The authors would like to acknowledge Dr. Birgit Werner for her help with producing this video article. This research was funded by an NSF CAREER Award.

Part 1: Experimental Preparation
Experimental procedures performed on horseshoe crabs have been approved by the Institutional Animal Care and Use Committee at Boston University. Animals are purchased from the Marine Biological Labs (Woods Hole, MA) or other vendor and housed in an aerated saltwater tank in a room exposed to a regulated light-dark cycle. The lighting regimen is important for entraining the crab's circadian clock and cycling the eye daily between its daytime and nighttime states. Immediately before starting any invasive procedures, the animal is chilled in an ice bucket for 10-15 minutes until its motion is slowed and then secured to a wooden platform with two stainless steel screws inserted in the prosoma and two in the opisthosoma. The platform is weighted underneath with granite so that it sinks in water. After terminal experiments the animal is euthanized by immersing into ice slurry again and pithing the brain, which lies above the mouth, with a scalpel.
Part 2: Solutions
Limulus Ringer's solution is composed of 430 mM NaCl, 9.56 mM KCl, 9.52 mM CaCl2, 9.97 mM MgCl2, 21.05 mM MgSO4, 50&mu;M TES, 50&mu;M HEPES, and 10ml/L Pen-Strep mix (Penicillin-Streptomycin, 10000 units/ml).
Part 3: Electroretinogram Recording
Tools needed for this procedure include a screwdriver, petroleum jelly, Ringer's solution, a transfer pipette, a green LED, a recording chamber, stainless steel screws, and a cotton swab.
<ol><li>A handmade chamber is used to record the ERG (Figure 1A). The chamber body is designed to hold a saline reservoir in contact with the eye. The lid contains a silver chloride wire for coupling the conductive solution to an amplifier and a small hole sized to accommodate an LED. An ultrabright LED with peak emission around 520nm works best.</li>
<li>To start, the underside of the chamber is coated with petroleum jelly and secured over the eye with two screws.</li>
<li>The chamber is then filled with saline and capped with the lid.</li>
<li>After chamber attachment the crab is placed in a light-tight cage in a tank filled with seawater over the gills.</li>
<li>The LED cable is inserted into the chamber lid.</li>
<li>The signal lead is clipped to the chloride wire, and the reference lead is clipped to one of the implanted screws.</li>
<li>Both leads are connected to the head stage of a high-impedance differential amplifier (X-cell 3x4, FHC Inc) for signal amplification and the amplifier filter is set to pass frequencies below 10Hz.</li>
<li>The amplifier output is fed to an oscilloscope for viewing and to a data acquisition board for computer analysis and storage.</li>
<li>After setup the cage door is closed to block room light from reaching the animal.</li>
<li>Data are collected with a custom program written in LabView. The program flashes the LED, records the evoked ERG signal, and measures the peak-to-peak response amplitude. The choice of flash paradigm depends on the experimental objective. For example, a brief (100ms) pulse of 5V applied every 10 minutes to the LED avoids effects of light adaptation when monitoring circadian changes in eye sensitivity.</li></ol>
Part 4: Optic nerve recording
Tools needed for this procedure include a screwdriver, thread, a trephine, Ringer's solution, rongeurs, vannas scissors, a recording chamber, curved forceps, fine needle probes, a dull scalpel, surgical scissors, and a suction electrode.
<ol>
<li>A handmade chamber is used to record optic nerve responses (Figure 1B). The interior of the chamber is 2cm-diameter open well that has a thin semicircular slotted opening in the bottom wall to accommodate the nerve and isolate it from surrounding tissue.</li>
<li>Before chamber attachment a 16-gauge needle is inserted between the hinge muscles into the heart and ~20cc of blood is drained from the animal. Exsanguination is not necessary but makes optic nerve dissection easier.</li>
<li>The location of the optic nerve is estimated by drawing on the carapace a slightly curved line between the lateral and median eyes.</li>
<li>A circular hole is then cut in the carapace with a trephine. The hole is the same diameter as the chamber well. The center of the hole is located 2-3 cm anterior to the lateral eye and slightly dorsal to the line so that the nerve runs along the ventral portion of the well.</li>
<li>Connective tissue is cleared until the nerve is fully visible and free from surrounding and underlying tissue.</li>
<li>A strand of thread is looped around the nerve and pulled into the chamber through the slot at the bottom. Through this same opening the nerve is gently guided into the chamber by pulling on the string. At the same time the chamber well is inserted into the hole. The chamber is then affixed to the carapace with screws and the well is filled with Ringer's solution.</li>
<li>After chamber attachment the animal is placed in a light-tight cage in a tank filled with seawater over the gills.</li>
<li>The well is visualized under a stereoscope (SMZ-168, Jed Pella Inc) and cotton is padded around the opening in the bottom to prevent leakage of blood into and saline out of the chamber.</li>
<li>Residual connective tissue around the nerve is removed with fine scissors and tweezers, and the chamber is refilled with fresh Ringer's solution.</li>
<li>A nick is made in the blood vessel that encapsulates the nerve and through this opening the nerve is carefully desheathed along its length using fine scissors, tweezers, and needle probes.</li>
<li>A tiny fiber bundle is separated from the nerve using the probe and cut at the end furthest from the eye for afferent fiber recording and at the end closest to the eye for efferent fiber recording. In the video the end for afferent fiber recordings is cut.</li>
<li>A microsuction electrode (A-M Systems Inc) filled with Ringers solution is used for optic nerve recording. The electrode tip is fitted to the nerve bundle by fire polishing the end of a 1mm diameter borosilicate glass capillary.</li>
<li>The electrode tip is lowered into the chamber well with a manual manipulator (WPI Inc), and the cut fiber bundle is sucked into the tip. Suction is provided by a Gilmont syringe connected to the electrode via tubing.</li>
<li>A BNC connection provides the signal lead and a silver chloride wire wrapped around the electrode to reduce noise provides the reference lead. The two leads are connected to the head stage of a differential amplifier for signal amplification and noise filtering (X-cell 3x4, FHC Inc). The amplifier output is sent to an oscilloscope for viewing and a data acquisition card for computer analysis and storage.</li>
