Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

Podsumowanie

The electrophysiological technique of intracellular recording is demonstrated and used to determine spectral sensitivities of single photoreceptor cells in the compound eye of a butterfly.

Streszczenie

Intracellular recording is a powerful technique used to determine how a single cell may respond to a given stimulus. In vision research, intracellular recording has historically been a common technique used to study sensitivities of individual photoreceptor cells to different light stimuli that is still being used today. However, there remains a dearth of detailed methodology in the literature for researchers wishing to replicate intracellular recording experiments in the eye. Here we present the insect as a model for examining eye physiology more generally. Insect photoreceptor cells are located near the surface of the eye and are therefore easy to reach, and many of the mechanisms involved in vision are conserved across animal phyla. We describe the basic procedure for in vivo intracellular recording of photoreceptor cells in the eye of a butterfly, with the goal of making this technique more accessible to researchers with little prior experience in electrophysiology. We introduce the basic equipment needed, how to prepare a live butterfly for recording, how to insert a glass microelectrode into a single cell, and finally the recording procedure itself. We also explain the basic analysis of raw response data for determining spectral sensitivity of individual cell types. Although our protocol focuses on determining spectral sensitivity, other stimuli (e.g., polarized light) and variations of the method are applicable to this setup.

Wprowadzenie

The electrical properties of cells such as neurons are observed by measuring ion flow across cell membranes as a change in voltage or current. A variety of electrophysiological techniques have been developed to measure bioelectric events in cells. Neurons found in the eyes of animals are accessible and their circuitry is often less complex than in the brain, making these cells good candidates for electrophysiological study. Common applications of electrophysiology in the eye include electroretinography (ERG)^1,2 and microelectrode intracellular recording. ERG involves placing an electrode in or on the eye of an animal, applying a light stimulus, and measuring the change in voltage as a sum of the responses of all nearby cells^3-6. If one is specifically interested in characterizing spectral sensitivities of individual photoreceptor cells, often multiple cell types simultaneously respond at different strengths to a given stimulus; thus it can be difficult to determine the sensitivities of specific cell types from ERG data especially if there are several different kinds of spectrally-similar photoreceptor cells in the eye. One potential solution is to create transgenic Drosophila with the photoreceptor (opsin) gene of interest expressed in the majority R1-6 cells in the eye and then perform ERGs⁷. Potential drawbacks of this method include no to low-expression of the photoreceptor protein⁸, and the long time frame for the generation and screening of transgenic animals. For eyes with fewer kinds of spectrally distinct photoreceptors, adaptation of the eye with colored filters can help with lowering the contribution of some cell types to the ERG, thereby permitting estimation of spectral sensitivity maxima⁹.

Intracellular recording is another technique where a fine electrode impales a cell and a stimulus is applied. The electrode records only that individual cell's response so that recording from and analyzing multiple individual cells can yield specific sensitivities of physiologically different cell types^10-14. Although our protocol focuses on analysis of spectral sensitivity, the basic principles of intracellular recording with sharp electrodes are modifiable for other applications. Using a different preparation of a specimen, for instance, and using sharp quartz electrodes, one may record from deeper in the optic lobe or other regions in the brain, depending on the question being asked. For example, response times of individual photoreceptor cells¹⁵, cell activity in the optic lobes¹⁶ (lamina, medulla or lobula¹⁷), brain¹⁸ or other ganglia¹⁹ can also be recorded with similar techniques, or color stimuli could be replaced with polarization^20-22 or motion stimuli^23,24.

Phototransduction, the process by which light energy is absorbed and converted into an electrochemical signal, is an ancient trait common to nearly all present day animal phyla²⁵. The visual pigment found in photoreceptor cells and responsible for initiating visual phototransduction is rhodopsin. Rhodopsins in all animals are made up of an opsin protein, a member of the 7 transmembrane G protein-coupled receptor family, and an associated chromophore which is derived from retinal or a similar molecule^26,27. Opsin amino acid sequence and chromophore structure affect the absorbance of rhodopsin to different wavelengths of light. When a photon is absorbed by the chromophore the rhodopsin becomes activated, initiating a G-protein cascade in the cell that ultimately leads to the opening of membrane-bound ion channels²⁸. Unlike most neurons, photoreceptor cells undergo graded potential changes that can be measured as a relative change in response amplitude with changing light stimulus. Typically a given photoreceptor type expresses only one opsin gene (though exceptions exist^8,10,29-31). Sophisticated color vision, of the kind found in many vertebrates and arthropods, is achieved with a complex eye of hundreds or thousands of photoreceptor cells each expressing one or occasionally more rhodopsin types. Visual information is captured by comparing responses over the photoreceptor mosaic via complex downstream neural signaling in the eye and brain, resulting in the perception of an image complete with color and motion.

