The authors thank Sanaz Vahidinia for introducing us to Dr. Jorgensen&#39;s colleagues and his work. This research was supported by grants from the Army Research Office (no. W911NF-10-0139), the Office of Naval Research (through MURI award no. N00014-09-1-1053), and NSF-INSPIRE award NSF-1343158.

This study is compliant with all the relevant ethical regulations of Yale University regarding vertebrate and invertebrate animal research.
1. Building the optical micro-probe
<ol>
	<li>Building the glass sleeve, material: Pasteur pipette, 5.75 in(see Table of Materials)
	<ol>
		<li>Using a mountable alligator clip (Table of Materials), mount the glass pipette by the wide end such that the tapered end is facing down toward the floor and the orientation of the pipette is perpendicular to the floor.</li>
		<li>Hang a 50 g plastic vice grip (Table of Materials) from the tapered end of the pipette. Place a pad of electrical tape between the pipette and the vice grip to help prevent slippage.</li>
		<li>Heat the tapered end of the pipette using a small butane torch (Table of Materials). Apply the flame 1 cm above the vice grip. Hold the torch such that the brightest part of the flame is 3 cm from the glass and the glass is at the tip of the flame. When the pipette&#39;s length has increased by about 5 in, immediately remove the flame.</li>
		<li>Use small scissors or a glass cutter to trim the pulled end of the pipette at the point where the new pulled diameter is roughly twice that of the optical fiber used in the next section (see step 1.2). File down any sharp areas of the trimmed end using carborundum paper (Table of Materials). Rinse away any resulting small glass shards or dust using a squirt bottle of isopropyl alcohol followed by compressed air.</li>
	</ol>
	</li>
	<li>Pulling the optical fiber (Figure 1D)
	<ol>
		<li>Use a razor blade to sever the optical fiber, removing one SMA connector of a 200 &#181;m diameter SMA-terminated optical fiber (Table of Materials). Cut close to one of the SMA terminations. Then, use the razor blade to remove the next 5 cm of plastic and fiberglass jacketing so that the bare optical fiber is exposed and protrudes from the rest of the intact assembly.</li>
		<li>Use the butane torch to burn off the polyimide polymer coating from the glass fiber. Rinse with isopropanol. Wipe the bare glass fiber clean using a lint-free wipe.</li>
		<li>The next step is to mount the bare fiber so it can also be pulled with a flame. Mount the bare end of the optical fiber directly in a plier clamp (Table of Materials) on a table or shelf edge, letting the SMA-terminated end of the fiber hang toward the floor. Add the weights for pulling in the form of two small clamps (total weight: 10 g) on the jacketed end of the fiber about 12 in down from where the bare optical fiber is held in the plier clamp.</li>
		<li>To pull the fiber, start with the butane torch flame on. With the flame on, hold the butane torch 1 cm from the bare fiber so the flame is perpendicular to the optical fiber and 3 cm down vertically from where the plier clamp holds the bare fiber. Allow the fiber to stretch, pull, separate, and drop to the floor.</li>
		<li>Examine the resulting pulled optical fiber end. Gently sand away any irregularities with carborundum paper, clean with isopropyl alcohol and a lint-free wipe, and dry with compressed air. 
		NOTE:&#160;At this point, one may use a microscope to characterize and document the size and shape of the pulled fiber.</li>
		<li>Use a film-opaquing pen to darken the sides of the fiber and prevent the entry of stray light (Table of Materials). Gently pull the fiber across the tip of the pen, leaving only a small area at the tip uncovered.</li>
	</ol>
	</li>
	<li>Mounting the pulled fiber inside the glass pipette sleeve (Figure 1A)
	<ol>
		<li>Working under a stereomicroscope, carefully insert the tapered optical fiber from step 1.2 into the wide end of the altered glass pipette from step 1.1, and push the fiber through until ~1 mm of bare fiber is sticking out of the tapered end of the pipette.</li>
		<li>Using electrical tape, secure the jacketed end of the optical fiber to the wide end of the pipette.</li>
		<li>Put a drop of cyanoacrylate adhesive onto a small-gauge needle (Table of Materials). Carefully touch the drop of adhesive to the cut edge of the pulled pipette, avoiding the bare end of the pulled fiber.</li>
		<li>Allow capillary action to wick the adhesive into the pipette, wetting the area between the wall of the pipette and the optical fiber. Allow the adhesive to cure for at least 15 min before moving the probe assembly.</li>
	</ol>
	</li>
	<li>Modifying the fiber tip with a scattering sphere (Figure 1C)
	<ol>
		<li>Create the raw material for the scattering ball tip by mixing a drop of UV-curable adhesive with titanium dioxide powder (Table of Materials) at ~1:1 v/v.</li>
		<li>Attach the probe to a micromanipulator with a mounting rod holder (Table of Materials) such that the probe is in a horizontal orientation. Prepare a working reservoir of the scattering material by dipping the tip of a wire or needle in the adhesive/TiO2 mixture prepared above such that a droplet of adhesive forms. Mount the wire or needle with the droplet near the tip of the horizontal probe. It is convenient to do this using an alligator clip mounted to the bench.</li>
		<li>Deposit a scattering sphere on the tip of the probe. Use the micromanipulator to slowly and carefully push the tip of the probe&#39;s optical fiber into the droplet of adhesive/TiO2. Then, quickly withdraw the tip from the glue. Repeat two to three times until a spherical droplet of adhesive of the desired size is deposited on the tip of the optical fiber. 