<li>Data are collected with a custom program written in LabView. The program controls light stimuli via a digital video processor (Bits++, Cambridge Research Systems Inc) and interfaces with a digital spike discriminator (APM, FHC Inc) that records spike trains. Light can be delivered to single cells in the eye via a fiber optic light pipe or to the entire eye via a computer-controlled video display.</li>
</ol>
Part 5: Intraretinal recording
Tools needed for this procedure include a screwdriver, a L-shaped lucite platform with threaded screw holes, a microelectrode holder with a glass pipette, stainless steel screws, tweezers, and a fine scalpel.
<ol>
<li>A handmade lucite plate with preset screw holes is used to record intracellular responses. The holes are spaced for mounting a motorized micromanipulator (PPM5000, WPI Inc). The plate is attached to the animal with two screws on the side and one on top.</li>
<li>After plate attachment the animal is placed in a light-tight cage in a tank filled with seawater over the gills.</li>
<li>The micropositioner is screwed into the plate, with its movable arm aligned over the eye.</li>
<li>A batch of glass micropipettes are pulled from 1-mm OD borosilicate glass and the tips are backfilled via capillary action by placing the micropipettes in a small vial of 3M KCl solution.</li>
<li>The rest of the pipettes are then filled manually with the salt solution and inserted into an electrode holder.</li>
<li>The electrode holder is connected to the head stage of an intracellular amplifier (IR-283, Cygnus Technology Inc) affixed to the micropositioner arm.</li>
<li>A tiny section of retina (~1mm2) is exposed by cutting away the corneal interface with a razor blade.</li>
<li>A drop of Ringer's solution is placed on the exposed retina to prevent drying and the micropipette tip is advanced through the opening into retinal tissue.</li>
<li>When the tip enters the solution, the current injection mode of the intracellular amplifier is engaged and electrode impedance is measured. Micropipettes with impedance outside the range of 20-70 MW are discarded. Those in this range are advanced in micron steps and impaled into cells by vibrating the pipette tip electronically or mechanically.</li>
<li>Three types of cells may be encountered in the eye. Retinular cells show only a depolarizing response to light, eccentric cells show a train of action potentials riding on a depolarizing response, and pigment cells show no light response at all.</li>
<li>Data are collected with a custom program written in Labview. The program controls light stimuli via a digital video processor (Bits++, Cambridge Research Systems). The stimuli are delivered to impaled cells with a fiber optic pipe or video display. Voltage responses are observed on the oscilloscope and digitized by the program to computer.</li>
</ol>
Part 6: Representative Results
A representative ERG is shown in Figure 2A. The waveform represents the summed electrical response to light of primarily the photoreceptive retinular cells, as they greatly outnumber the eccentric cells and are electrically coupled to them. There are not multiple waveform components from different retinal cell types as with the mammalian ERG. Circadian feedback from a clock in the Limulus brain modulates the physiological properties of retinal cells on a daily basis, causing the amplitude and time course of the ERG to vary over time (1). As shown in Figure 2B, ERG amplitude is highest during the animal's subjective night and lowest during the subjective day.
A representative trace of a single optic nerve fiber response to light is shown in Figure 3. Eccentric cells all behave roughly the same, showing a transient increase in spike discharge rate followed by a decay to a sustained level. The rate decay reflects the combined action of light adaptation and spike-dependent self-inhibition on eccentric cells (2). Other spike patterns, such as light-dependent decreases in rate, are seen in the Limulus brain but not the eye (3).
Representative traces of the voltage response of a pigment cell, retinular cell, and eccentric cell to light are shown in Figure 4. Only the latter two retinal cell types are visual. The amplitude and time course of their response depends on the quality of the recording and the location of the electrode within the cell. Usually, the electrode penetrates pigment cells or retinular cells because of their large size and number. If the latter, a transient depolarization that decays to a sustained level is recorded. The decay is due to light adaptation by retinular cells (2). If the electrode enters an eccentric cell, large action potentials are recorded in the axon (40-70mV) and small action potentials riding on a depolarizing waveform (&lt;25mV) near the soma.
<img src="/files/ftp_upload/1384/1384fig1.jpg" alt="figure 1" /> Figure 1. Schematic diagrams of chambers used for electroretinogram recording (A) and optic nerve recording (B). Bar equals 7mm in A and 5mm in B.
<img src="/files/ftp_upload/1384/1384fig2.jpg" alt="figure 2" /> Figure 2. Example trace of a Limulus ERG evoked by a 100ms LED pulse of 5V in the dark (A) and the peak-to-peak fluctuation in ERG amplitude over time in constant darkness (B). Darkness was initiated at the time indicated by the white triangle. The growth in ERG amplitude through the time indicated by the black triangle is due to light adaptation, which increases eye sensitivity in the dark. The cyclic variation in ERG amplitude thereafter is due to the animal's internal circadian clock. Time points in B are every 5min.
<img src="/files/ftp_upload/1384/1384fig3.jpg" alt="figure 3" /> Figure 3. Example trace of an optic nerve fiber response to light flashed on a single ommatidial receptor. Waveform above the trace shows stimulus timing. Eccentric cells, whose axons give rise to nerve fibers, all show a similar pattern of firing as this under the illumination conditions of the experiment (5sec flash in total darkness). Retinal coding with spikes is a property Limulus has in common with mammals, unlike other invertebrates.
<img src="/files/ftp_upload/1384/1384fig4.jpg" alt="figure 4" /> Figure 4. Example traces of the voltage response to light of the three cell types present in the Limulus lateral eye: pigment cell (A), retinular cell (B), and eccentric cell (C). The first cell is non-visual. The latter two depolarize upon a light flash. The depolarization is largest for retinular cells because they transduce the light and send the signal through gap junctions to the eccentric cell, with some loss along the way. In the eccentric cell, action potentials fired by the axon are seen riding on the depolarization due to spike backpropagation into the soma. The amplitude of action potentials has been attenuated in the figure for better viewing of the depolarizing potential. The resting potential of the cells is -50mV.

