encouraging laminar flow across the chamber.

Furthermore, the device contains a gas inlet port for gas introduction in case of hypoxia studies. Gas flows around the grooves and is heated, ultimately entering the central chamber through radial grooves. To avoid drug adhesion, the heating plate is coated with polytetrafluoroethylene (PTFE). In addition, the low-profile chamber design keeps the unit from interfering with electrodes, objective lens, probes, etc. The compact unit allows the heater output to be counterbalanced with heat exchanger mass and likely perfusate demands. 

The temperature controller uses Proportional Integral Derivative (PID) to monitor perfusate temperature within +/- 0.1oC. Another temperature feedback probe placed in the central chamber is connected to the controller allowing chamber temperature and the temperature offset between perfusate and chamber to establish. The controller has analog data output, and all power outputs from it are DC reducing the interference of electromagnetic noise emission with other instrumentation. 

The suction capillary tube maintains a steady flow of waste perfusate from the central chamber while keeping a smooth, constant perfusate level in the center chamber. The suction tube can be adjusted according to perfusate depth and does not interfere with other instrumentation probes. 

The heat exchange system can accept a 22mm slip carrier or a 35mm Petri plate. It is mounted on the microscope stage using an insulator adapter ring suitable for most inverted and upright microscopes. 

Applications

Whole-Cell Patch-Clamp for Maintaining physiological temperature during brain slicing

Huang and Uusisaari (2013) tested the assumption that brain slice preparations must be cooled to near-freezing temperatures (less than 5oC) to get healthy slices. The researchers took mice (strain C57BL/6J 6w) of either gender pertaining to two age groups, i.e., immature (age: P17-P30) and mature (age: 2.5-7 months). The subjects were anesthetized using isoflurane and decapitated. Using a coronal cut, they separated the cerebellum from the forebrain and immediately glued it to a cutting stage immersed in artificial cerebrospinal fluid (ACSF). The researchers cut brain slices using a microtome blade. Bath temperature was maintained by adding cold or warm water to the external slicer chamber. A glass/mercury thermometer was used to monitor the temperature. Then they placed slices in a holding chamber containing oxygenated ACSF for 30 minutes. After this, they proceeded for patch-clamp electrophysiology. Brain slices were superfused in an oxygenated ACSF bath in the imaging chamber. 

Following this, researchers visualized neurons via interference contrast video microscopy. They recorded whole-cell patch clamps using borosilicate glass electrodes filled with an internal solution containing 140mM potassium gluconate, 10mM KCl, 10mM EGTA, and 10mM HEPES, 8mM biocytin, 4mM MgATP, 0.4mM NaGTP, and 10mM phosphocreatine. The experimenters identified Purkinje neurons and, following the identification, proceeded with biocytin imaging and slice staining. After analyzing electrophysiological data via statistical procedures, they concluded that when slices are cut with cutting solutions pre-warmed to near-physiological temperature, i.e., 34-35oC, the slice surface appears even and smooth compared to when they are prepared using the conventional ice-cold method.

Evaluation of the Impact of Protein Synthesis Inhibitors on Rat Hippocampal Slices

Puskarjov et al. (2012) demonstrated that applying protein synthesis inhibitors like cycloheximide and emetine does not affect KCC2 protein level and K-Cl transporter function. In the experiment, coronal hippocampal slices were prepared from immature male (P15-P20) Wistar rats and immersed in the conventional sucrose-based ice-cold cutting solution. For quality control, subsets of brain slices immersed in cutting solution prewarmed to near-physiological temperature. Following quantitative immunoblots, in vitro calpain cleavage assay, and analyzing the surface expression, the researchers proceeded for electrophysiological recordings. 

The researchers visually identified cell-attached and whole-cell patch-clamp recordings at 32oC. Borosilicate patch pipettes with resistance 4.5-6.5MΩ were used. The perfusate consisted of (in mM): 95 K-gluconate, 10D-glucose, 2 Mg-ATP, 5 HEPES, 1 EGTA, 20 sucrose, 0.1 Alexa Fluor 488, 30 N-methyl-D-glucamine-HCl, 2NaOH, and 5.4 KOH (pH 7.3). The slices were placed in a recording chamber and continually perfused at a 3.5 ml/min rate with an extracellular solution. Membrane potential values were corrected for calculated liquid junction potentials. The omission of Mg-2 from the extracellular solution during recordings and after recovery stimulated some slices to generate interictal-like activity. Using video microscopy, the scientists measured KCC2-mediated Cl– extrusion in one cell per slice via visual patching and imaging. EGABA quantified the chloride ion extrusion, i.e., the difference between the reversal potential of GABAA at the soma and 50µm away along the apical dendrite. They concluded that “total block protein synthesis does not affect KCC2 protein level or function in rat hippocampal slices for several hours.”