After measuring the raw responses of a photoreceptor cell to different wavelengths of light via intracellular recording, it is possible to calculate its spectral sensitivity. This calculation is based on the Principle of Univariance, which states that a photoreceptor cell's response is dependent on the number of photons it absorbs, but not on the particular properties of the photons it absorbs³². Any photon that is absorbed by rhodopsin will induce the same kind of response. In practice, this means that a cell's raw response amplitude will increase due to either an increase in light intensity (more photons to absorb), or to a shift in wavelength toward its peak sensitivity (higher probability of rhodopsin absorbing that wavelength). We make use of this principle in relating cellular responses at known intensity and the same wavelength to responses at different wavelengths and the same intensity but unknown relative sensitivity. Cell types are often identified by the wavelength at which their sensitivity peaks.

Here we show one method for intracellular recording and analysis of spectral sensitivity of photoreceptors in the eye of a butterfly, with a focus on making this method more accessible to the wider research community. Although intracellular recording remains common in the literature, particularly with respect to color vision in insects, we have found that descriptions of materials and methods are usually too brief to allow for reproduction of the technique. We present this method in video format with the aim of permitting its easier replication. We also describe the technique using easily obtainable and affordable equipment. We address common caveats that often are not reported, which slow down research when optimizing a new and complex technique.

Protokół

All animals were treated as humanely as possible. Insects were shipped as pupae from Costa Rica Entomological Supply, Costa Rica.

1. Heliconius Pupae Care

1. Hang all pupae spaced 2-3 cm apart in a humidified chamber using insect pins.
2. After eclosion, allow wings to dry then keep butterflies alive for at least 1 day in a humidified chamber and feed a dilute honey solution daily before recording.
   1. Dilute honey with water to about a 20% honey solution by volume, and pour into a shallow Petri dish.
   2. Bring individual butterflies to the Petri dish, one by one. Upon touching the solution with their front tarsi, the butterflies will automatically extend their proboscides and drink from the Petri dish. If their proboscis does not automatically extend, use forceps to pull the proboscis out and introduce it to the honey solution.