<ol>
	<li>Barlow, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+rhythms+in+the+Limulus+visual+system.">Circadian rhythms in the Limulus visual system.</a> J. Neurosci. 3, 856-870 (1983).</li><li>Passaglia, C. L., Dodge, F. A., Barlow, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+based+model+of+the+Limulus+lateral+eye.">Cell based model of the Limulus lateral eye.</a> J. Neurophysiol. 80, 1800-1815 (1998).</li><li>Snodderly, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+of+visual+inputs+by+the+brain+of+Limulus.">Processing of visual inputs by the brain of Limulus.</a> J. Neurophysiol. 34, 588-611 (1971).</li><li>Passaglia, C., Dodge, F., Herzog, E., Jackson, S., Barlow, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deciphering+a+neural+code+for+vision.">Deciphering a neural code for vision.</a> Proc. Natl. Acad. Sci. 94, 12649-12654 (1997).</li><li>Barlow, R. B., Kaplan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limulus+lateral+eye:+properties+of+receptor+units+in+the+unexcised+eye.">Limulus lateral eye: properties of receptor units in the unexcised eye.</a> Science. 174, 1027-1029 (1971).</li><li>Bolbecker, A. R., Lewis, A. R., Swan, A. A., Carlson, K., Fleet, J. R., Beck, K. E., Wasserman, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+Bellows+Cup+Electrode+Demonstrates+Low-frequency+Properties+of+Long-term+Electroretinographic+Recordings+in+the+Limulus+Lateral+Eye.">Stable Bellows Cup Electrode Demonstrates Low-frequency Properties of Long-term Electroretinographic Recordings in the Limulus Lateral Eye.</a> J. Neurosci. Meth. 159, 252-260 (2007).</li></ol>