		NOTE: The scattering sphere will be stable at diameters from two to eight times greater than the diameter of the tapered optical fiber. The final optimal size depends on the final desired application.</li>
		<li>Cure the sphere by connecting the SMA-terminated end of the optical fiber to a fiber-coupled UV light source (Table of Materials). 
		&#8203;NOTE: The light source used in this study had a power of 5.3 mW. At this power, the recommended cure time is at least 12 h or overnight.&#160;After curing is a good time to characterize and measure the size of the final optical probe tip.&#160;</li>
	</ol>
	</li>
</ol>
2. Preparing and mounting the tissue for optical measurements (Figure 2 and Figure 3)
<ol>
	<li>Prepare the dishes to mount the samples for an experiment. Heat the large end of a Pasteur pipette using a butane torch to make a punch to melt a 0.5 cm diameter hole in the bottom of a 35 mm x 10 mm plastic Petri dish. Seal the hole on the bottom side of the dish with electrical tape or lab tape. Make several at a time for efficiency.</li>
	<li>Prepare liquid gelatin (Table of Materials) according to the instructions on the package. 
	NOTE: Commercial food-grade flavorless gelatin from the grocery store is better for this purpose than chemical-grade gelatin from chemical suppliers because the chemical-grade gelatin is less optically clear and less mechanically reliable than the food product.</li>
	<li>Perform the dissection and preparation of the tissue that will be used. 
	NOTE: From experience, any flat tissue up to 1 cm thick can be successfully measured in this experiment using any dissection technique appropriate for the system of interest. For giant clam tissue, an 8 mm biopsy punch was used to make a flat, regular circle of clam tissue for use in the experiment12. For this system, only one sample could effectively be prepared at a time given the length of time required to complete a measurement to ensure that the sample was still in lifelike condition at the end of the experiment.</li>
	<li>Fill two Petri dishes from step 2.1 a quarter of the way with the liquid gelatin, and let cool to room temperature (RT) just short of gelation. In one dish, place the biopsy in the cushion of viscous gelatin that forms in the hole in the bottom of the dish. Use a Pasteur pipette to gently add RT gelatin around the biopsy until the Petri dish is full (Figure 3). Fill the second dish with gelatin to make a blank sample.</li>
	<li>Place the sample dishes into a refrigerator for about 10-30 min until the gelatin is fully cured and slightly elastic to the touch. 
	NOTE: In a cool room, this may not be necessary; when working in the tropics, this step is required to fully cure the gelatin.</li>
	<li>Making a mount for the Petri dish on the micromanipulator (Figure 2)
	<ol>
		<li>Cut a 6 cm x 6 cm square of 1/4 in thick plexiglass (Table of Materials).</li>
		<li>Drill or melt a hole in the plastic square the same diameter as the Petri dish (~35 mm). Ensure that the Petri dish fits snugly in this hole and is held in place with friction. Add tape to the edges of the dish to slightly increase the size and frictional contact.</li>
		<li>Drill or melt a 1/4 in diameter hole in the corner of the plastic square. Use this hole to attach the square to the micromanipulator using a 1/4 in screw and bolt.</li>
		<li>Cover the plastic square with black electrical tape to reduce the stray light reaching the probe. Similarly, use tape to create a skirt around the sides of the square extending below the bottom of the inserted dish (&#62;10 mm) to reduce stray light (Figure 2B).</li>
		<li>Use a bolt and appropriate hardware to attach the Petri dish holder to a micromanipulator that allows for three-dimensional adjustments and well-resolved vertical movement over an extent greater than the thickness of the samples (part 1 and part 2 of the manipulator mentioned in the Table of Materials are good examples). Affix this micromanipulator to an optical breadboard.</li>
	</ol>
	</li>
</ol>
3. Measurement setup for tissue biopsies
<ol>
	<li>Mount a light source above the area where the measurement takes place such that the light is collimated when it reaches the plane where the sample will be placed (Figure 2A). For example, attach a 5 cm collimating lens (Table of Materials) to a 5 mm liquid light guide (Table of Materials), and elevate above the sample by &#62;0.6 m using a vice and frame attached to an optical breadboard (Table of Materials). Then, connect the light guide to a broadband light source, such as the plasma light source listed in the Table of Materials.</li>
	<li>Attach the probe to a vertically oriented mount such that it points toward the light source. Secure the probe with clamps, hose clamps, or tape to an optical table post. 
	NOTE: In the giant clam experiment, a specifically designed rod holder, such as the micromanipulator listed in the Table of Materials, is used. In this work, it was secured by magnets to an optical breadboard (Table of Materials). The additional degrees of freedom for alignment obtained by having both the probe and the sample on manipulators are convenient but not required. Be careful not to over-clamp to the post, as this can break the pipette housing.</li>
	<li>Position the sample manipulator near the vertically mounted probe. Use the sample manipulator to adjust the position of the sample stage such that the probe is centered in the 0.5 cm opening at the bottom of the Petri dish. Use extreme caution to avoid touching or bumping the tip of the mounted probe. 
	NOTE: A boom-mounted stereomicroscope positioned over the sample area can be useful. This way, the top-down alignment of the probe tip, stage, and sample can be viewed before starting an experiment (Figure 2A).</li>