2. Optical Track, Calibration, and Measurement of Experimental Light Conditions

1. Place a 150 W Xenon arc lamp with housing and universal power supply and an attached condenser lens assembly on one end of a table at least one meter long to deliver bright white light.
   CAUTION: Xenon arc lamps produce extremely bright light with strong UV intensities. Protective eyewear should be worn at all times and the lamp should be used as directed by the manufacturer to prevent accumulation of ozone caused by interaction of UV light with atmospheric oxygen.
2. Set up an optical track one meter in length for the light exiting the housing assembly to pass through (Figure 1).
   1. Place in the following order on the optical track with approximate distances apart: 1) a convex silica or quartz lens 40 cm from the condenser assembly, 2) a neutral density filter wheel (with no filters currently in the light path) 22 cm further along the track, 3) a shutter with drive unit 14 cm from the ND filters, 4) a concave silica or quartz lens immediately adjacent following the shutter, and 5) a collimating beam probe 6 cm further along the far end of the track.
   2. Affix a 600 µm diameter fiber optic cable to the collimating beam probe.
      Note: Depending on light intensity, a 5-10 mm diameter fiber optic cable may be required to deliver enough light to other preps and may be substituted for this.
   3. Adjust the distance, height and angle of each optical element so that the light beam exiting the assembly is at the highest intensity possible.
   4. As optical track elements may differ slightly with different applications, ensure that all elements transmit light in the UVA and visible range (315-700 nm).
3. Once the optical track is assembled, measure the light that passes through the setup using a spectrometer. Calibrate the spectrometer first using a calibration lamp with a known spectrum and the manufacturer's software.
   Note: We describe the following set up using Ocean Optics products for clarity but other manufacturers (e.g., Avantes) sell comparable products.
   1. Turn on the tungsten calibration lamp at least 45 min before taking measurements.
   2. To calibrate, attach the spectrometer via USB to a computer with the associated software installed. Then connect the spectrometer to the tungsten calibration lamp via a UV-visible transmitting cosine corrector.
   3. Select "New Absolute Irradiance Measurement" from the "File" tab, and select the spectrometer as the "Source."
   4. Follow the prompts to create a new calibration "cal" file. When prompted, load the provided data file for the known spectrum of the tungsten calibration lamp in the visible light range (300-800 nm) into the software, which automatically calculates the corrected spectrum from the spectrometer output.
   5. Save the calibration file. Load this file when initializing the software for all future measurements of light spectra using the spectrometer.
4. Once the spectrometer is calibrated, use this to record the light spectra from the experimental setup. Hereafter when the software is opened, select "New Absolute Irradiance Measurement" and load the previously saved calibration file. Next take a dark spectrum by blocking all light to the spectrometer.
   1. With the spectrometer currently measuring the desired experimental light conditions, adjust the integration time (4 msec), scans to average (5), and boxcar width (5), so the spectrum is properly scaled and smoothed. Keep the settings the same for all spectral measurements, so that light intensities from different measurements can be compared.
5. Measure spectra for unattenuated white light, for all neutral density filters to be used during experiments, and for each bandpass interference filter (Figure 2).
   1. Measure the white light spectrum without any filters in the light path by affixing the free end of the fiber optic cable from step 2.2.2 to the spectrometer. With the calibration file loaded from step 2.3, save the white light spectrum using the spectrometer's software as a text file.
      Note: Spectra saved as text files list the wavelength (x coordinates) in one column and the intensity of light (y coordinates) in the second column, so that the data may be loaded into a spreadsheet for step 2.6.
   2. Using the same setup as step 2.5.1, record the spectrum from each optical density (OD) (0-3.5 OD) used during experiments by rotating the neutral density (ND) filter wheel in the optical track, and save the text file for each OD.
   3. Using the same setup as step 2.5.1, place the 10 nm half bandwidth interference filters one by one into the light path and record the spectrum observed for each filter. Repeat this procedure for each of 41 different interference filters with peak transmittances spaced every 10 nm from 300 to 700 nm. Filters spaced further apart (20 nm) are acceptable for most applications (for spectra, see Figure 2).
6. Correct for differences in intensity of light when interference filters are placed in the light path. Each interference filter allows a different total number of photons to pass, and the low transmission of some filters makes it difficult to further attenuate intensity so that all filters allow equal numbers of photons.
   1. To calculate the relative intensity (I) for each 10 nm bandwidth interference filter, solve for I in the expression, I = T/sec, where T is the area under the spectral curve of each 10 nm interference filter (from 2.5.3), and s is the maximum absolute irradiance (y value of saved text file from 2.5.1) of white light at the peak wavelength of each filter (See Figure 2 for an example at 520 nm).
   2. Divide all calculated intensities by the max intensity value calculated in 2.6.1 to normalize to one, and take the reciprocal of the relative normalized values for use as a correction factor applied to the raw sensitivity at each wavelength (see Step 6.4).
7. Perform steps 2.1 through 2.6 only once before a set of experiments. Over the course of an experiment periodically record the absolute irradiance of the Xenon arc lamp under bright light and neutral density filters, to make sure the intensity of the light stimulus does not change.
8. During the course of an experiment, if any cellular response to light transmitted through the interference filters approaches the maximum response amplitude, use the ND filters to attenuate the signal. If ND filters are used during an experiment, account for the corresponding decrease in intensity during the calculation of spectral sensitivity.
9. Set up optical track, calibration, and filters days or weeks before experiments begin. Keep filters covered to prevent dust accumulation.

