This protocol is significant because it simplifies and permits radiance measurements in thick materials, allowing us to gather important information when studying living systems. The main advantage of this technique is that it allows for spatially resolved spectral measurements in highly absorbing materials using equipment that can be constructed and experiments that can be run in field labs. This method can be adapted and applied to any living system.
The technique of making the probes is nuanced, but practice makes perfect. Adjust the measurement technique depending on the material being probed and make changes that result in more secure tissue position and lower probe friction. To begin build the glass sleeve with a 5.75-inch Pasteur pipette.
Using a mountable alligator clip, mount the glass pipette by the wide end such that the tapered end is facing down to toward the floor and the orientation of the pipette is perpendicular to the floor. Place a pad of electrical tape on a 50-gram plastic vice grip to help prevent slippage and hang the vice grip from the tapered end of the pipette. Using a small butane torch, heat the tapered end of the pipette as described in the manuscript.
When the pipette length has increased by about five inches, immediately remove the flame. Next, use small scissors or a glass cutter to trim the pulled end of the pipette. File down any sharp areas of the trimmed end using carborundum paper and rinse away any tiny glass shards or dust using isopropyl alcohol followed by compressed air.
Use a razor blade to sever the optical fiber and remove one SMA connector of an SMA-terminated optical fiber. Ensure to cut close to one of the SMA terminations. Then remove the following five centimeters of plastic and fiberglass jacketing so that the bare optical fiber is exposed and protrudes from the rest of the entire assembly.
Use the butane torch to burn off the polyamide polymer coating from the glass fiber. Rinse with isopropanol and wipe the bare glass fiber using a lint-free wipe. Now mount the bare end of the optical fiber directly in a plier clamp on a table or shelf edge, letting the SMA-terminated end of the fiber hang toward the floor.
About four to six inches from where the bare optical fiber is held in the plier clamp, add the weights for pulling using two small clamps on the jacketed end of the fiber. To pull the fiber, start with the butane torch flame on. Hold the butane torch one centimeter from the bare fiber as described in the manuscript.
And allow the fiber to stretch, pull, separate, and drop to the floor. Check the fiber under the microscope and trim the end of the fiber with small dissection scissors if necessary. Use a foam opaque pen to darken the sides of the fiber and prevent the entry of stray light.
Gently pull the pen's tip across the fiber, leaving only a small area at the tip uncovered. Under a stereo microscope, carefully insert the tapered optical fiber into the wide end of the altered glass pipette and push the fiber until approximately one millimeter of bare fiber is sticking out of the tapered end of the pipette. Using electrical tape, secure the jacketed end of the optical fiber to the wide end of the pipette.
Put a drop of cyanoacrylate adhesive onto a small gauge needle. Carefully touch the drop of adhesive to the cut edge of the pulled pipette, avoiding the bare end of the pulled fiber. To modify the fiber tip with a scattering sphere, create the raw material by mixing a drop of UV curable adhesive with titanium dioxide powder in an equal ratio.
Attach the probe to a micromanipulator with a mounting rod holder 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 prepared mixture such that a droplet of adhesive forms. Mount the wire or needle with the droplet near the tip of the horizontal probe.
Deposit a scattering sphere on the tip of the probe. Use the micromanipulator to push the tip of the probe's optical fiber slowly and carefully into the prepared mixture. Then quickly would withdraw the tip from the glue and view the scattering ball in a microscope.
Repeat until a spherical droplet of adhesive of the desired size is deposited on the tip of the optical fiber. Prepare the dishes to mount the samples. Using a butane torch, heat the large end of a Pasteur pipette to melt a 0.5-centimeter diameter hole in the bottom of a plastic Petri dish.
Seal the hole on the bottom side of the dish with electrical tape or lab tape. Then fill one Petri dish a quarter of the way and a second Petri dish all the way with the liquid gelatin and let it cool to room temperature just short of gelation. In one dish, place the biopsy in the cushion of viscous gelatin that formed in the hole in the bottom of the dish.
Using a Pasteur pipette, gently add room temperature gelatin around the biopsy until the Petri dish is full. For the blank sample, use the dish filled with gelatin only. Attach the probe to a vertically-oriented mount such that it points toward the light source and secure the probe with clamps, hose clamps, or tape to an optical table post.
Connect the SMA-terminated end of the probe's optical fiber to a USB fiber optic spectrometer and connect the spectrometer to the computer using a USB cable. Keep the probe and manipulators in the exact aligned position where the data will be collected. Use a cotton swab, fine gauge needle, or dropper to carefully apply a small amount of silicone lubricant 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.
Now, remove the tape covering the hole in the sample Petri dish and place it in the sample holder, ensuring it is held securely in place by friction. Use the micromanipulator to lower the sample onto the probe and allow the probe to enter the gelatin via the hole on the underside of the Petri dish. Continue until the probe is approximately five millimeters from the bottom of the gelatin layer.
Turn the light source on. Using the spectrometer software, adjust the spectrometer integration time until the signal is as high as possible, but not saturating. Set the number of average scans between two and five, and the smoothing pixels'value to six.
Usable integration times at this stage can vary between one to 15 milliseconds for this baseline. Verify that the integration time is appropriate for the measurement by ensuring that the spectrum is neither too noisy nor saturated. Change the integration time if necessary.
Move the sample down a specified distance using the micromanipulator and perform the measurements. Continue to save the spectra at each vertical position within the tissue. The microscopic appearance of the probe coming up through the tissue is visualized.
The percentage of light at different tissue depths relative to the baseline is shown. The most important thing to remember is that these probes are fragile, so take your time and make sure the probe junctions are secure before proceeding. After making the probe, the measurement techniques can be adapted.
For instance, without the scattering ball, probes can be used to measure radiance reaching the detector from a single direction. After developing this probe, researchers were able to measure light inside both brain and adipose tissue in mice using a variation on our measurement apparatus. This helped answer the question of whether there was enough light to activate photoreceptors present deep within the tissue.