	<li>Connect the SMA-terminated end of the probe&#39;s optical fiber to a USB fiber-optic spectrometer (Table of Materials), and connect the spectrometer to the computer using a USB cable.</li>
</ol>
4. Data collection
<ol>
	<li>Experimental setup
	<ol>
		<li>Have the probe and manipulators in the exact aligned position in which the data will be collected, as in step 3.4.</li>
		<li>Use a cotton swab or fine-gauge needle to carefully apply a small amount of silicone lubricant (Table of Materials) to the part of the probe that will be inserted into the tissue to lower the friction between the sides of the probe and the tissue sample. Reapply the silicone lubricant before each baseline or tissue measurement.</li>
		<li>Remove the tape covering the hole in the blank sample Petri dish, and place it in the sample holder (step 2.7), making sure it is held securely in place by friction.</li>
		<li>Use the micromanipulator to lower the sample onto the probe. Allow the probe to enter the gelatin via the hole on the underside of the petri dish. Continue until the probe is approximately 5 mm from the bottom of the gelatin layer.</li>
		<li>Turn on the light source. Using the spectrometer software, adjust the spectrometer integration time until the signal is as high as possible but not saturating. Set the number of averaged scans between two and five and the smoothing pixels value to six. Usable integration times at this stage can vary between 1-50 ms for this baseline.</li>
	</ol>
	</li>
	<li>Collecting the reference spectra
	<ol>
		<li>Collect a dark measurement.
		<ol>
			<li>Once the spectrometer parameters have been set with the blank sample, detach the probe fiber from the spectrometer, and cover the port with the opaque metal cover that came with the spectrometer.</li>
			<li>Obtain a dark measurement from the spectrometer (often using the dark lightbulb icon in the GUI) to characterize the noise of the spectrometer with no input light. Save this measurement according to the data acquisition strategy.</li>
		</ol>
		</li>
		<li>Collect a lamp measurement. Reconnect the probe fiber to the spectrometer, wait for the signal to stabilize, and acquire another spectrum. This measurement characterizes the light reaching the probe through the gelatin in the absence of tissue.</li>
	</ol>
	</li>
	<li>Collecting the tissue spectra
	<ol>
		<li>Remove the tape on the bottom of the sample Petri dish, and place it in the sample holder.</li>
		<li>Align the sample dish with the probe using three-dimensional manipulation so that the tissue biopsy is centered above the tip of the probe.</li>
		<li>Apply silicone gel to the probe (see 4.1.2), and slowly lower the sample onto the probe until the probe tip has passed through the gelatin supporting the tissue sample and has just entered the tissue sample. 
		NOTE: Some spectrometers and computer programs allow real-time monitoring of the detected light. Use this to determine when the probe enters the tissue. The spectrum intensity will abruptly decrease as soon as the probe tip is in direct contact with the tissue.</li>
		<li>Verify that the integration time is appropriate for the measurement by ensuring that the measured spectrum is neither too noisy nor saturated. Change the integration time if necessary.
		<ol>
			<li>Any time the integration time is adjusted, perform and store a new dark measurement (step 4.2.1). Do not change the number of averaged scans or the number of smoothed pixels.</li>
			<li>After finding suitable measurement parameters, take the measurements and save the spectrum.</li>
		</ol>
		</li>
		<li>Move the sample down a specified distance using the micromanipulator. For the manipulator used for this study, each small tick mark was 0.001 in or 25.4 &#181;m. The sample was moved down through five tick marks, or 127 &#181;m, for each measurement. Repeat step 4.3.4.</li>
		<li>Continue to save the spectra at each vertical position within the tissue. 
		NOTE: For the system described here, the required integration times varied between 1 ms to 5 s for the measurements within a single tissue sample. Eventually, the probe tip will exit the top of the tissue and be visible there (Figure 4A). This is the final measurement and characterizes the light incident at the top of the tissue.</li>
	</ol>
	</li>
	<li>Loading and processing the data
	<ol>
		<li>Use the spectrometer&#39;s software to convert all the collected spectra (dark spectra, lamp spectra, and tissue measurements) to delimited text files.</li>
		<li>Using Matlab or a coding language of choice, load all the measurement files for a sample. For each tissue measurement spectrum, subtract the dark spectrum with the matching integration time, and then divide it by the integration time. 
		NOTE: In this study, a Matlab script was used for loading and processing the data (Supplementary File 1).</li>
		<li>Calculate the percentage of incident light reaching each position in the tissue by dividing the spectra obtained with the probe inside the tissue by the baseline spectrum obtained in empty gelatin. 
		NOTE: The measurement at the top of the tissue (step 4.3.6) should be very similar to the baseline measurement in gelatin (step 4.1) and, when divided, should be close to 100%. Depending on the experimental context, either lamp spectrum (the spectrum from empty gelatin or that from the very top of the tissue) can be used as a reference. However, it is still important to collect a lamp spectrum before starting the experiment. This is because, if the probe breaks or the experiment otherwise fails before reaching the top of the tissue, the data obtained before the failure are still usable, which is not the case if there is no corresponding initial lamp reference spectrum.</li>
		<li>Visualize the data; contour plots can be helpful (Figure 4B).</li>
	</ol>
	</li>
</ol>