3. Recording Equipment Setup

1. Feed the same fiber optic cable used for calibration through a Faraday cage and mount on a goniometric device such as a Cardan arm perimeter (see Figure 3 for diagram). The cable will be about 10 cm away from the eye of the specimen.
2. Place a metal stage on a vibrationally isolated table with an electrode holder mounted directly above the stage under control of a micromanipulator (Figure 4). Place the Cardan arm so that the specimen's head is at the center of the sphere created by the arm's rotational movement.
3. Using an intracellular preamplifier system, which includes an amplifier (outside the Faraday cage) and preamplifier (headstage, near the prep inside the Faraday cage) mount the headstage above the metal stage where the specimen will be placed.
   1. Connect a coaxial cable to the headstage via a BNC connection. Split open only the tip on the other end of the coaxial cable, and separate the outer metal sheath of the cable from the inner wire.
   2. Solder the outer sheath (kept at ground potential) to one end of an insulated copper wire with an alligator clip on the other end. This alligator clip will attach to the metal reference electrode on the specimen platform (Step 5.1.4).
   3. Solder the inner wire of the coaxial cable to a thin silver wire, to serve as the recording electrode. This wire should be thin enough to be fed into the solution-filled glass electrode in Step 5.2.3.
4. Place a stereomicroscope attached to a swinging arm and base on the wooden bench outside the Faraday cage, so that it may be swung in to lower the electrode into the eye, and swung back out again once the electrode is in the eye.
5. Make sure everything metal inside the Faraday cage is properly grounded.
6. Outside the Faraday cage, attach the preamplifer to the input of a 50-60 Hz noise reducer (optional), and connect the output to one channel of an oscilloscope using a BNC T-adapter.
7. Using the other end of the T-adapter, connect the signal passing through the oscilloscope to one channel of the hardware. Attach this hardware to a computer by a USB cable, which will allow responses recorded with the preamplifier to be read by software on the computer.
8. Attach the shutter driver from the optical track to the second channel of the oscilloscope using another T-adapter and connect this to a pulse generator that will control the frequency and duration of light flashes delivered to the eye (Step 5.5).
   Note: Setup of the rig itself should only need to be done once. Break here until ready to begin recordings.

4. Prep on the Day of Recording

1. Turn on the Xenon lamp at least 45 min before the experiment and turn on the glass microelectrode puller at least 30 min before pulling glass electrodes.
2. Turn on all recording equipment (shutter, amplifier, noise eliminator, pulse generator, oscilloscope, and data acquisition hardware) and make sure the shutter is closed by default so no light passes through the fiber optic cable.
3. Pull fine borosilicate (or aluminosilicate) glass microelectrodes (100-250 MΩ resistance is ideal) using a glass microelectrode puller. Use glass electrodes within only a few hr of being pulled.
4. Backfill the electrodes with 3 M Potassium chloride (KCl). Note that this solution may be modified according to the researcher's needs, e.g. dye injection.