We have illustrated how to perform ERG recordings, optic nerve recordings, and intraretinal recordings on horseshoe crabs in vivo. The recording techniques each provide different insights into the neural basis of vision, and they can all be used to study retinal function in live animals thanks to the crab's large eyes and hard carapace. Optic nerve activity can even be recorded from freely behaving animals in the ocean with proper construction of electrodes (4). These techniques can also be performed on excised eyes with...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>LED</td>
<td>Light source</td>
<td>Newark Inc</td>
<td>33C1292</td>
<td>&nbsp;</td>
<tr>
<td>Suction electrode</td>
<td>Electrode</td>
<td>A-M Systems, Inc</td>
<td>573000</td>
<td>&nbsp;</td>
<tr>
<td>XCell 3*4-Channel Extracellular Amplifier</td>
<td>Amplifier</td>
<td>FHC Inc.</td>
<td>40-40-8B</td>
<td>&nbsp;</td>
<tr>
<td>Intracellular Recording</td>
<td>Amplifier</td>
<td>Cygnus</td>
<td>IR-283A</td>
<td>&nbsp;</td>
<tr>
<td>APM</td>
<td>Neural Spike Discriminator</td>
<td>FHC Inc.</td>
<td>APM</td>
<td>&nbsp;</td>
<tr>
<td>Bits++</td>
<td>Video Board</td>
<td>Cambridge Research Systems Inc</td>
<td>Bits++</td>
<td>&nbsp;</td>
<tr>
<td>Piezopatch Manipulator</td>
<td>Micropositioner</td>
<td>WPI Inc</td>
<td>PPM5000</td>
<td>&nbsp;</td>
<tr>
<td>Square Pulse Stimulator</td>
<td>Nerve Stimulator</td>
<td>Grass Instrument Division, Astro-Med, Inc.</td>
<td>Model S48</td>
<td>&nbsp;</td>
<tr>
<td>P-97</td>
<td>Micropipette Puller</td>
<td>Sutter Instruments</td>
<td>Model P-97</td>
<td>&nbsp;</td>
<tr>
<td>Borosilicate Glass Capillary</td>
<td>Electrode glass</td>
<td>WPI Inc</td>
<td>1B150-4</td>
<td>&nbsp;</td>
<tr>
<td>Horseshoe crab (Limulus polyphemus)</td>
<td>Animal</td>
<td>Marine Biological Laboratories</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Micropipette Puller</td>
<td>Glass Puller</td>
<td>Sutter Instruments</td>
<td>P-97</td>
<td>&nbsp;</td>
<tr>
<td>Zoom Stereoscope</td>
<td>Microscope</td>
<td>Jed Pella Inc.</td>
<td>SMZ-168</td>
<td>&nbsp;</td>		</tbody>
		</table>
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using the horseshoe crab, limulus polyphemus, in vision research

