Photodiode-Based Optical Imaging for Recording Network Dynamics with Single-Neuron Resolution in Non-Transgenic Invertebrates

Summary

This protocol presents a method for imaging neuronal population activity with single-cell resolution in non-transgenic invertebrate species using absorbance voltage-sensitive dyes and a photodiode array. This approach enables a rapid workflow, wherein imaging and analysis can be pursued over the course of a single day.

Abstract

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light represents a revolutionary advance for studies of the neural basis of behavior. However, a downside of this development is its tendency to focus investigators on a very small number of “designer” organisms (e.g., C. elegans and Drosophila), potentially negatively impacting the pursuit of comparative studies across many species, which is needed for identifying general principles of network function. The present article illustrates how optical recording with voltage-sensitive dyes in the brains of non-transgenic gastropod species can be used to rapidly (i.e., within the time course of single experiments) reveal features of the functional organization of their neural networks with single-cell resolution. We outline in detail the dissection, staining, and recording methods used by our laboratory to obtain action potential traces from dozens to ~150 neurons during behaviorally relevant motor programs in the CNS of multiple gastropod species, including one new to neuroscience – the nudibranch Berghia stephanieae. Imaging is performed with absorbance voltage-sensitive dyes and a 464-element photodiode array that samples at 1,600 frames/second, fast enough to capture all action potentials generated by the recorded neurons. Multiple several-minute recordings can be obtained per preparation with little to no signal bleaching or phototoxicity. The raw optical data collected through the methods described can subsequently be analyzed through a variety of illustrated methods. Our optical recording approach can be readily used to probe network activity in a variety of non-transgenic species, making it well suited for comparative studies of how brains generate behavior.

Introduction

The development of transgenic lines of invertebrates such as Drosophila and C. elegans has provided powerful systems in which the neural bases of behavior may be optically interrogated and manipulated. However, these special preparations may have the downside of reducing enthusiasm for neural circuit studies of non-transgenic species, particularly with respect to the introduction of new species into neuroscience research. Focusing exclusively on one or two model systems is detrimental to the search for general principles of network function, because comparative studies represent an essential route by which such principles are discovered^1,2,3,4. Our aim here is to demonstrate a large-scale imaging approach for obtaining rapid insight into the functional structure of gastropod neural networks, in an effort to facilitate comparative studies of neural network function. 

Gastropod mollusks such as Aplysia, Lymnaea, Tritonia, Pleurobranchaea and others have long been used to investigate principles of neural network function, in large part because their behaviors are mediated by large, often individually identifiable neurons located on the surface of ganglia, making  them readily accessible to recording techniques⁵.  In the 1970s, voltage-sensitive dyes (VSDs) that can integrate into the plasma membrane were developed that soon enabled the first electrode-free recordings of the action potentials generated by multiple neurons⁶. Here, we demonstrate our use of VSDs to examine network activity in several species of gastropods, including one new to neuroscience, Berghia stephanieae. The imaging device is a commercially available 464-element photodiode array (PDA) that samples at 1,600 frames/second (Figure 1), which, when used with fast absorbance VSDs, reveals the action potentials of all recorded neurons⁷. The signals recorded by all diodes are displayed immediately after acquisition and superimposed on an image of the ganglion in the PDA acquisition software, making it possible to investigate neurons of interest with sharp electrodes in the same preparation^8,9. 

In the raw PDA data, many diodes redundantly record the larger neurons, and many also contain mixed signals from multiple neurons. A turning point was the development of an automated spike-sorting method using independent component analysis  to rapidly process each raw 464-channel PDA data set into a new set of traces, in which every recorded neuron appears in a separate trace containing just its action potentials^10,11.   

In this article we outline the essential steps involved in obtaining large-scale action potential recordings from gastropod nervous systems with a photodiode array and fast absorbance VSDs.  We moreover illustrate analytical methods that can be employed for clustering and mapping the optically recorded neurons with respect to their functional ensembles, and for characterizing population-level features that often are not apparent through simple inspection of the firing traces^12,13.     