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There are no conflicts of interest.

This protocol describes a technique for systematically characterizing the optical environment through a large volume of living tissue with a spatial resolution approximately on the scale of single cells. This inexpensive, flexible, and field-tractable method could be useful to any researchers studying the propagation of light within living systems. From experience, compared to existing methods7, these probes require a little more practice and skill to build but result in less tissue damage and the...

Surprisingly, land animals and shallow ocean dwellers have enough light within their body for visual physiology and even photosynthesis. For example, the light levels in the center of a mouse&#39;s head (outside of the strong hemoglobin absorbance bands) are attenuated by three or four orders of magnitude relative to the outside world. This is roughly the difference between the light levels indoors and outside. So, the opacity of a tissue or material due to strong scattering is not the same as opacity due to strong light absorption. Light can keep propagating over long distances in a strongly forward-scattering system, similar to light propagating through aquatic systems with high concentrations of cells and particles1.&#160;This observation is particularly salient in light of the fact that opsin proteins are near-ubiquitously expressed in all tissues of all animals. Thus, it is important to understand how and where light is attenuated and scattered within living tissue. However, unlike aquatic systems, with living tissue, it is impossible to immerse an instrument in the water column and obtain radiance and irradiance measurements, and a new technique is necessary.
Other methods previously used to characterize the absorption and scattering properties of living tissue include measuring tissue reflectance probes and/or integrating spheres2,3, microscopic methods such as scanning confocal microscopy4, measuring the diffusion of laser light on the surface5, and modeling techniques such as Monte Carlo radiative transfer6. The experimental methods mentioned often require specific, large, and expensive equipment or detailed knowledge about tissue structure and are generally limited in their ability to characterize the spatial structure of light deep within the tissue.
There are also similar probe-based methods that use a hypodermic needle to insert an optical fiber through tissue7,8,9. In our experience, modified needles are effective at puncturing tissue but require considerable force and generally tear delicate tissues when passaging through densely packed cells. Therefore, these needles generally require a surgical procedure to insert more than a millimeter or so into a tissue layer. The method described here, using a lubricated, pulled glass support, is capable of sliding between cells with minimal wounding of the tissue and without additional surgery.
This manuscript presents a method inspired by the work of Jorgenson and colleagues on measuring light within algal mats10,11, using glass-supported optical microprobes and portable electronics that are amenable to probing deep into dense tissue and to construction and use in the field. These probes can be constructed to characterize scalar irradiance (light hitting a point from all directions) and downwelling radiance (light intersecting a horizontal plane) inside living tissue at high spatial resolutions. These probes were originally developed to measure radiative transfer within the tissue of photosymbiotic giant clams12. Standard measurements of absorption and transmission of the total tissue were not enough to characterize the photosynthetic performance of the tissue, since it makes a big difference whether all the incident light is absorbed by a few cells experiencing high intensity at the surface of the tissue or many cells experiencing low intensities throughout the volume of the tissue. In a second project, these probes were used to measure in vivo irradiance within a mouse&#39;s brain13,14, thereby characterizing the light environment of opsins expressed deep within the brain. These micro-probes are both small and sensitive enough to measure irradiance within mouse brain tissue with all the fur, skin, and bone intact and demonstrate that physiological light levels are easily high enough to stimulate deep-brain opsins.
This micro-optical probe and measuring setup could be useful to researchers needing to quantify and characterize the light internal to biological tissue, particularly for a more nuanced understanding of photosynthesis or the functions of visual pigments expressed outside of the eyes. This method can be used alone or in conjunction with other techniques to fully characterize the optical properties and light propagation within living tissue at a low cost, with small portable equipment built in-house and with task-dependent adjustable parameters.