The American horseshoe crab, Limulus Polyphemus is one of the oldest creatures on earth, and the animal continues to play an indispensable role in biomedical research. Not only does their blood contain special cells that scientists use to detect bacteriotoxins in our medicines, but their eyes also contain a neural network that has provided much insight about physiological processes operating in our visual system, such as light adaptation and lateral inhibition. The horseshoe crab remains an attractive model for vision research because the animal is large and hardy for an invertebrate, its retinal neurons are big and easily accessible, its visual system is compact and extensively studied, and its visual behavior is well defined. Moreover, the structure and function of the eyes are modulated on a daily basis by a circadian clock in the animal s brain. In short, the visual system of horseshoe crabs is simple enough to be understood yet complex enough to be interesting.
In this video we present three electrophysiological paradigms for investigating the neural basis of vision that can be performed in vivo with Limulus. They are electroretinogram recording, optic nerve recording, and intraretinal recording. Electroretinogram (ERG) recordings measure with a surface electrode the summed electrical response of all cells in the eye to a flash of light. They can be used to monitor the overall sensitivity of the eye for prolong periods of time. Optic nerve recordings measure the spiking activity of single nerve fibers with an extracellular microsuction electrode. They can be used to study visual messages conveyed from the eye to the brain as well as circadian-clock messages fed back from the brain to the eye. Intraretinal recordings measure with an intracellular microelectrode the voltage fluctuations induced by light in individual cells of the eye. They can be used to elucidate cellular mechanisms of retinal processing.

Watch this Scientific Journal Video about Using the Horseshoe Crab, Limulus Polyphemus, in Vision Research at JoVE.com

Using the Horseshoe Crab, Limulus Polyphemus, in Vision Research

In this video we perform electroretinogram recording, optic nerve recording, and intraretinal recording with the American horseshoe crab, Limulus Polyphemus. These electrophysiological paradigms can be used for investigating the neural basis of vision in a research or teaching lab.

Using the Horseshoe Crab, Limulus Polyphemus, in Vision Research

The American horseshoe crab, Limulus Polyphemus is one of the oldest creatures on earth, and the animal continues to play an indispensable role in ...

using-the-horseshoe-crab-limulus-polyphemus-in-vision-research

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JoVE Journal

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14.2K Views.  Boston University. In this video we perform electroretinogram recording, optic nerve recording, and intraretinal recording with the American horseshoe crab, Limulus Polyphemus. These electrophysiological paradigms can be used for investigating the neural basis of vision in a research or teaching lab.

