The overall goal of this procedure is functional characterization of in vivo tissue by combining high-resolution structural information with quantitative spectral data. This method can be used to answer key questions in the field of cancer biology such as quantifying the effects of therapy on tissue microstructure and perfusion. The main advantage of this technique is that a single probe can be used to collect both image and spectroscopy data from the same location.
The implications of this technique extend towards gastrointestinal cancer therapy because the probe is compatible with conventional endoscopy techniques. Generally, individuals new to this method will struggle because small irregularities in probe placement can significantly affect data accuracy. We first had the idea for this method when we realized the importance of combining a high-resolution imaging technique with a quantitative spectroscopy method.
Visual demonstration of this method is critical as the preparation steps are difficult to learn because of the complexity of both the construction and calibration. To begin assembling the high-resolution fluorescence microscopy modality of the multimodal micro endoscope, mount a 470 nanometer dichroic mirror in a 30 millimeter cage cube. Attach cage assembly rods to the front, right, and left sides of the cage cube.
Attach a stress-free retaining ring to a threaded cage plate and screw a lens tube into the retaining ring. Attach another cage plate to the lens tube aligned with the first cage plate. Next, secure a UV-enhanced aluminum mirror in a right angle mirror mount.
Attach four rods to the front of the mirror mount and two rods diagonally to the right side of the mount. Slide the lens tube assembly onto the left side of the cage cube. Connect the right side of the mirror mount assembly to the lens tube assembly.
Slide a Z-axis translation mount onto the right side of the assembly. Attach a 10x achromatic objective lens to the translation mount. Next, secure a fiber adapter plate to an XY-axis translation lens mount.
Slide the mount onto the assembly in front of the objective lens. Using retaining rings, secure an appropriate excitation filter and emission filter in lens tubes. Screw the excitation filter lens tube into the front of the cage cube.
And the emission filter lens tube into the front of the right angle mirror mount. Using epoxy, attach a 455 nanometer LED to the cage plate in front of the excitation filter. Slide a cage plate in front of each filter lens tube.
Using a retaining ring, secure an achromatic doublet tube lens into another lens tube so that the arrow on the outside of the lens faces the externally threaded side of the lens tube. Attach the tube lens to the left cage plate, place another cage plate in front of the tube lens. Using a stress-free retaining ring, attach a USB monochrome camera to the cage plate.
To begin assembling the sub-diffuse reflection spectroscopy modality, attach a threaded cage plate to the front of a tungsten halogen light source with epoxy. Insert four cage assembly rods into the cage plate and slide a Z-axis translation mount onto the rods. Screw a 20x achromatic objective lens into the Z-axis translation mount.
Connect a fiber adapter plate to an XY-axis translation mount and slide the mount onto the assembly in front of the objective lens. Finally, secure both assemblies to an optical table close together using appropriate mounting devices. To begin constructing the source detector separation switching device, screw an externally threaded SMA fiber adapter plate into an aluminum motor arm.
Attach a motor arm adapter to the back of the motor arm. Then, screw a stepper motor into an aluminum motor housing. Thread the motor arm assembly onto the motor rod and tighten the locking screw.
Next, screw three fiber adapter plates into an optical switch and then screw the face plate onto the optical switch. Thread the stepper motor rod through the center hole of the optical switch. Place a stepper motor driver across the center line of a sodderless red board.
Connect the driver to an appropriate power supply and the stepper motor. Secure the optical switch to the optical table. Attach a 550 micrometer 0.22 na patch cable to the motor arm.
Connect the other end to a USB spectrometer. To connect the fiber optic probe, attach the central 1 millimeter image fiber cable to the high-resolution fluorescence microscopy modality assembly. Attach the left 200 micrometer multi-mode cable to the sub-diffuse reflection spectroscopy modality assembly.
Continuing from the left, connect the remaining multi-mode cables in order to the left, middle, and right adapters of the optical switching device. To begin calibration for the experiment, connect all USB peripherals to the computer. Turn on all instrumentation and ensure the tungsten halogen lamp shutter is open.
Then, turn off the room lights and close other sources of ambient light. Open the data acquisition software. Allow the equipment to run for 30 minutes before proceeding to calibration.
Place a 20%diffuse reflection standard into the bottom section of the calibration device. Insert the fiber optic probe into the left slot of the device and set the motorized optical switch to the left to select the 374 micrometer as DS setting. In the software, set the integration time to 500 milliseconds.
Then, click acquire spectrum to begin acquisition of RMAX for this SDS. When acquisition of RMAX is finished, save the spectrum in a designated folder. Close the tungsten halogen lamp shutter and acquire the R dark spectrum for this SDS.
Open the shutter, move the probe to the right slot in the calibration device and set the motorized optical switch to the middle position for the 730 micrometer SDS setting. Acquire RMAX and RDARK for this SDS to complete the calibration. First, determine the appropriate skin area for data collection.
Using a yellow highlighter as a Pyranine source, lightly stain the chosen area. To begin the high-resolution fluorescence microscopy, turn on the 455 nanometer LED and close the shutter of the tungsten halogen lamp. Place the fiber optic probe in gentle contact with the skin.
Move the probe across the Pyranine-stained tissue to display the apical current insight architecture. In the software set an appropriate exposure time and gain to avoid image saturation. Then, click acquire image to obtain a static image.
Keep the probe in place for the sub-diffuse reflectance spectroscopy. Turn off the 455 nanometer LED and open the tungsten halogen lamp shutter. Then, switch to the 374 micrometer SDS setting on the left.
Click acquire spectra to acquire the R-tissue spectrum for this SDS. Then, switch to the middle position and acquire the 730 micrometer SDS R-tissue spectrum. Open the post-processing software and click run.
When prompted, select the calibration spectra, the in vivo spectra and the high-resolution fluorescence image to obtain the tissue image. The high resolution fluorescence microscopy modality provides an in focus high-resolution image of the tissue site. Pyranine staining shows individual Keratinocyte outlines.
The post-processing software uses the sub-diffuse reflectance spectroscopy data to calculate hemoglobin concentration, melanin concentration, and tissue oxygen saturation. A benign melanocytic nevus was compared to neighboring normal skin tissue using this multimodal micro endoscope. The melanocytic nevus displayed a slightly lower hemoglobin concentration and a slightly elevated melanin concentration compared to normal tissue.
Once mastered, this technique can be done in less than an hour if performed properly. While attempting this procedure, it's important to let the tungsten halogen lamp warm up for 15 minutes and to calibrate the instrumentation with a reflectance standard prior to data collection. Following this procedure, other techniques such as confocal microscopy or multiphoton microscopy can be used to explore subcellular changes in protein structure or metabolism.
After its development this technique has paved the way for researchers to explore the relationship between tissue perfusion and microstructure in response to chemotherapy. After watching this video, you should have a good understanding of how to construct and use a hybrid micro endoscopic imaging and spectroscopy device.