Benzethonium chloride (BEC) and Benzalkonium chloride (BAC) Patch Tests

Benjamin et al. (2012) experimented with differentiating irritants BAC and BEC from allergic patch test reactions to quaternary ammonium compounds. The researchers recruited ten patients (age group 34-65) to conduct patch tests with eight test articles. They placed occlusive skin patches and left them for 48h on the subject’s forearm. The eight test articles included “BAC (0.15% aq), BAC (0.15% pet), BEC (0.05% aq), BEC (0.15% pet), BEC (0.15% aq), BEC (0.5% aq).” Sodium lauryl sulfate (SLS) was used as positive irritant control and deionized water as a negative control. A random sample of each article was analyzed using HPLC. Skin responses to each patch test were recorded on the 4th and 7th days using the IDCRG scoring system, and visual reads were recorded. 

After patching, the researchers proceeded to skin site imaging using RCM (Reflectance-Mode Confocal Scanning Laser Microscopy). Hold the immersion fluid in place, and reduce blurring due to motion; a skin contact device consisting of a metal ring was fixed to the patient’s skin with medical-grade double-sided tape to stabilize the skin site. The ring was magnetically coupled to microscope housing. The scientist performed systemic horizontal mapping in case of reactive patch tests and captured four images in the axial section corresponding to four skin layers. The scientists also took three virtual “punch biopsies” in three different areas of each reactive test by combining multiple images taken in the z plane. Their experiments’ visual patching and imaging results revealed that two patients had definite allergic reactions, and one indicated BAC and BEC cross-reaction.

Whole-Cell Patching and Imaging for studying Iconic Mechanisms Encoding Sound Termination

“Rodent auditory brainstem possesses circuits involved in gap detection and sound duration encoding in which superior paraolivary nucleus (SPN) and trapezoid body’s medial nucleus are key components.” Scheinpflug et al. (2011) explained that offset responses upon sound termination in rodents are critical for perceptual grouping and gap detection. In addition, offset firing arises in the brainstem SPN, which receives powerful inhibition during sound and converts it into action potential (AP) upon sound termination. The experimenters used whole-cell patching and recording to compute sound termination. The neurons showed offset firing upon hyperpolarization; however, a minority of neurons did not show offset firing yet sustained onset firing in vivo. Visual patching and imaging also indicated IPSPs rather than EPSPs triggered offset firing. They also showed that action potential firing could occur from inhibition via integration of large IPSPs, driven by extremely negative chloride reversal potential (ECI). Moreover, sudden repolarization stimulates offset Apps that match onset time accuracy. 

Precautions

Underlying parameters should be ensured to ensure the unnecessary variability between the tests.

1. Maintain the perfusate level in the chamber bath at a flat, ideal level, with minimal volume undulation. This can be achieved by ensuring correct laminar flow with a four to one air to perfusate extraction by extraction needle.
2. Place the bath probe in the center and 0.5mm raised from the bottom of the bath. This step ensures the temperature measurement of perfusate and not the chamber’s bottom. 
3. The researchers can use a handheld temperature probe according to the experiment requirements. There is a negligible difference between the measurement of the handheld probe and the other temperature probe. 

Strengths and Limitations

Visual patching and imaging have several advantages. The first and foremost advantage is keeping the specimens at near-to-natural physiological temperature during experiments. Second, the specimens prepared with cutting solutions prewarmed to near-physiological temperatures have smooth and flat surfaces. Third, this approach can preserve fine image details and sharp edges. 

However, there are a few disadvantages as well. For instance, the similarity between patches helps in estimating flat regions. So, it might be time-consuming to compare similar patches. But the advantages of visual imaging and patch testing outweigh the disadvantages (Alkinani and El-Sakka, 2017). 

Summary

1. Visual patching and imaging chamber is a temperature control device employed to maintain the specimen temperature, thereby preserving it in optimal conditions for a longer duration during imaging or electrophysiological experiments.
2. The device comprises a heat exchange system to maintain accurate temperature and feedback control to ensure bath temperature stability.
3. It is mounted on a microscope stage, and the bath chamber is filled with the perfusate.
4. The patch recording technique has various applications, including maintaining near-physiological temperature during brain slice preparations and mechanisms encoding sound termination.
5. The major advantage of the technique is that the specimens prepared with cutting solutions prewarmed to near-physiological temperatures have smooth and flat surfaces.

References

1. Limef.com. (2022). Retrieved 21 April 2022, from http://www.limef.com/downloads/ci7800-paper.pdf. 
2. Huang, S., & Uusisaari, M. Y. (2013). Physiological temperature during brain slicing enhances the quality of acute slice preparations. Frontiers in cellular neuroscience, 7, 48.
3. Benjamin, B., Chris, F., Salvador, G., Melissa, G., & Susan, N. (2012). Visual and confocal microscopic interpretation of patch tests to benzethonium chloride and benzalkonium chloride. Skin Research and Technology, 18(3), 272-277.
4. Kopp-Scheinpflug, C., Tozer, A. J., Robinson, S. W., Tempel, B. L., Hennig, M. H., & Forsythe, I. D. (2011). The sound of silence: ionic mechanisms encoding sound termination. Neuron, 71(5), 911-925.
5. Alkinani, M. H., & El-Sakka, M. R. (2017). Patch-based models and algorithms for image denoising: a comparative review between patch-based images denoising methods for additive noise reduction. EURASIP Journal on Image and Video Processing, 2017(1), 1-27.