5. Specimen Prep and Recording Procedure

1. Prepare the Specimen
   1. Affix an individual butterfly inside a small plastic tube with hot wax so the head is immobile and protruding from one end of the tube. Wax down proboscis, antennae, and wings (Figure 5).
   2. Hold down the abdomen with a dry piece of wax and keep the tube humidified by placing a wet tissue inside the tube behind the abdomen. Make sure the specimen is completely immobile.
   3. Mount the tube using a small piece of wax onto a small platform with a ball-and-socket joint that is attached to a magnetic base.
   4. Under a dissecting microscope, insert a silver wire of 0.125 mm diameter into the head via the mouthparts to be used as the reference electrode. Before the experiment, permanently fix the wire to the platform in such a way that the copper wire in Step 3.3.2 may clip on to it once the platform is placed on the stage for recording.
   5. Once the reference electrode is in a suitable position it may be kept in place by quickly melting and then cooling wax around the wire.
   6. Using a breakable carbon steel razor blade, grip part of the blade with a blade holder and break off a small piece to use for cutting the cornea.
   7. Cut a small hole (~10 ommatidia in diameter) in the left cornea using the razorblade and seal the hole with Vaseline to prevent desiccation.
2. Once the cornea is cut, insert the recording electrode into the eye as quickly as possible because hemolymph in the eye will quickly harden and make it impossible to insert an electrode. If possible perform the dissection in the rig where the recording will take place.
   Note: Vaseline should not be smeared on the rest of the eye as this will defocus the optics.
   1. If not already on the stage, place the mounted specimen and platform onto the stage in the recording rig. Connect the headstage ground wire from step 3.3.2 to the reference electrode on the specimen platform using alligator clips.
      Note: If possible use a red filter to illuminate the animal.
   2. Use a light source with gooseneck attachments to briefly light the specimen under a stereoscope while lowering the recording electrode into the eye.
   3. Insert the silver wire connected to the headstage from step 3.3.3 into the KCl solution in the back of a glass microelectrode. Mount the glass electrode on the electrode holder.
   4. Adjust the electrode holder so the microelectrode is directly over the hole previously cut in the cornea, about a millimeter above the cornea. Lower the microelectrode into the eye using the micromanipulator until a circuit is completed, as shown by a large change in potential (mV) on the oscilloscope.
3. Once in the eye, swing the stereoscope outside the Faraday cage, and turn off the light source illuminating the specimen. The room should be kept dark so the eye becomes dark adapted.
4. Check the resistance of the electrode by applying a 1 nA current from the amplifier and noting the change in voltage. Resistance should typically be in the range between 100-250 MΩ. Higher resistances are indicative of blockage or bending of the electrode, and low resistances of electrode breakage.
5. Activate the pulse generator so the shutter opens allowing a flash of light with a 50 msec duration every 0.5 sec, and allow it to continue flashing for the duration of the experiment.
   1. Adjust the pulse generator so it allows flashes of up to 50 msec duration. This duration and 0.5 sec pause between flashes keeps the specimen as near to dark adapted as possible during the experiment. Fifty msec is close to the shortest flash duration that will elicit the same amplitude in response as longer flash durations.
   2. Re-measure responses at both the beginning and end of the experiment (Step 5.16). Over the course of about a twenty minute experiment, these flash settings do not degrade the response over time. Different preps may require adjustments to these flash settings.
6. Position the Cardan arm so that the fiber optic cable is directed toward the eye.
7. Check the oscilloscope for voltage change with each light flash. A negative change in voltage signifies that the electrode has not yet entered a cell.
8. Move the Cardan arm around the specimen until it is positioned at an angle to the eye at which there is a maximum voltage response.
9. Rotate the micromanipulator back and forth, causing very small vertical movements of the electrode in both directions while lightly tapping the base of the electrode holder or using the Buzz function on the preamplifier. Continue making small adjustments until a depolarizing light response appears on the oscilloscope (Figure 6).
10. Adjust the Cardan arm again to find the angle of incidence where a flash of light produces the largest depolarizing signal. Make small adjustments with the micromanipulator and use the Buzz function on the amplifier as needed to make sure the electrode is stably recording the cell and that it will stay in the cell for the whole experiment (See Step 5.11).
11. Once the setup is stable, begin recording. A stable recording should have little to no change in resting potential, low background noise, and a consistently large depolarizing response (at least a 10:1 signal to noise ratio).
    1. Run the software on the computer, and begin a "new experiment," which will open a pop up window with four channels.
    2. Adjust the voltage scale at the top right corner of the software window to 500 mV. The first channel will display the responses recorded from the electrode in real time, while the second channel will record the square wave produced by the function generator, if the signal is fed to the data acquisition hardware via the oscilloscope, showing when the shutter is open. The other two channels are unneeded.
    3. Click "Start" at the bottom right hand corner to begin recording, and allow the software to run for the duration of the experiment. Adjust the zoom of the x (time) and y (voltage) axes so that the responses are clear.
12. First, with white light, record up to 10 individual responses with the ND filter wheel at 3.5 OD (about 5-10 sec).
13. Next record the same number of responses at 3.3 OD, then 3.1, 3.0, 2.5, 2.3, 2.1, etc. in every combination until 0.0 OD. These response amplitudes to the ND filter series will provide the response-log intensity curve in Section 6. If bleaching occurs, use fewer flashes of bright stimuli during the course of the experiment.
14. Record the response of the cell to all wavelengths, using the interference filters.
    1. First find the peak wavelength. Without ND filters in the light path (0.0 OD), place a UV transmitting filter in the light path and briefly observe the response amplitude. Repeat with a blue transmitting filter, a green transmitting filter, and a red transmitting filter, which should give some idea of where the peak response will be.
    2. Use filters at about 350, 450, 550, 650 nm to find the general region of peak sensitivity in step 5.14.1. The exact wavelength does not matter in this initial search phase because all wavelengths will be recorded in the next step. If estimates exist of peak sensitivities, or they have been previously recorded, use known wavelengths to quickly identify the peak response.
    3. Once the peak response or close to it is identified, record at this wavelength for 10 responses (about 5 sec).
    4. After recording at the wavelength of peak response, record with the other interference filters, from 300-700 nm at 10 nm steps. Start from the peak and step out toward both shorter and longer wavelengths by swapping the filters out from the light path one by one (e.g. if the peak response is at 520 nm, record responses at this wavelength first, then 510 nm, followed by 530 nm, 500 nm, 540 nm, 490 nm, 550 nm, and so on until no there is no response).
    5. Allow for up to 10 responses per filter (5 sec each). When swapping interference filters, allow the cell to respond to 1-2 flashes of white light without any filter in the light path, which is helpful to monitor whether the peak response is degrading over time. Reduce the number of responses or increase the OD if bleaching occurs.
15. If the response under any interference filter is too close to the maximum response under white light at 0 OD, then attenuate with ND filters. The interference filters and size of the fiber optic cable used in this experiment greatly attenuate the intensity of light and so ND filters are typically not needed.
16. If the recording remains stable, re-record wavelengths around the peak response, which serves as a pseudoreplicate for confirming previous response amplitudes and helps to ensure the response has not degraded over time. Once all wavelengths are recorded, re-record the responses under the ND series, as in step 5.12.
17. Once recording is complete click "Stop" on the software, and save the recording for analysis.
18. After an experiment, sacrifice the individual by freezing, or cooling for several minutes followed by swiftly severing the head and crushing the thorax.
19. Shut down all equipment. Break here if needed before doing the analysis.