<table><tbody><tr><td>1&quot; travel ball bearing center+D11+A2:D31+A2:A2:D31</td><td>Edmond Optics</td><td>37-935</td><td>Part 2 of manipulator for lowering sample</td></tr><tr><td>1/4&quot; thick acrylic sheet</td><td>McMaster-Carr</td><td>8505K754</td><td>For making Petri dish holder</td></tr><tr><td>3/4&quot; mini spring clamp</td><td>Anvil</td><td>99693</td><td>Use as weight for pulling optical fiber</td></tr><tr><td>8 mm biopsy punch</td><td>Fisher Scientific</td><td>NC9324386</td><td>For tissue sample</td></tr><tr><td>Butane Torch</td><td>McMaster-Carr</td><td>MT-51</td><td>Heat source for pulling fiber and pipette</td></tr><tr><td>Collimating lens</td><td>Thorlabs</td><td>LLG5A1-A</td><td>To collimate light source through liquid light guide</td></tr><tr><td>Compressed air</td><td>McMaster-Carr</td><td>7437K35</td><td>For drying pulled fiber and pipette</td></tr><tr><td>Cyanoacrylate glue - liquid</td><td>McMaster-Carr</td><td>66635A31</td><td>For securing tapered fiber end at top of pulled pipette</td></tr><tr><td>Electrical tape</td><td>McMaster-Carr</td><td>76455A21</td><td>For securing fiber in pipette and for adding grip to clamps</td></tr><tr><td>Fine grade carborundum paper</td><td>McMaster-Carr</td><td>4649A24</td><td>Small triangle on exacto knife holder works well</td></tr><tr><td>Gelatin</td><td>Knox</td><td>10043000048679</td><td>For securing the tissue biopsy in the petri dish</td></tr><tr><td>Glass Pasteur Pipete</td><td>Fisher Scientific</td><td>13-678-20B</td><td>Disposable glass pipette 5.75&quot; in length</td></tr><tr><td>Insulin syringes, 31G needle</td><td>BD</td><td>320440</td><td>For applying glue</td></tr><tr><td>Isopropanol</td><td>McMaster-Carr</td><td>54845T42</td><td>For cleaning pulled fiber and pipette</td></tr><tr><td>Kimwipe</td><td>Cole-Parmer</td><td>SKU 33670-04</td><td>For wiping optical fiber and glass pipette clean</td></tr><tr><td>LED driver</td><td>Thorlabs</td><td>LEDD1B</td><td>For powering the UV LEDs</td></tr><tr><td>Light source for measurements</td><td>Cole-Parmer</td><td>UX-78905-05</td><td>Low heat white light source for measurements</td></tr><tr><td>Linear metric X-Y-Z axis rack and pinion stage</td><td>Edmond Optics</td><td>55-023</td><td>Part 1 of manipulator for lowering sample</td></tr><tr><td>Liquid light guide</td><td>Thorlabs</td><td>LLG5-4T</td><td>For light source in measurements</td></tr><tr><td>Magnetic feet</td><td>Siskiyou</td><td>MGB 8-32</td><td>For use with magnetic strips</td></tr><tr><td>Magnetic strips</td><td>Siskiyou</td><td>MS-6.0</td><td>For mounting magnetically to breadboard</td></tr><tr><td>Manipulator #1</td><td>Siskiyou</td><td>MX10R</td><td>4-axis manipulator with pipette holder</td></tr><tr><td>Opaquer pen, small</td><td>WindowTint</td><td>TOP01</td><td>For opaquing side of optical fiber to prevent stray light from enter probe</td></tr><tr><td>Optical breadboard</td><td>Edmond Optics</td><td>03-640</td><td>For stable affixation of probe holder, sample, microscope and light source</td></tr><tr><td>Optical fibers</td><td>Ocean Optics</td><td>P-100-2-UV-VIS</td><td>About 4 fibers are good to have</td></tr><tr><td>Plasma light source</td><td>Thorlabs</td><td>HPLS345</td><td>For tissue radiometry measurements</td></tr><tr><td>Plastic plier clamp</td><td>McMaster-Carr</td><td>5070A11</td><td>Plier clamp used for weight in pulling pipette</td></tr><tr><td>Polystyrene Petri dishes</td><td>Thomas scientific</td><td>3488N10</td><td>Sample holders, enough volume to hold sample thickness plus ~10 mm of gelatin on top</td></tr><tr><td>Razor blades</td><td>McMaster-Carr</td><td>3962A3</td><td>For stripping jacketing from optical fiber</td></tr><tr><td>Silicone oil lubricant</td><td>Thomas scientific</td><td>1232E30</td><td>For reducing friction between probe and tissue</td></tr><tr><td>Software for analyzing data</td><td>Matlab</td><td></td><td>Chosen software for data analysis</td></tr><tr><td>Spectrometer + spectrometer software</td><td>Avantes</td><td>AvaSpec-2048L</td><td>Spectrometer can be any brand, this one is compatible with sma-terminated optical fibers and comes with its own software for running the spectrometer</td></tr><tr><td>Titanium dioxide powder</td><td>Sigma Aldrich</td><td>718467-100G</td><td>For making scattering sphere</td></tr><tr><td>Toolour tabletop clip</td><td>Toolour</td><td>Toolour0004</td><td>For holding pipette while pulling and for holding finished probes</td></tr><tr><td>Trigger-action bar clamps</td><td>mcMaster-Carr</td><td>51755A2</td><td>Good for holding optical fibers while pulling or curing</td></tr><tr><td>UV curable adhesive</td><td>Delo Photobond</td><td>GB368</td><td>For making scattering sphere</td></tr><tr><td>UV light source</td><td>Thorlabs</td><td>M365FP1</td><td>Light source for curing adhesive in scattering ball, this one is sma-fiber compatible, higher intensity = less cure time</td></tr><tr><td>White LED light source</td><td>Thorlabs</td><td>MCWHF2</td><td>For characterizing pulled fiber and scattering sphere</td></tr></tbody></table>

an intra-tissue radiometry microprobe for measuring radiance in situ in living tissue

Organisms appear opaque largely because their outer tissue layers are strongly scattering to incident light; strongly absorbing pigments, such as blood, typically have narrow absorbances, such that the mean free path of light outside the absorbance peaks can be quite long. As people cannot see through tissue, they generally imagine that tissues like the brain, fat, and bone contain little or no light. However, photoresponsive opsin proteins are expressed within many of these tissues, and their functions are poorly understood. Radiance internal to tissue is also important for understanding photosynthesis. For example, giant clams are strongly absorbing yet maintain a dense population of algae deep in the tissue. Light propagation through systems like sediments and biofilms can be complex, and these communities can be major contributors to ecosystem productivity. Therefore, a method for constructing optical micro-probes for measuring scalar irradiance (photon flux intersecting a point) and downwelling irradiance (photon flux crossing a plane perpendicularly) to better understand these phenomena inside living tissue has been developed. This technique is also tractable in field laboratories. These micro-probes are made from heat-pulled optical fibers that are then secured in pulled glass pipettes. To change the angular acceptance of the probe, a 10-100 &#181;m sized sphere of UV-curable epoxy mixed with titanium dioxide is then secured to the end of a pulled, trimmed fiber. The probe is inserted into living tissue, and its position is controlled using a micromanipulator. These probes are capable of measuring in situ tissue radiance at spatial resolutions of 10-100 &#181;m or on the scale of single cells. These probes were used to characterize the light reaching the adipose and brain cells 4 mm below the skin of a living mouse and to characterize the light reaching similar depths within living algae-rich giant clam tissue.