Video: Using the Horseshoe Crab, Limulus Polyphemus, in Vision Research

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate. The study of immunity in horseshoe crabs has been facilitated by the ease in collecting large volumes of blood and from the simplicity of the blood. Horseshoe crabs show only a single cell type in the general circulation, the granular amebocyte. The plasma has the salt content of sea water and only three abundant proteins, hemocyanin, the respiratory protein, the C-reactive proteins, which function in the cytolytic destruction of foreign cells, including bacterial cells, and &alpha;2-macroglobulin, which inhibits the proteases of invading pathogens. Blood is collected by direct cardiac puncture under conditions that minimize contamination by lipopolysaccharide (a.k.a., endotoxin, LPS), a product of the Gram-negative bacteria. A large animal can yield 200 - 400 mL of blood. For the study of the plasma, blood cells are immediately removed from the plasma by centrifugation and the plasma can then be fractionated into its constituent proteins. The blood cells are conveniently studied microscopically by collecting small volumes of blood into LPS-free isotonic saline (0.5 M NaCl) under conditions that permit direct microscopic examination by placing one of more LPS-free coverglasses on the culture dish surface, then mounting those coverglasses in simple observation chambers following cell attachment. A second preparation for direct observation is to collect 3 - 5 mL of blood in a LPS-free embryo dish and then explanting fragments of aggregated amebocytes to a chamber that sandwiches the tissue between a slide and a coverglass. In this preparation, the motile amebocytes migrate onto the coverglass surface, where they can readily be observed. The blood clotting system involves aggregation of amebocytes and the formation of an extracellular clot of a protein, coagulin, which is released from the secretory granules of the blood cells. Biochemical analysis of washed blood cells requires that aggregation and degranulation does not occur, which can be accomplished by collecting blood into 0.1 volumes of 2% Tween-20, 0.5 M LPS-free NaCl, followed by centrifugation of the cells and washing with 0.5 M NaCl.

The American horseshoe crab, Limulus polyphemus, is arguably the most convenient source for large quantities of blood of any invertebrate. The blood is simple in composition, with only one cell-type in the general circulation, the granular amebocyte, and only three abundant proteins in the plasma, hemocyanin, the C-reactive proteins, and &alpha;2-macroglobulin. Blood is collected from the heart and the blood cells and plasma are separated by centrifugation.

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate.  The study of immunity in horseshoe crabs has been facilitated ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Sample Preparation for Metabolic Profiling using MALDI Mass Spectrometry Imaging

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to investigate the biological activities during development and diseases. Metabolomics approaches&#160;are widely employed in the study of cancer, nutrition/diet, diabetes, and other physiological and pathological conditions involving metabolic processes. An advantageous tool that aids in metabolomic profiling advocated in this paper is matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI). Its ability to detect metabolites in situ without labeling, structural modifications, or other specialized reagents, such as those used in immunostaining, makes MALDI MSI a unique tool in advancing methodologies relevant in the field of metabolomics. An appropriate sample preparation process is critical to yield optimal results and will be the focus of this paper.

The goal of this protocol is to provide detailed guidance on the sample preparation when planning for experiments using MALDI MSI to maximize metabolic and molecular detection in biological samples.

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to ...

Determining the Toxicity of UV Radiation and Chemicals on Primary and Immortalized Human Corneal Epithelial Cells

This article describes the methods of measuring the toxicity of ultraviolet (UV) radiation and ocular toxins on primary (pHCEC) and immortalized (iHCEC) human corneal epithelial cell cultures. Cells were exposed to UV radiation and toxic doses of benzalkonium chloride (BAK), hydrogen peroxide (H2O2), and sodium dodecyl sulfate (SDS). Metabolic activity was measured using a metabolic assay. The release of inflammatory cytokines was measured using a multi-plex interleukin (IL)-1&#946;, IL-6, IL-8, and tumor necrosis factor-alpha (TNF-&#945;) assay, and cells were evaluated for viability using fluorescent dyes. 
The damaging effects of UV on cell metabolic activity and cytokine release occurred at 5 min of UV exposure for iHCEC and 20 min for pHCEC. Similar percent drops in metabolic activity of the iHCEC and pHCEC occurred after exposure to BAK, H2O2, or SDS, and the most significant changes in cytokine release occurred for IL-6 and IL-8. Microscopy of fluorescently stained iHCEC and pHCEC BAK-exposed cells showed cell death at 0.005% BAK exposure, although the degree of ethidium staining was greater in the iHCECs than pHCECs. Utilizing multiple methods of assessing toxic effects using microscopy, assessments of metabolic activity, and cytokine production, the toxicity of UV radiation and chemical toxins could be determined for both primary and immortalized cell lines.

This article describes the procedures used to evaluate the toxicity of UV radiation and chemical toxins on a primary and immortalized cell line.

This article describes the methods of measuring the toxicity of ultraviolet (UV) radiation and ocular toxins on primary (pHCEC) and immortalized (iHCEC) ...