6. Spectral Sensitivity Analysis

1. With the software used to record raw responses, calculate the mean response amplitude of 10 individual responses for each filter in the ND series and for each interference filter.
2. Create a response-log intensity (VlogI) function from the ND filter series recorded in Steps 5.12-5.13 (Figure 7). Do this by plotting log units of intensity (OD) on the X axis, and response to each intensity on the y axis.
   1. To derive spectral sensitivity of the cell at different wavelengths, typically fit the Naka-Rushton equation to the data from step 6.2, and use this equation to relate experimentally obtained spectral responses of different wavelengths to relative photons required to elicit that response under a constant wavelength (in this case white light).
      Note: The Naka-Rushton equation is: V/V_max = Iⁿ/(Iⁿ + Kⁿ), where I is the stimulus intensity, V is the response amplitude, V_max is the maximum response amplitude, K is the stimulus intensity giving ½ V_max, and n is the exponential slope. Various methods can be used to fit this equation to the VlogI data, including curve fitting software, or code-based statistical packages.
   2. To fit the Naka-Rushton equation using simple calculations and a spreadsheet program, transform the VlogI response data for each stimulus intensity: log[(V_max/V) - 1]. Then perform linear regression on the transformed data to get the equation of the line of best fit.
      Note: V_max must be greater than any measured responses; to keep this consistent, this method estimates V_max as 1% greater than the highest measured response.
   3. From the equation of the regression line, estimate the exponent (n) by taking the negative slope, and log(K) = y-intercept/n.
3. Once the parameters for V_max, n, and K have been estimated, determine the relative number of photons required to elicit the spectral response of the cell at each wavelength by plugging in the measured spectral response at a given wavelength as (V) and solving for I.
4. Multiply the calculated stimulus intensity (I) from step 6.3 by the correction factor for each interference filter (from step 2.4.3) at each wavelength.
5. To get sensitivity, all intensities must be related to the V log-I curve so they can be compared. Do this by relating each wavelength intensity to ½ V_max or K, calculated in Step 6.2.3.
   1. Subtract each corrected wavelength intensity (Step 6.4) from K. 
   2. Then for each wavelength intensity, add this “distance from K” value to K, and multiply by (-1). 
   3. Next bring all data points positive by adding the absolute value of the lowest data point in the series to each wavelength.
6. Find sensitivity at each wavelength by taking the reciprocal of all newly calculated intensities from Step 6.5.1. Transform the data so that the sensitivity spectrum falls between 0 and 1.
7. After recording from more than one cell of the same type average the final responses and plot with standard error bars or 95% confidence intervals (Figure 8). 

Wyniki

For many elements of the recording setup, a written description does not provide enough detail. Figure 1 is a schematic of the components involved in the complete recording setup. In Figure 2, spectra are plotted for white light and each interference filter to give a sense of why a correction factor is needed and what is needed to calculate this correction. Figure 3 shows photos and a diagram of the Cardan arm that is used for these experiments. Figure 4...

Dyskusje

Intracellular recording can be a difficult technique to master due to the many technical steps involved. For successful experiments several important points must be considered. First, it is important to have a properly vibrationally-isolated table on which the experiment is performed. Many researchers use air tables, which completely separate the tabletop from the base, giving superior vibration isolation. Our setup involves a thick marble table with a sandbox on top, into which is placed the micromanipulator/electrode h...

Materiały

Name	Company	Catalog Number	Comments
Butterfly pupae			Several local species available, need USDA permits for shipping. Carolina Bio Supply has several insect species that may be ordered within the U.S. without the need for additional permits
Large plastic cylinder			Any chamber that remains humidified will work
Insect pins, size 2	BioQuip	1208B2
100% Desert Mesquite Honey	Trader Joe's		Any honey or sucrose solution will work
Xenon Arc Lamp	Oriel Instruments	66003	Oriel is now a part of Newport Corporation
Universal Power Supply	Oriel Instruments	68805	Oriel is now a part of Newport Corporation
Optical Track	Oriel Instruments	11190	Oriel is now a part of Newport Corporation
Rail Carrier, Large (2x)	Oriel Instruments	11641	Oriel is now a part of Newport Corporation
Rail Carrier, Small (4x)	Oriel Instruments	11647	Oriel is now a part of Newport Corporation
Thread Adaptor, 8-32 Male to 1/4-20 Male, pack of 10	Newport Corporation	TA-8Q20-10
Optical Mounting Post, 1.0 in., 0.5 in. Dia. Stainless, 8-32 & 1/4-20 (5x)	Newport Corporation	SP-1
No Slip Optical Post Holder, 2 in., 0.5 in. Diameter Posts, 1/4-20 (5x)	Newport Corporation	VPH-2
Fixed lens mount, 50.8 mm	Newport Corporation	LH-2
Fixed lens mount, 25.4 mm	Newport Corporation	LH-1
Condenser lens assembly	Newport Corporation	60006
Convex silica lens, 50.8 mm	Newport Corporation	SPX055
Six Position Filter Wheel, x2	Newport Corporation	FW1X6
Filter Wheel Mount Hub	Newport Corporation	FWM
Concave silica lens, 25.4 mm	Newport Corporation	SPC034
Collimator holder	Newport Corporation	77612
Collimating beam probe	Newport Corporation	77644
Ferrule Converter, SMA Termination to 11 mm Standard Ferrule	Newport Corporation	77670	This adapter allows the fiber optic to fit into the collimator holder
600 μm diameter UV-vis fiber obtic cable	Oriel Instruments	78367	Oriel is now a part of Newport Corporation
Shutter with drive unit	Uniblitz	100-2B
UV Fused Silica Metallic ND Filter, 0.1 OD	Newport	FRQ-ND01
UV Fused Silica Metallic ND Filter, 0.3 OD	Newport	FRQ-ND03
UV Fused Silica Metallic ND Filter, 0.5 OD	Newport	FRQ-ND05
UV Fused Silica Metallic ND Filter, 1.0 OD	Newport	FRQ-ND10
UV Fused Silica Metallic ND Filter, 2.0 OD	Newport	FRQ-ND30
UV Fused Silica Metallic ND Filter, 3.0 OD	Newport	FRQ-ND50
LS-1-Cal lamp	Ocean Optics	LS-1-Cal
Spectrometer	Ocean Optics	USB-2000
SpectraSuite Software	Ocean Optics
Interference bandpass filter, 300 nm	Edmund Optics	67749
Interference bandpass filter, 310 nm	Edmund Optics	67752
Interference bandpass filter, 320 nm	Edmund Optics	67754
Interference bandpass filter, 330 nm	Edmund Optics	67756
Interference bandpass filter, 340 nm	Edmund Optics	65614
Interference bandpass filter, 350 nm	Edmund Optics	67757
Interference bandpass filter, 360 nm	Edmund Optics	67760
Interference bandpass filter, 370 nm	Edmund Optics	67761
Interference bandpass filter, 380 nm	Edmund Optics	67762
Interference bandpass filter, 390 nm	Edmund Optics	67763
Interference bandpass filter, 400 nm	Edmund Optics	65732
Interference bandpass filter, 410 nm	Edmund Optics	65619
Interference bandpass filter, 420 nm	Edmund Optics	65621
Interference bandpass filter, 430 nm	Edmund Optics	65622
Interference bandpass filter, 440 nm	Edmund Optics	67764
Interference bandpass filter, 450 nm	Edmund Optics	65625
Interference bandpass filter, 460 nm	Edmund Optics	67765
Interference bandpass filter, 470 nm	Edmund Optics	65629
Interference bandpass filter, 480 nm	Edmund Optics	65630
Interference bandpass filter, 492 nm	Edmund Optics	65633
Interference bandpass filter, 500 nm	Edmund Optics	65634
Interference bandpass filter, 510 nm	Edmund Optics	65637
Interference bandpass filter, 520 nm	Edmund Optics	65639
Interference bandpass filter, 532 nm	Edmund Optics	65640
Interference bandpass filter, 540 nm	Edmund Optics	65642
Interference bandpass filter, 550 nm	Edmund Optics	65644
Interference bandpass filter, 560 nm	Edmund Optics	67766
Interference bandpass filter, 570 nm	Edmund Optics	67767
Interference bandpass filter, 580 nm	Edmund Optics	65646
Interference bandpass filter, 589 nm	Edmund Optics	65647
Interference bandpass filter, 600 nm	Edmund Optics	65648
Interference bandpass filter, 610 nm	Edmund Optics	65649
Interference bandpass filter, 620 nm	Edmund Optics	65650
Interference bandpass filter, 632 nm	Edmund Optics	65651
Interference bandpass filter, 640 nm	Edmund Optics	65653
Interference bandpass filter, 650 nm	Edmund Optics	65655
Interference bandpass filter, 660 nm	Edmund Optics	67769
Interference bandpass filter, 671 nm	Edmund Optics	65657
Interference bandpass filter, 680 nm	Edmund Optics	67770
Interference bandpass filter, 690 nm	Edmund Optics	65659
Interference bandpass filter, 700 nm	Edmund Optics	67771
Faraday cage			Any metal structure will work that can be grounded and that fits the experimental setup.
Stereomicroscope, 6X, 12X, 25X, 50X magnification	Wild Heerbrugg	Wild M5	Any Stereomicroscope will do
Microscope stand with swinging arm and heavy base	McBain Instruments		Any heavy base with arm will do
Cardan arm			Custom built, See Figure 4
Fiber-lite high intensity illuminator	Dolan-Jenner	MI-150	For lighting specimen
Fiber-lite goose-neck light guide	Dolan-Jenner	EEG 2823	Any goose-neck light guide will do
Marble table
Raised wooden table			Hole should be cut through this table so that the sandbox can rest on the marble table underneath
Wooden box filled with sand			custom built, any box with sand
Manipulator	Carl Zeiss - Jena
Electrode holder
Specimen stage
Alligator clip wires for grounding
Insulated copper wire
Silver wire, 0.125 mm diameter	World Precision Instruments	AGW0510
BNC cables
Preamplifier with headstage	Dagan Corporation	IX2-700
Humbug Noise reducer	Quest Scientific	Humbug
Oscilloscope, 30 MHz, 2 CH, Dual Trace, Alt-triggering, without probe	EZ Digital	os-5030
BNC T-adapter
Powerlab hardware 2/20	ADI instruments	ML820
Labchart software	ADI instruments	Chart 5
10 MHz Pulse Generator	BK Precision	4030
Glass pipette puller	Sutter Instruments	P-87
Borosillicate glass capillaries with filament	World Precision Instruments	1B120F-4
Potassium chloride, 3 M
Slotted plastic tube
Low melting temperature wax
Soldering Iron	Weller
Platform with ball-and-socket magnetic base	Hama photo and video
Double edge carbon steel, breakable razor blade	Electron Microscopy Sciences	72004
Vaseline
Microsoft Excel	Microsoft