This protocol describes the procedure for constructing a micro-optical probe that can be used to measure the downwelling irradiance (the light reaching a point from one direction) or, with the addition of a light-scattering spherical tip, to measure the scalar irradiance (the light reaching a point from all directions). These probes can measure irradiance at spatial resolutions approaching the length scales of single cells inside living tissue. This protocol also describes a representative method for preparing a tissue s...

Watch this Scientific Journal Video about An Intra-Tissue Radiometry Microprobe for Measuring Radiance In Situ in Living Tissue at JoVE.com

An Intra-Tissue Radiometry Microprobe for Measuring Radiance In Situ in Living Tissue

In this paper, a method for measuring radiance in situ in living tissue is described. This work includes details of the construction of micro-scale probes for different measurements of radiance and irradiance, provides guidance for mounting tissue for the characterization of radiance, and outlines computational methods for analyzing the resulting data.

An Intra-Tissue Radiometry Microprobe for Measuring Radiance In Situ in Living Tissue

Organisms appear opaque largely because their outer tissue layers are strongly scattering to incident light; strongly absorbing pigments, such as blood, ...

an-intra-tissue-radiometry-microprobe-for-measuring-radiance-situ

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

537 Views.  Yale University. In this paper, a method for measuring radiance in situ in living tissue is described. This work includes details of the construction of micro-scale probes for different measurements of radiance and irradiance, provides guidance for mounting tissue for the characterization of radiance, and outlines computational methods for analyzing the resulting data. 

Video: An Intra-Tissue Radiometry Microprobe for Measuring Radiance In Situ in Living Tissue

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

Biology

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

Medicine

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The cell line was originally induced by intracranial injection of 3-methyl-cholantrene into a C57BL/6 syngeneic mouse strain 1; therefore, immunologically competent C57BL/6 mice can be used. While we use GL261, the following protocol can be used for the implantation and monitoring of any intracranial mouse tumor model. GL261 cells were engineered to stably express firefly luciferase (GL261-luc). We also created the brighter GL261-luc2 cell line by stable transfection of the luc2 gene expressed from the CMV promoter. C57BL/6-cBrd/cBrd/Cr mice (albino variant of C57BL/6) from the National Cancer Institute, Frederick, MD were used to eliminate the light attenuation caused by black skin and fur. With the use of albino C57BL/6 mice; in vivo imaging using the IVIS Spectrum in vivo imaging system is possible from the day of implantation (Caliper Life Sciences, Hopkinton, MA). The GL261-luc and GL261-luc2 cell lines showed the same in vivo behavior as the parental GL261 cells. Some of the shared histological features present in human GBMs and this mouse model include: tumor necrosis, pseudopalisades, neovascularization, invasion, hypercellularity, and inflammation 1.
Prior to implantation animals were anesthetized by an intraperitoneal injection of ketamine (50 mg/kg), xylazine (5 mg/kg) and buprenorphine (0.05 mg/kg), placed in a stereotactic apparatus and an incision was made with a scalpel over the cranial midline. A burrhole was made 0.1mm posterior to the bregma and 2.3mm to the right of the midline. A needle was inserted to a depth of 3mm and withdrawn 0.4mm to a depth of 2.6mm. Two &mu;l of GL261-luc or GL261-luc2 cells (107 cells/ml) were infused over the course of 3 minutes. The burrhole was closed with bonewax and the incision was sutured.
Following stereotactic implantation the bioluminescent cells are detectable from the day of implantation and the tumor can be analyzed using the 3D image reconstruction feature of the IVIS Spectrum instrument. Animals receive a subcutaneous injection of 150&mu;g luciferin /kg body weight 20 min prior to imaging. Tumor burden is quantified using mean tumor bioluminescence over time. Tumor-bearing mice were observed daily to assess morbidity and were euthanized when one or more of the following symptoms are present: lethargy, failure to ambulate, hunched posture, failure to groom, anorexia resulting in &gt;10% loss of weight. Tumors were evident in all of the animals on necropsy.

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Intracranial implantation of GL261 cells into C57BL/6 mice produces malignant gliomas that recapitulate many of the hallmarks of human glioblastoma multiforme. We used GL261 cells stably expressing luciferase to allow us to use in vivo imaging to follow tumor progression. The surgery and 3D in vivo imaging are demonstrated.

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The ...