Protocol

NOTE: The workflow outlined below is summarized in Figure 2.

1. Minimize vibration

1. If possible, ensure the rig is on the ground floor, and use a spring-based isolation table, which dampens a wider range of vibration frequencies than air tables.
2. If using a spring-based table, make sure it is floating (it needs to be adjusted every time one adds or takes something off the table).
3. Reduce vibration-based noise in the imaging room as much as possible, even to the extent of shutting off airflow during imaging if necessary. Minimize any vibration stemming from fluid turbulence in perfusion systems.
   NOTE: The neural preparation must not move during acquisition. Movements of any kind produce shifts of contrast edges across the diodes, leading to artifactual signals. If the procedure involves a stimulus during acquisition, it must not induce preparation movement.

2. Run a pinhole test to enable proper alignment of ganglion photos with the PDA data

1. Place a piece of aluminum foil with 3 small holes poked in it on a microscope slide. Take an image of the three holes with the digital camera mounted on its parfocal photoport.
2. Using the imaging software that comes with the PDA, acquire a short file (e.g., 5 s). Partway through the acquisition, tap the table to induce vibration artifacts that will be very visible around the edges of the pinholes, allowing for the image of the pinholes to be precisely aligned with the optical data.
3. Use the Superimpose function in the imaging software, found in menu item “Display | Page Superimpose | Superimpose Traces w/External Image | Superimpose Image,” to overlay the diode data onto the photo of the pinholes, and then iteratively adjust the x, y, and magnification settings for the photo until the pinholes sit directly atop the pinhole artifacts in the diode data.
   1. Save these numbers to align preparation images taken with the camera with the diode data in future experiments.
      NOTE: Pinhole alignment of the PDA only needs to be performed once after the PDA is mounted on the microscope, until it is rotated or removed, at which time it must be done again.