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform (RpEGEN) for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent retinal and vascular tissues. At present, the limited reparative capacity of mammalian RPE, which is restricted to small injuries, has hindered progress to understanding in vivo RPE regenerative processes. Here, a detailed methodology is provided to facilitate the study of in vivo RPE repair utilizing the zebrafish, a&#160;vertebrate model capable of robust tissue regeneration. This protocol describes a transgenic nitroreductase/metronidazole (NTR/MTZ)-mediated injury paradigm (rpe65a:nfsB-eGFP), which results in ablation of the central two-thirds of the RPE after 24 h treatment with MTZ, with subsequent tissue recovery. Focus is placed on RPE ablations in larval zebrafish and methods for testing the effects of pharmacological compounds on RPE regeneration are also outlined. Generation and validation of RpEGEN, a MATLAB script created to automate quantification of RPE regeneration based on pigmentation, is also discussed. Beyond active RPE repair mechanisms, this protocol can be expanded to studies of RPE degeneration and injury responses as well as the effects of RPE damage on adjacent retinal and vascular tissues, among other cellular and molecular processes. This zebrafish system holds significant promise in identifying genes, networks, and processes that drive RPE regeneration and RPE disease-related mechanisms, with the long-term goal of applying this knowledge to mammalian systems and, ultimately, toward therapeutic development.

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform RpEGEN for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

This protocol describes the methodology to genetically ablate the retinal pigment epithelium (RPE) using a transgenic zebrafish model. Adapting the protocol to incorporate signaling pathway modulation using pharmacological compounds is extensively detailed. A MATLAB platform for quantifying RPE regeneration based on pigmentation was developed and is presented and discussed.

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent ...

Investigating Zinc Transport Mechanism in Mammalian Cells with FluoZin-3 Assay

Characterizing Mammalian Zinc Transporters Using an In Vitro Zinc Transport Assay

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured indirectly by determining the expression level of the transporter under different concentrations of Zn2+. This was done by utilizing immunohistochemistry, measuring mRNA in the tissue, or determining the cellular Zn2+ levels. With the development of intracellular Zn2+ sensors, the activities of zinc transporters are currently primarily determined by correlating changes in intracellular Zn2+, detected using fluorescent probes, with the expression of the Zn2+ transporters. However, even today, only a few labs monitor dynamic changes in intracellular Zn2+ and use it to measure the activity of zinc transporters directly. Part of the problem is that out of the 10 zinc transporters of the ZnT family, except for ZnT10 (transports manganese), only zinc transporter 1 (ZnT1) is localized at the plasma membrane. Therefore, linking the transport activity to changes in the intracellular Zn2+ concentration is hard. This article describes a direct way to determine the zinc transport kinetics using an assay based on a zinc-specific fluorescent dye, FluoZin-3. This dye is loaded into mammalian cells in its ester form and then trapped in the cytosol due to cellular di-esterase activity. The cells are loaded with Zn2+ by utilizing the Zn2+ ionophore pyrithione. The ZnT1 activity is assessed from the linear part of the reduction in fluorescence following the cell washout. The fluorescence measured at an excitation of 470 nm and emission of 520 nm is proportional to the free intracellular Zn2+. Selecting the cells expressing ZnT1 tagged with the mCherry fluorophore allows for monitoring only the cells expressing the transporter. This assay is used to investigate the contribution of different domains of ZnT1 protein to the transport mechanism of human ZnT1, a eukaryotic transmembrane protein that extrudes excess zinc from the cell.

Author Spotlight: Exploring Cellular Zinc Regulation Through ZnT1 Functionality 

Zinc transport has proven challenging to measure due to the weak causal links to protein function and the low temporal resolution. This protocol describes a method for monitoring, with high temporal resolution, Zn2+ extrusion from living cells by utilizing a Zn2+ sensitive fluorescent dye, thus providing a direct measure of Zn2+ efflux.

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured ...

TEM Workflows, Results Analysis, and Data Management Using a Machine-Vision Approach

A Machine-Vision Approach to Transmission Electron Microscopy Workflows, Results Analysis and Data Management

Transmission electron microscopy (TEM) enables users to study materials at their fundamental, atomic scale. Complex experiments routinely generate thousands of images with numerous parameters that require time-consuming and complicated analysis. AXON synchronicity is a machine-vision synchronization (MVS) software solution designed to address the pain points inherent to TEM studies. Once installed on the microscope, it enables the continuous synchronization of images and metadata generated by the microscope, detector, and in situ systems during an experiment. This connectivity enables the application of machine-vision algorithms that apply a combination of spatial, beam, and digital corrections to center and track a region of interest within the field of view and provide immediate image stabilization. In addition to the substantial improvement in resolution afforded by such stabilization, metadata synchronization enables the application of computational and image analysis algorithms that calculate&#8239;variables between images. This calculated metadata can be used to analyze trends or identify key areas of interest within a dataset, leading to new insights and the development of more sophisticated machine-vision capabilities in the future. One such module that builds on this calculated metadata is dose calibration and management. The dose module provides state-of-the-art calibration, tracking, and management of both the electron fluence (e-/&#197;2&#183;s-1) and cumulative dose (e-/&#197;2) that is delivered to specific areas of the sample on a pixel-by-pixel basis. This enables a comprehensive overview of the interaction between the electron beam and the sample. Experiment analysis is streamlined through a dedicated analysis software in which datasets consisting of images and corresponding metadata are easily visualized, sorted, filtered, and exported. Combined, these tools facilitate efficient collaborations and experimental analysis, encourage data mining and enhance the microscopy experience.

Author Spotlight: A Machine-Vision Approach to Transmission Electron Microscopy Workflows, Results Analysis and Data Management  

Here, we present a protocol to utilize machine-vision software to stabilize dynamic processes during TEM imaging, while concurrently indexing multiple streams of metadata to each image into a navigable timeline. We demonstrate how this platform enables automated calibration and mapping of the electron dose over the course of an experiment.

Transmission electron microscopy (TEM) enables users to study materials at their fundamental, atomic scale. Complex experiments routinely generate ...

Dose Calibration of the Transmission Electron Microscope for TEM and Scanning TEM (STEM) Imaging Modes Using a Machine-Vision Approach

Begin the dose calibration by clicking the Connect icon in the MVS software installed on TAEM. Select the microscope and activate the connection between the TEM and the MVS software to visualize the images from the camera or detector in the image viewer. Navigate to the Dose tab and click on Start Dose Area Calibration.Click OK to confirm the calibration hardware and follow the software prompts to enter the requested user configurable values. Then click Start Dose Current Calibration. Follow the software prompts and enter the requested user configurable values to complete the calibration.

Determination of Dose Threshold Using the Machine-Vision Synchronization (MVS) and Dose Software

Begin by opening the MVS software application on the TEM instrument and selecting other. Connect the microscope to the MVS software. Navigate to the image metadata tab and select magnification, max dose and dose rate to overlay on the image stream seen in the live display.Other metadata may be included if the user desires. Open the dose tab. Select dose management and enable dose monitoring.Then select show dose layer and show position uncertainty to activate automated electron dose tracking and display the dose color overlay. Set the values for the low dose level and high dose level before pressing save. Then navigate to the settings tab.Select dose and set the dose navigation map opacity and dose image overlay opacity values. Activate drift correction in the live image viewer window by clicking on drift correct. Next, navigate to the data view tab and plot the metadata values defocus and focus quotient on the y axis.Activate focus assist then select calibrate focus to run the calibration of the automated focus assist. Once the calibrate focus routine is complete close the data view tab. In the MVS software, open the image analysis tab and activate the live FFT and quadrants one and two options.Using the microscope software controls adjust the beam conditions so that the electron flux is approximately 600 angstrom square per second. Move to a new region in the sample and center the sample ROI in the live view of the MVS software. Maintain a constant dose rate while continuously imaging the same ROI until the spots corresponding to the atomic structure and the FFT have disappeared.