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system is valuable for the pre-clinical in vivo evaluation of putative antitumor compounds.
The 4T1 cell line has been engineered to constitutively express the firefly luciferase gene (luc2). When mice carrying 4T1-luc2 tumors are injected with Luciferin the tumors emit a visual light signal that can be monitored using a sensitive optical imaging system like the IVIS Spectrum. The photon flux from the tumor is proportional to the number of light emitting cells and the signal can be measured to monitor tumor growth and development. IVIS is calibrated to enable absolute quantitation of the bioluminescent signal and longitudinal studies can be performed over many months and over several orders of signal magnitude without compromising the quantitative result.
Tumor growth can be monitored for several days by bioluminescence before the tumor size becomes palpable or measurable by traditional physical means. This rapid monitoring can provide insight into early events in tumor development or lead to shorter experimental procedures.
Tumor cell death and necrosis due to hypoxia or drug treatment is indicated early by a reduction in the bioluminescent signal. This cell death might not be accompanied by a reduction in tumor size as measured by physical means. The ability to see early events in tumor necrosis has significant impact on the selection and development of therapeutic agents.
Quantitative imaging of tumor growth using IVIS provides precise quantitation and accelerates the experimental process to generate results.

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

Mammary tumor cells expressing luciferase are implanted subcutaneously in mice and visualized using optical imaging to monitor tumor growth and development non-invasively in a longitudinal study.

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system ...

Intact Histological Characterization of Brain-implanted Microdevices and Surrounding Tissue

Research into the design and utilization of brain-implanted microdevices, such as microelectrode arrays, aims to produce clinically relevant devices that interface chronically with surrounding brain tissue. Tissue surrounding these implants is thought to react to the presence of the devices over time, which includes the formation of an insulating "glial scar" around the devices. However, histological analysis of these tissue changes is typically performed after explanting the device, in a process that can disrupt the morphology of the tissue of interest.
Here we demonstrate a protocol in which cortical-implanted devices are collected intact in surrounding rodent brain tissue. We describe how, once perfused with fixative, brains are removed and sliced in such a way as to avoid explanting devices. We outline fluorescent antibody labeling and optical clearing methods useful for producing an informative, yet thick tissue section. Finally, we demonstrate the mounting and imaging of these tissue sections in order to investigate the biological interface around brain-implanted devices.

Here we present a histological method for capturing, labeling, optically clearing, and imaging the intact brain tissue interface around chronically implanted microdevices in rodent brain tissue. Results from the techniques comprising this method are useful for understanding the impact of various penetrating brain-implants on their surrounding tissue.

Research  into the design and utilization of brain-implanted microdevices, such as  microelectrode arrays, aims to produce clinically relevant devices ...

Neuroscience

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of spectroscopy techniques. Combining imaging and spectroscopy techniques into a single optical probe may provide a more complete analysis of tissue health. In this article, two dissimilar modalities are combined, high-resolution fluorescence microendoscopy imaging and diffuse reflectance spectroscopy, into a single optical probe. High-resolution fluorescence microendoscopy imaging is a technique used to visualize apical tissue micro-architecture and, although mostly a qualitative technique, has demonstrated effective real-time differentiation between neoplastic and non-neoplastic tissue. Diffuse reflectance spectroscopy is a technique which can extract tissue physiological parameters including local hemoglobin concentration, melanin concentration, and oxygen saturation. This article describes the specifications required to construct the fiber-optic probe, how to build the instrumentation, and then demonstrates the technique on in vivo human skin. This work revealed that tissue micro-architecture, specifically apical skin keratinocytes, can be co-registered with its associated physiological parameters. The instrumentation and fiber-bundle probe presented here can be optimized as either a handheld or endoscopically-compatible device for use in a variety of organ systems. Additional clinical research is needed to test the viability of this technique for different epithelial disease states.

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

The assembly and use of a multimodal microendoscope is described which can co-register superficial tissue image data with tissue physiological parameters including hemoglobin concentration, melanin concentration, and oxygen saturation. This technique can be useful for evaluating tissue structure and perfusion, and can be optimized for individual needs of the investigator.

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of ...

Radiation Response in Patient-Derived Tumor Organoids Using a Clonogenic Assay

Live Imaging to Quantify Cellular Radiosensitivity in Patient-Derived Tumor Organoids

Radiation therapy (RT) is one of the mainstays of modern clinical cancer management. However, not all cancer types are equally sensitive to irradiation, often (but not always) because of differences in the ability of malignant cells to repair oxidative DNA damage as elicited by ionizing rays. Clonogenic assays have been employed for decades to assess the sensitivity of cultured cancer cells to ionizing irradiation, largely because irradiated cancer cells often die in a delayed manner that is difficult to quantify with short-term flow cytometry- or microscopy-assisted techniques. Unfortunately, clonogenic assays cannot be employed as such for more complex tumor models, such as patient-derived tumor organoids (PDTOs). Indeed, irradiating established PDTOs may not necessarily abrogate their growth as multicellular units, unless their stem-like compartment is completely eradicated. Moreover, irradiating PDTO-derived single-cell suspensions may not properly recapitulate the sensitivity of malignant cells to RT in the context of established PDTOs. Here, we detail an adaptation of conventional clonogenic assays that involves exposure of established PDTOs to ionizing radiation, followed by single-cell dissociation, replating in suitable culture conditions and live imaging. Non-irradiated (control) PDTO-derived stem-like cells reform growing PDTOs with a PDTO-specific efficiency, which is negatively influenced by irradiation in a dose-dependent manner. In these conditions, PDTO-forming efficiency and growth rate can be quantified as a measure of radiosensitivity on time-lapse images collected until control PDTOs achieve a predefined space occupancy.

Author Spotlight: Radiotherapy and Clonogenic Assays for Advancing Cancer Research and Personalized Medicine

Here, we detail a straightforward live imaging approach for quantifying the sensitivity of patient-derived tumor organoids to ionizing radiation.