3. Dissections for three marine gastropod species

1. For species that grow to large size, such as Tritonia and Aplysia, start with smaller individuals, which have thinner, less opaque ganglia, easing obtaining sufficient light for optimal signal-to-noise.
2. Have ready filtered artificial seawater to be used as saline for the dissections and imaging experiments.
   NOTE: In all subsequent steps of the protocol, “saline” denotes artificial seawater.
3. Dissection of Tritonia diomedea
   1. Place an animal in the refrigerator for about 20 min to anesthetize it.
   2. For larger animals, expose the brain by holding the animal in one hand, letting the head end drape over the forefinger to expose the “neck.” For smaller animals, pin them dorsal side up in a wax-lined dissection dish before exposing the brain.
   3. Using dissection scissors, make a 3-4 cm midline incision on the dorsal side of the animal, above the buccal mass (which can be felt through the body wall).
      NOTE: The needed portion of the CNS, consisting of fused, bilateral cerebropleural and pedal ganglia, is orange and distinct in appearance from the surrounding tissue; it lies immediately posterior to the rhinophores and atop the buccal mass.
   4. Excise the CNS by severing those nerves innervating the animal’s body using forceps and microdissection scissors, keeping intact all those nerves connecting the central ganglia. Leave a long length of pedal nerve 3 (PdN3), or whichever nerve will be stimulated.
   5. Use minutien pins to affix the CNS to the bottom of a saline-filled elastomer-lined dish for further dissection. Maintain the preparation temperature at 11 °C by perfusing the dish with saline delivered with a feedback-controlled, in-line Peltier cooling system using a peristaltic pump.
   6. Using forceps and microdissection scissors, carefully remove the loosely adhering layer of the connective tissue from around the CNS. Leave the fine sheath closely adhering to the ganglia.
   7. Briefly (~10 s) dip the ganglia in a solution of 0.5% glutaraldehyde in saline. Place the ganglia back in the saline-perfused elastomer-lined dish, allowing saline to wash away the glutaraldehyde before beginning VSD staining.
      NOTE: This light fix of the connective tissue and its intrinsic muscles will help prevent movement during imaging.
4. Dissection of Aplysia californica
   1. Anesthetize an approximately 40 g animal by injecting ~20 mL of 350 mM MgCl₂ into the body through the ventral surface (foot).
   2. Use pins to position the animal ventral side up in a wax-lined dissection dish.
   3. Using dissection scissors, make a 2-3 cm midline incision along the most anterior extent of the foot. Pin down the flaps of the foot on either side of the incision to reveal part of the CNS and buccal mass.
      NOTE: The needed portion of the CNS, consisting of fused cerebral ganglia, and closely apposed, bilateral pleural and pedal ganglia, is yellow-orange and distinct in appearance from the surrounding tissue; it sits dorsal and posterolateral to the bulbous muscular buccal mass.
   4. Use forceps and dissection scissors to carefully dissect away the buccal mass, revealing the cerebral ganglia.
   5. Excise the CNS by severing those nerves innervating the animal’s body using forceps and microdissection scissors, keeping intact all those nerves connecting the central ganglia. Leave a long length of pedal nerve 9 (PdN9), or whichever nerve will be stimulated.
   6. Use minutien pins to position the CNS in a saline-filled elastomer-lined dish. Maintain the preparation temperature at 15-16 °C by perfusing the dish with saline passing through a Peltier cooling device.
   7. Using forceps and microdissection scissors, remove excessive connective tissue from the CNS, and dissect away a superficial portion of the sheath on the ganglion or ganglia to be imaged. Be careful during this process to not make a hole in the sheath, which would result in neurons spilling out from within.
   8. Briefly (~20 s) dip the ganglia in a solution of 0.5% glutaraldehyde in saline. Place the ganglia back in the saline-perfused elastomer-lined dish, allowing saline to wash away the glutaraldehyde before beginning VSD staining.
5. Dissection of Berghia stephanieae
   1. Place an animal in the refrigerator for about 20 min to anesthetize it.
   2. Using an elastomer-lined dish filled with room-temperature saline, place minutien pins in both the head and tail.
   3. Using microdissection scissors, make a 5-7 mm dorsal incision superficial to the CNS.
      NOTE: The eyes, dark spots that reside within the animal beside the CNS, conveniently mark the position of the needed portion CNS, which consists of bilaterally fused cerebropleural and pedal ganglia and sits atop the buccal mass.
   4. Excise the CNS by severing those nerves innervating the animal’s body using forceps and microdissection scissors. Leave any nerve being stimulated sufficiently long for a suction electrode.

4. Stain the preparation with a voltage-sensitive dye

1. Prepare stock solutions of either RH155 (also known as NK3041) or RH482 (also known as NK3630 or JPW1132).
   1. RH155: Dissolve 5.4 mg of solid dye in 1 mL of 100% EtOH, pipetting 29 µL into each of 34 microcentrifuge tubes. With the contents of each tube exposed to the air, let them dry overnight in the dark. Cap and place the resulting solid aliquots of RH155, each containing 0.15 mg, in a -20 °C freezer.
   2. RH482: Dissolve 2 mg of solid dye in 100 µL of DMSO, divide the solution into 20 aliquots of 5 µL, each containing 0.1 mg of RH482, and store in a -20 °C freezer.
      NOTE: For Tritonia and Aplysia, either bath perfusion or pressure application may be used to load the VSD RH155 into the membranes of the neurons in the preparation. Pressure application has the advantage of exposing just the ganglion being imaged to the VSD.
2. For bath perfusion, add 5 mL of saline to each of two of the above aliquots of solid RH155 and vortex into solution, producing a combined solution of 10 mL containing 0.03 mg/mL RH155.
   1. Perfuse in the dark (to avoid photobleaching) for 1 to 1.5 h at 11 °C for Tritonia and at 16 °C for Aplysia. Maintain the temperature by passing the perfusion solution through a Peltier cooling system.
3. For pressure application, add 500 µL saline to one aliquot of RH155, and vortex to produce a dye concentration of 0.3 mg/mL.
   1. Draw about 200 µL of the solution into polyethylene tubing using a handheld microdispenser, ensuring that there is a good match between the tube diameter and the diameter of the ganglion to be stained.
   2. Use a micromanipulator to carefully place the end of the tube over the target ganglion, lowering it until it forms a snug seal on the ganglion. Use the type of cooling system described above to keep the ganglia at the desired temperature.
   3. Dim the room lights to avoid photobleaching and turn the microdispenser applicator knob every 5 min to force more dye onto the ganglion.
   4. Check at 30 min to confirm that good staining is occurring, and then continue for a total staining time of about 1 h.
4. For staining in Berghia, add 1 mL of saline to a frozen aliquot of RH482, and vortex to dissolve.
   1. Transfer 200 µL of this solution into a microcentrifuge tube containing 800 µL of saline and vortex into solution, producing a final staining solution of 0.02 mg/ml RH482 in saline with 0.1% DMSO.
   2. Place the entire CNS into the microcentrifuge tube, wrapping the tube in aluminum foil to avoid photobleaching, and shake by hand every 5-6 min for about 1 h. Store the remaining 800 µL of the first solution in the refrigerator, and use for up to 3 days to stain subsequent preparations.

5. Flatten the preparation and set up for nerve stimulation

NOTE: The steps in this section should be performed under minimal illumination or with green light to minimize photobleaching.

1. After staining, immerse the CNS in saline within the imaging chamber and place under a dissecting microscope.
2. Place pieces of silicone (for Tritonia or Aplysia) or blobs of petroleum jelly (for Berghia) to the left and right of the ganglion/ganglia to be imaged.
3. Press an appropriately sized piece of a glass or plastic coverslip down onto the preparation to flatten it. Press firmly but not so hard as to damage the neurons.
   NOTE: Flattening the convex surface of the preparation in this manner will render a greater number of neurons parfocal, thereby increasing the number of neurons recorded, and will furthermore help to immobilize the preparation during imaging.
4. If stimulating a nerve to elicit a fictive motor program, prepare a suction electrode whose front tip is approximately as wide as the nerve diameter. Accomplish this by carefully melting a segment of PE-100 polyethylene tubing over a flame while gently pulling both ends of the tubing segment and then cutting the resulting taper at the desired point.
   1. Draw a small volume of saline through the tapered end of a polyethylene suction electrode, followed by the end of the nerve to be stimulated, by attaching a length of thick-walled, flexible polymer tubing to the back end of the electrode and using mouth suction to apply negative pressure.
   2. Confirm that the saline in the electrode lacks bubbles that could interrupt electrical conduction.

6. Preparation and optimization for imaging

1. Move the chamber to the imaging rig. Start the saline perfusion through the recording chamber and place the temperature probe near the preparation. Set the temperature controller for the temperature desired for the species being imaged (for Tritonia, 11 °C, for Aplysia, 15-16 °C, or for Berghia, 26-27 °C).
2. Place one chlorided silver wire down the suction electrode, making sure that it contacts the saline in the electrode, and put the other Ag-AgCl wire (the return path) into the bath saline, near the suction electrode.
3. Lower the water immersion lens into the saline. Close the base diaphragm, and then raise or lower the substage condenser and adjust the focus until the edges of the diaphragm are in sharp focus, creating Köhler illumination.
   1. Focus on the region of the preparation to be imaged. Bias focusing on smaller neurons over larger ones, as optical signals originating in larger neurons are more likely than smaller ones to be registered even if they are slightly out of focus.
   2. Take a picture of the ganglion to be imaged with the parfocal digital camera.
   3. With the control panel gain switch set to 1x, inspect the resting light intensity (RLI) in the imaging software by clicking on the “RLI” button and checking the average RLI of the diodes. Adjust the voltage level sent from a stimulator to the LED lamp power supply and continue checking the average RLI level until it is in the desired range (usually around 3-4 V).
      NOTE: High RLIs, corresponding to approximately 3-4 V on the PDA, are desirable. The higher the light, the better the signal-to-noise ratio of the optical signals, however, this must be balanced against a faster rate of photobleaching at higher RLIs. This risk is minimized by using high-NA objective lenses. The water immersion objective lenses used are 10x/0.6 NA, 20x/0.95 NA, 40x/0.8 NA, and 40x/1.15 NA.
4. Set the control panel gain switch to 100x for the recording.
5. If stimulating a nerve, set the desired voltage, frequency, and duration on a separate stimulator from that used to set the light level. Confirm that TTL triggering between the control panel and stimulator is properly configured.
   NOTE: Sample nerve stimulus parameters in each species are as follows: Tritonia PdN3, 2 s, 10 Hz pulse train of 5 ms, 10 V pulses; Aplysia PdN9, 2.5 s, 20 Hz pulse train of 5 ms, 8 V pulses; a Berghia pedal nerve, 2 s, 10 Hz pulse train of 5 ms, 5 V pulses.
6. Double-check that the spring or air table is floating.

7. Optical recording

1. Turn off or dim the room lights, including any overhead fluorescent lighting.
2. Set the desired file duration, the pathway, and file name, and then click on the “Take Data” button in the imaging software to acquire files up to the capacity of the computer’s available RAM. Remain still during the optical recording, as small vibrations can introduce large artifacts into the optical recording data.
   NOTE: For acquisitions that would exceed the computer’s available RAM, a custom-made C++ acquisition program is available through Dr. Jian-young Wu of Georgetown University.
3. To view data immediately after acquisition, use the Superimpose function in the imaging software to superimpose the data collected by all 464 diodes over the image of the ganglion taken earlier of the preparation⁷. Click on any of the diodes represented in the software to expand what they recorded on a separate trace screen.
   1. Achieve exact alignment of the diodes with respect to the preparation by entering the x, y, and magnification factors as previously determined by the pinhole test.
   2. To maximize action potential visibility and improve the neuron yield for subsequent spike-sorting¹⁴, impose a bandpass Butterworth filter with 5 Hz and 100 Hz cutoffs (available in imaging software) to remove both low- and high-frequency noise.
   3. To save filtered optical data as a text file for further analysis in a scientific computing platform, first select the “TP filter” box just under the “Page” screen in the imaging software. Then, select “Save Page as ASCII” from the “Output” tab and enter the desired filename into the dialog box that appears.

Discussion

One of the most important details in implementing our large-scale VSD imaging approach is to minimize vibration, which produces movements of contrast edges across the diodes, resulting in large artifactual signals. Because absorbance VSDs produce very small-percentage changes in light intensity with action potentials, vibration artifacts, if not prevented, can obscure the neuronal signals of interest. We employ several methods to minimize vibration artifacts. First, our imaging room is situated on the ground floor, which isolates the preparation from vibrations related to building air-handling equipment and many other sources. Second, a spring-based isolation table was used, which other PDA users have confirmed provides better vibration dampening than the more common air table¹⁶. Third, water immersion objectives were used, which eliminate image fluctuations arising from surface ripples. Fourth, the preparation being imaged was lightly pressed between the chamber coverslip bottom and a coverslip fragment pressed down from above that is held in place by silicone plugs or petroleum jelly, further stabilizing the preparation. This also flattens the convex surface of the ganglion or ganglia being imaged, resulting in more neurons in the plane of focus of the objective, which increases the number of neurons recorded.

To maximize the signal-to-noise ratio for the very small changes in the degree of VSD light absorbance resulting from an action potential, it is essential to achieve near-saturating light through the preparation to the PDA, while at the same time minimizing photobleaching of the dye. To this end, we typically work at 3-4 V of resting light intensity, as measured with the PDA control panel gain switch in the 1x position (the PDA’s 464 amplifiers saturate at 10 V of light). During data acquisition this gain factor is changed to 100x. Getting sufficient light to reach 3-4 V as measured by the PDA can be accomplished in several ways. First, use an ultrabright LED light source that delivers a wavelength appropriate to the absorption properties of the absorbance dye in use. Accordingly, a 735-nm LED collimated lamp was used, which overlaps with the optimal absorption wavelengths of RH155 and RH482. Second, if necessary, use a flip-top substage condenser that concentrates the light from the LED light source to a smaller area. Third, adjust the condenser height to achieve Köhler illumination, which ensures high, even brightness and maximal image quality. Fourth, ensure there are no heat filters in the optical pathway, which can attenuate the LED lamp’s 735-nm wavelength. Fifth, remove diffusers, if more light is needed, from the optical pathway. Sixth, use high-NA objectives, which provide high spatial resolution, and allow sufficient levels of light to reach the PDA at lower lamp intensities. This has allowed us to minimize photobleaching to the extent that we can obtain several acquisition files of 10-20 min duration per preparation using the same light intensity across all files and without a significant loss of signal amplitude or the need for re-staining. Crucially, if the experimenter wishes to track neurons across these longer files, ensure that the focal plane does not change, and that the preparation does not move. Finally, an additional way to route sufficient light to the PDA is to use younger animals, which have thinner, and thus less opaque ganglia.

From time to time we find that the signal-to-noise ratio of the optical signals deteriorates and/or the motor program rhythms are suboptimal (e.g., slow or abnormal). When this begins to occur consistently, we mix fresh solutions of VSD. Aliquots of VSD typically remain viable for about 6 months in a -20 °C freezer. Relatedly, it is worth noting that for Berghia, the best results have so far been obtained with the absorbance VSD RH482. As RH482 is more lipophilic than RH155, it may better stain Berghia’s comparatively smaller neurons or remain in the neuronal membranes more effectively at the higher recording saline temperature used for this tropical species.

One limitation of PDA-based imaging of neural activity relates to the AC coupling of the voltage signals in hardware before the 100x preamplification step: although this represents a necessary feature to remove the large DC offset produced by the high resting light level required by this technique, the AC coupling intrinsic to the PDA precludes the measurement of slow changes in membrane potential, such as those associated with synaptic inputs. If recording slow or steady-state potential changes is desired, a DC-coupled CMOS camera imaging system can be utilized to capture subthreshold activity. Byrne and colleagues recently used such a setup with RH155 to image the activity of neurons in the buccal ganglion of Aplysia^17,18. We have used both systems and found that the CMOS camera, due to its much higher density of detectors (128 x 128), generates 50x larger data files for the same imaging time⁷. The PDA’s smaller files facilitate faster processing and analysis. This also enables extended single-trial recordings (Figure 4) and studies of learning, in which data from multiple trials are concatenated into one large file before spike-sorting, allowing network organization to be tracked as learning develops¹⁹.

In other camera-based investigations, fluorescent VSDs have been used by Kristan and colleagues to examine network function in the segmental ganglia of the leech. In one influential study this led to the identification of a neuron involved in the animal’s decision to swim or to crawl²⁰. In another study, Kristan et al. examined the extent to which the leech’s swimming and crawling behaviors are driven by multifunctional vs. dedicated circuits²¹. More recently, Wagenaar and colleagues used a two-sided microscope for voltage imaging that allows them to record from almost all neurons in a leech segmental ganglion²². In contrast to many camera-based imaging methods, an advantage of our PDA-based imaging method is rapid and unbiased spike-sorting by ICA, a form of blind source separation that involves no decisions about neuronal boundaries for results processing.

With respect to the choice of VSDs, one advantage of the absorbance dyes RH155 and RH482 is the little-to-no phototoxicity associated with them^23,24, enabling longer recording times than is typical for fluorescent VSDs. Moreover, the fast absorbance VSDs we use are well suited for recording the overshooting somatic action potentials in gastropod preparations, which are typically 80 mV in amplitude. As shown in Figure 3G, our optical method can record action potential undershoots (none of our recordings are trace-averaged): this suggests that the VSDs we use should be able to discern action potentials in other model systems that attenuate to some degree and thus are not overshooting by the time they reach the soma. Nevertheless, our optical approach may not be ideal for species that are known to exhibit highly attenuated action potentials when recorded in the soma.

Much current research on neural networks is being focused on a small number of designer transgenic species. However, neuroscience benefits from the study of a wide variety of phylogenetically distinct species. Studying many different species provides insights into how circuits evolve^25,26, and illuminates principles of network function that may be common across phyla^1,2,3,4,27. We have so far applied our imaging method to a number of gastropod species, including Aplysia californica^{8,11,12,13,14,28}, Tritonia diomedea^{8,9,11,14,19,28}, Tritonia festiva²⁸, Pleurobranchaea californica (unpublished data), and most recently Berghia stephanieae (Figure 5). An appeal of this approach is that it can be readily applied to many species, with no need for transgenic animals. We wish to acknowledge that our use of VSD imaging with fast absorbance dyes and a PDA follows in the footsteps of pioneering work that accomplished this in semi-intact, behaving Navanax²⁹ and Aplysia³⁰ preparations. Our emphasis on the rapidity of our approach is in part an answer to concerns that many investigators may be increasingly reluctant to initiate network studies in new species due to fears that years of study will be necessary to characterize basic network organization before being able to explore scientific questions of broad interest to neuroscience³¹. Accordingly, our goal here is to demonstrate a technique that greatly speeds the process – to the point that significant same-day insights into network organization may be obtained from single preparations.

Materials

Name	Company	Catalog Number	Comments
Achromat 0.9 NA swing condenser	Nikon	N/A
Bipolar temperature controller	Warner Instruments	CL-100 with SC-20	Controls perfusion saline temperature
Chamber thermometer	Physitemp	BAT-12 with IT-18 microprobe
Digital camera	Optronics	S97808
Dissecting forceps	Dumont	#5
Dissecting scissors	American Diagnostic Corp.	ADC-3410Q
Imaging microscope	Olympus	BX51WIF
Imaging perfusion chamber	Siskiyou	PC-H
Instant Ocean	Instant Ocean	SS6-25	Makes 25 gallons at a time
Master-8 pulse stimulator	A.M.P.I.	Master-8
Microdispenser	Drummond Scientific	3-000-752	Dye applicator for pressure staining
Microdissection scissors	Moria	15371-92
Minutien pins (0.1 mm)	Fine Science Tools	NC9677548	For positioning and stabilizing CNS
Motorized microscope platform	Thorlabs	GHB-BX	Gibraltar platform
NeuroPlex imaging software	RedShirtImaging	NeuroPlex	Compatible with the WuTech photodiode array
Objective lenses	Olympus	XLPLN10XSVMP, XLUMPLFLN20XW, LUMPLFLN40XW, UAPON40XW340
PE-100 polyethylene tubing	VWR	63018-726	Tubing to make suction electrodes
Perfusion pump	Instech	P720 with DBS062SDBSU tube set
Petroleum jelly	Equate	NDC 49035-038-54
Photodiode array with control panel	WuTech Instruments	469-IV photodiode array	Contact jianwu2nd@gmail.com for ordering information
RH155	Santa Cruz Biotechnology	sc-499432	Voltage-sensitive dye
RH482	Univ of Conn. Health Center	JPW-1132	Voltage-sensitive dye; special order from Leslie Leow
Silicone earplugs	Mack's	Model 7	To be use for preparation compression
Staining PE tubing	VWR	63018-xxx	Different sizes depending on fit
Sylgard 184 silicone elastomer kit	Dow Corning	Sylgard 184 silicone elastomer kit
Thorlabs LED and driver	Thorlabs	M735L2-C1, DC2100	LED lamp and driver
Tygon tubing	Fisher Scientific	14-171-xxx
Vibration isolation table	Kinetic Systems	MK26	Spring-based