Radiation therapy (RT) is one of the mainstays of modern clinical cancer management. However, not all cancer types are equally sensitive to irradiation, ...

Cancer Research

High Spatial Resolution Chemical Imaging of Implant-Associated Infections with X-ray Excited Luminescence Chemical Imaging Through Tissue

Microbial infections associated with implantable medical devices are a major concern in fracture fixation failure. Early diagnosis of such infection will allow successful eradication with antibiotics without an extra cost for a second surgery. Herein, we describe XELCI as a technique with high X-ray resolution, implant specificity, and chemical sensitivity to noninvasively image chemical concentrations near the surface of implanted medical devices. The devices are coated with chemically reporting surfaces. This chemically responsive surface consists of two layers coated on an implantable medical device; a pH-sensitive layer (bromothymol blue or bromocresol green incorporated hydrogel) which is coated over a red-light emitting scintillator (Gd2O2S: Eu) layer for monitoring. A focused X-ray beam irradiates a spot on the implant, and the red light generated by the scintillator (with 620 nm and 700 nm peaks) is transmitted through the sensing layer which alters the spectral ratio depending on the pH. An image is generated by scanning the X-ray beam across the implant and measuring the spectral ratio of light passing through the tissue point-by-point. We used this imaging technique for monitoring implant-associated infections previously on the bone surface of the femur with a modified implantable plate sensor. Now we are studying pH changes that occur from tibial intramedullary rod infections. Two different types of intramedullary rod designs are used in pre-pilot rabbit studies, and we learned that the XELCI technique could be used to monitor any chemical changes that occur not only on the bone surface but also inside the bone. Thus, this enables noninvasive, high spatial resolution, low background local pH imaging to study implant-associated infection biochemistry.

Here, we present a protocol for high-resolution optical detection of chemical information around implanted medical devices with X-ray excited luminescence chemical imaging (XELCI). This novel imaging technique is developed in our lab which enables studying implant-associated infection biochemistry.

Microbial infections associated with implantable medical devices are a major concern in fracture fixation failure. Early diagnosis of such infection will ...

Chemistry

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various pathologies such as cancer, atherosclerosis and diabetes. We have previously shown that dysfunction of the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/ Kelch-like erythroid cell-derived protein 1 (Keap1) signaling pathway leads to extreme ROS imbalance during cutaneous wound healing in diabetes. Since ROS levels are an important indicator of progression of wound healing, specific and accurate quantification techniques are valuable. Several in vitro assays to measure ROS in cells and tissues have been described; however, they only provide a single cumulative measurement per sample. More recently, the development of protein-based indicators and imaging modalities have allowed for unique spatiotemporal analyses. L-012 (C13H8ClN4NaO2) is a luminol derivative that can be used for both in vivo and in vitro chemiluminescent detection of ROS generated by NAPDH oxidase. L-012 emits a stronger signal than other fluorescent probes and has been shown to be both sensitive and reliable for detecting ROS. The time lapse applicability of L-012-facilitated imaging provides valuable information about inflammatory processes while reducing the need for sacrifice and overall reducing the number of study animals. Here, we describe a protocol utilizing L-012-facilitated in vivo imaging to quantify oxidative stress in a model of excisional wound healing using diabetic mice with locally dysfunctional Nrf2/Keap1.

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

We describe a non-invasive in vivo imaging protocol that is streamlined and cost-effective, utilizing L-012, a chemiluminescent luminol-analog, to visualize and quantify reactive oxygen species (ROS) generated in a mouse excisional wound model.

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various ...

Immunology and Infection

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic molecular vibration, thus providing a perfect tool for in vivo study of biological processes at subcellular resolution. By integrating two-photon excited fluorescence (TPEF) imaging into the SRS microscope, the dual-modal in vivo imaging of tissues can acquire critical biochemical and biophysical information from multiple perspectives which helps understand the dynamic processes involved in cellular metabolism, immune response and tissue remodeling, etc. In this video protocol, the setup of a TPEF-SRS microscope system as well as the in vivo imaging method of the animal spinal cord is introduced. The spinal cord, as part of the central nervous system, plays a critical role in the communication between the brain and peripheral nervous system. Myelin sheath, abundant in phospholipids, surrounds and insulates the axon to permit saltatory conduction of action potentials. In vivo imaging of myelin sheaths in the spinal cord is important to study the progression of neurodegenerative diseases and spinal cord injury. The protocol also describes animal preparation and in vivo TPEF-SRS imaging methods to acquire high-resolution biological images.

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows label-free imaging of biomolecules based on their intrinsic vibration of specific chemical bonds. In this protocol, the instrumental setup of an integrated SRS and two-photon fluorescence microscope is described to visualize cellular structures in the spinal cord of live mice.

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic ...

An Intra-Tissue Radiometry Microprobe for Measuring Radiance In Situ in Living Tissue

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Rozdziały w tym wideo

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

In vivo Dual Substrate Bioluminescent Imaging

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

Intact Histological Characterization of Brain-implanted Microdevices and Surrounding Tissue

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

Live Imaging to Quantify Cellular Radiosensitivity in Patient-Derived Tumor Organoids

High Spatial Resolution Chemical Imaging of Implant-Associated Infections with X-ray Excited Luminescence Chemical Imaging Through Tissue

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy