This material is based on work supported by the National Institutes of Health (1R03-CA182052, 1R15-CA202662), the National Science Foundation Graduate Research Fellowship Program (G.G., DGE-1450079), the Arkansas Biosciences Institute, and the University of Arkansas Doctoral Academy Fellowship. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the acknowledged funding agencies.

Institutional Review Board approval (IRB #15-09-149) was obtained from the Human Subjects Research program at the University of Arkansas for all aspects of this study. The methods described were carried out in accordance with the approved guidelines, and informed consent was obtained from all participants.
1. Assembly of the High-resolution Fluorescence Microendoscopy Modality
Note: The outlined steps for assembly of the high-resolution fluorescence microendoscopy modality can be visualized in Figure 2.
<ol>
	<li>Place a 470 nm Dichroic Mirror Inside a 30 mm Cage Cube.
	<ol>
		<li>Obtain a 30 mm cage cube and remove the dichroic filter mount.</li>
		<li>Place a 470 nm dichroic mirror in the dichroic filter mount.</li>
		<li>Re-insert and secure the dichroic filter mount back inside the cage cube.</li>
	</ol>
	</li>
	<li>Attach Cage Assembly Rods to the 30 mm Cage Cube.
	<ol>
		<li>Secure four 1.5 inch cage assembly rods to the front of the cage cube.</li>
		<li>Secure four 3.0 inch cage assembly rods to the right side of the cage cube.</li>
		<li>Secure two 2.0 inch cage assembly rods diagonally on the left side of the cage cube.</li>
	</ol>
	</li>
	<li>Build a Cage Plate/Lens Tube Assembly.
	<ol>
		<li>Obtain a 1.0 inch threaded 30 mm cage plate and attach a stress free retaining ring to the inside of the cage plate using the provided threading.</li>
		<li>Screw in a 1.0 inch lens tube to the stress-free retaining ring.</li>
		<li>Attach a second 1.0 inch threaded 30 mm cage plate to the 1.0 inch lens tube and adjust the standard retaining rings so that the two cage plates are flush.</li>
	</ol>
	</li>
	<li>Slide the 1.0 inch Cage Plate/Lens Tube Assembly onto the Left Side of the 30 mm Cage Cube.</li>
	<li>Build the right-angle mirror mount assembly.
	<ol>
		<li>Obtain a right-angle mirror mount and a 1.0 inch UV-enhanced aluminum mirror.</li>
		<li>Place the 1.0 inch UV-enhanced aluminum mirror into the mirror mount and tighten.</li>
		<li>Secure four 2.0 inch cage assembly rods to the front of mirror mount</li>
		<li>Secure two 2.0 inch cage assembly rods diagonally on the right side of the cage cube.</li>
	</ol>
	</li>
	<li>Connect the right-angle mirror mount assembly onto the left side of the 1.0 inch cage plate/lens tube assembly by placing the opposing cage assembly rods through the respective openings of the 30 mm cage plate.</li>
	<li>Thread a z-axis translation mount through the 3.0 inch cage assembly rods on the right side of the assembly.</li>
	<li>Attach a 10X achromatic objective lens to the z-axis translation mount.</li>
	<li>Build a 1.0 inch fiber adaptor plate/xy-axis translation lens mount assembly.
	<ol style="margin-left: 40px;">
		<li>Obtain an xy-axis translation mount and a 1.0 inch fiber adaptor plate.</li>
		<li>Secure the 1.0 inch fiber adaptor plate into the xy-axis translation lens mount.</li>
	</ol>
	</li>
	<li>Slide the 1.0 inch fiber adaptor/xy-axis translation lens mount assembly in front of the objective lens.</li>
	<li>Obtain two 0.5 inch long, 1.0 inch diameter lens tubes, one 440/40 nm bandpass filter (excitation filter) and one 525/36 nm bandpass filter (emission filter).</li>
	<li>Place each filter inside a 0.5 inch long, 1.0 inch diameter lens tube, such that the arrow on the outside of the filter is facing the side of the lens tube with the external threads.</li>
	<li>Attach the filters to the assembly.
	<ol>
		<li>Obtain two standard retaining rings.</li>
		<li>Secure the filters inside the 0.5 inch long, 1.0 inch diameter lens tubes with the standard retaining rings.</li>
		<li>Screw in the lens tube with the excitation filter to the front of the 30 mm cage cube and screw in the lens tube with the emission filter to the right-angle mirror mount.</li>
		<li>Screw in the 0.5 inch lens tube with the emission filter to the front of the right-angle mirror mount.</li>
	</ol>
	</li>
	<li>Obtain two 1.0 inch threaded 30 mm cage plates and place them in front of the 0.5 inch long, 1.0 inch diameter lens tubes containing the filters.</li>
	<li>Using epoxy or strong adhesive, attach a 455 nm LED to the cage plate connected to the excitation filter.</li>
	<li>Obtain one 0.5 inch long, 1.0 inch diameter lens tube and a 1.0 inches achromatic doublet tube lens with focal length of 50 mm.</li>
	<li>Place the tube lens inside the lens tube such that the arrow on the outside of lens is facing the side of the lens tube with the external threads.</li>
	<li>Screw in the tube lens to the assembly.
	<ol>
		<li>Obtain one standard retaining ring.</li>
		<li>Secure the lens inside the 0.5 inch long, 1.0 inch diameter lens tube with the standard retaining ring.</li>
		<li>Attach the lens tube with the tube lens to the left-most cage plate.</li>
	</ol>
	</li>
	<li>Place a 30 mm cage plate in front of the 0.5 inch long, 1.0 inches diameter lens tube containing the tube lens.</li>
	<li>Attach a stress free retaining ring to the inside of the 30 mm cage plate.</li>
	<li>Attach a USB monochrome camera to the cage plate with the stress free retaining ring.</li>
	<li>Construct the optical post mounting devices.
	<ol>
		<li>Obtain four 0.5 inch post holders, four 0.5 inches optical posts, and four mounting bases.</li>
		<li>Secure the 0.5 inch optical posts inside the 0.5 inch post holders.</li>
		<li>Secure the 0.5 inch post holders onto the mounting bases.</li>
	</ol>
	</li>
	<li>Screw in the four optical post mounting devices to the screw holes located under the 30 mm cage cube, the right-angle mirror mount, the cage plate connected to the LED, and the cage plate connected to the camera.</li>
	<li>Screw in the four the optical post mounting devices to either an optical breadboard or optical table to finish construction of the high-resolution fluorescence microendoscopy modality.</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/54564/54564fig2.jpg" /> 
Figure 2: Assembly of the high-resolution fluorescence microendoscopy modality. The high-resolution fluorescence microendoscopy modality can be constructed by building a shell of 1.0 inch diameter-sized components, with special care taken in handling the dichroic mirror, objective lens, excitation/emission filters, and tube lens. Glass surfaces of these components must be carefully handled using lens paper. <a href="http://cloudfront.jove.com/files/ftp_upload/54564/54564fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Assembly of the Sub-diffuse Reflectance Spectroscopy Modality
Note: The outlined steps for assembly of the sub-diffuse reflectance spectroscopy modality can be visualized in Figure 3.
<ol>
	<li>Obtain a tungsten-halogen light source and, using epoxy or a strong adhesive, secure a 1.0 inch threaded 30 mm cage plate onto the front.</li>
	<li>Secure four 3.0 inch cage assembly rods to the cage plate.</li>
	<li>Attach a z-axis translation mount to the cage assembly rods.</li>
	<li>Screw in a 20X achromatic objective lens to the z-axis translation mount.</li>
	<li>Build a fiber adaptor plate/xy-axis translation lens mount assembly.
	<ol>
		<li>Obtain an xy-axis translation mount and a 1.0 inches fiber adaptor plate.</li>
		<li>Secure the fiber adaptor plate into the xy-axis translation lens mount.</li>
	</ol>
	</li>
	<li>Slide the 1.0 inch fiber adaptor/xy-translation mount assembly in front of the objective lens.</li>
	<li>Build the motor arm assembly.
	<ol>
		<li>Obtain the custom-built aluminum motor arm and one SMA fiber adaptor plate.</li>
		<li>Screw in the fiber adaptor plate (with external threading) into the aluminum motor arm (with internal threading).</li>
		<li>Attach the custom-built aluminum motor arm adaptor to the motor arm with four #4-40 0.5 in. screws.</li>
	</ol>
	</li>
	<li>Build the motor/motor arm/motor housing assembly.
	<ol>
		<li>Obtain the custom-built aluminum motor housing and the 400-step stepper motor.</li>
		<li>Line up the screw holes on the stepper motor and motor housing and then secure with four #4-40 0.5 inch screws.</li>
		<li>Feed the rotating motor rod of the stepper motor through the opening of the motor arm assembly and tighten the locking screw on the aluminum motor arm adaptor.</li>
	</ol>
	</li>
	<li>Build the optical switch assembly.
	<ol>
		<li>Obtain the custom-built aluminum optical switch and three 1.0 inch fiber adaptor plates.</li>
		<li>Thread the adaptor plates into the threaded holes in the optical switch.</li>
		<li>Attach the custom-built aluminum optical switch face-plate onto the optical switch with four #4-40 0.5 inch screws.</li>
	</ol>
	</li>
	<li>Attach the motor/motor arm/motor housing assembly to the optical switch by feeding the rotating motor rod of the stepper motor through the central hole of the optical switch.</li>
	<li>Obtain an electric circuit board and stepper motor driver, and then place the stepper motor driver across the central groove of the breadboard.</li>
	<li>Observe the electrical connection schematic (Figure 3, 2.12) for the stepper motor driver, 12 V power supply, and stepper motor.</li>
	<li>Connect the stepper motor driver, 12 V power supply, and stepper motor as specified in the circuit diagram (Figure 3, 2.12) to complete construction of the motorized optical switch.</li>
	<li>Screw in the optical switch components and tungsten-halogen light source to an optical breadboard or optical table near the previously constructed (Figure 2, 1.24) high-resolution fluorescence microendoscopy assembly.</li>
	<li>Attach one end of a 550 &#181;m, 0.22 NA patch cable to the 1.0 inch fiber adaptor plate of the motor arm assembly.</li>
	<li>Attach the other end of the 550 &#181;m, 0.22 NA patch cable to the fiber connector of the USB spectrometer.</li>
	<li>Screw in the five distal probe cables to the respective 1.0 inch fiber adaptor plates on the instrumentation to finish completion of the multimodal high-resolution imaging and sub-diffuse reflectance spectroscopy fiber-bundle microendoscope.
	<ol>
		<li>Screw in the central 1 mm diameter image fiber cable to the 1.0 inch fiber adaptor plate mentioned in step 1.9.2.</li>
		<li>Screw in the leftmost 200 &#181;m multimode fiber cable to the 1.0 inch fiber adaptor plate mentioned in step 2.6.</li>
		<li>Screw in the 2nd 200 &#181;m multimode fiber cable to the leftmost 1.0 inch fiber adaptor attached to the tungsten-halogen lamp mentioned in step 2.9.2.</li>
		<li>Screw in the 3rd 200 &#181;m multimode fiber cable to the middle 1.0 inch fiber adaptor plate mentioned in step 2.9.2.</li>
		<li>Screw in the 4th 200 &#181;m multimode fiber cable to the rightmost 1.0 inch fiber adaptor plate mentioned in step 2.9.2.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 3" src="/files/ftp_upload/54564/54564fig3.jpg" /> 
Figure 3: Assembly of the sub-diffuse reflectance spectroscopy modality. The sub-diffuse reflectance spectroscopy modality can be constructed using a basic tungsten-halogen lamp coupled to an objective lens to focus light through the 200 &#181;m multimode delivery fiber, and a spectrometer. Additionally, a custom-built motorized optical switch can be constructed within the lamp-fiber-spectrometer path to switch between each SDS. Investigators using multiple spectrometers to acquire from multiple SDSs can bypass the optical switch component. <a href="http://cloudfront.jove.com/files/ftp_upload/54564/54564fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Calibration of the Sub-diffuse Reflectance Spectroscopy Modality
Note: The following steps (section 3) must be completed prior to spectral data collection (section 4).
<ol>
	<li>Turn on all components of the instrumentation, including the 455 nm LED, broadband tungsten-halogen lamp, CMOS camera, USB spectrometer, stepper motor, and motor control board. Ensure the shutter on the tungsten-halogen lamp is open.</li>
	<li>Turn off all ambient light.</li>
	<li>Open the custom data acquisition software.</li>
	<li>Keep equipment running for 30 min for the lamp reach an appropriate temperature and for inherent noise from the spectrometer to stabilize.</li>
	<li>Place a 20% diffuse reflectance standard inside the bottom opening of the custom-built, 3D printed calibration standard device.</li>
	<li>Place the fiber-optic probe inside the leftmost slot of the custom, 3D printed fiber-holder, demonstrated in Figure 4. The leftmost slot fixes the perpendicular distance from the fiber-optic probe tip to the reflectance standard at 2.1 mm, which is the optimum distance in which the signal reaching the spectrometer is maximized for the first SDS of 374 &#181;m.</li>
	<li>Adjust the motorized optical switch to the left-most position such that the spectrometer is connected to the first SDS of 374 &#181;m.</li>
	<li>Set the integration time to 500 msec. This integration time must be chosen as to not saturate the spectrometer but maintain a practically low integration time.</li>
	<li>Acquire a spectrum, Rmax,374&#181;m, by clicking &#34;Acquire Spectrum&#34; in the software.</li>
	<li>Close the shutter on the tungsten-halogen lamp and record a spectrum, Rdark,374&#181;m, of background noise, by clicking &#34;Acquire Spectrum&#34; in the software. Once acquired, open the shutter once again.</li>
	<li>Place the fiber-optic probe inside the rightmost slot of the custom, 3D printed fiber-holder, demonstrated in Figure 4. The rightmost slot fixes the perpendicular distance from the fiber-optic probe tip to the reflectance standard at 3.9 mm, which is the optimum distance in which the signal reaching the spectrometer is maximized for the second SDS of 730 &#181;m.</li>
	<li>Adjust the motorized optical switch to the middle position such that the spectrometer is connected to the second SDS of 730 &#181;m.</li>
	<li>Acquire a spectrum, Rmax,730&#181;m, by clicking &#34;Acquire Spectrum&#34; in the software.</li>
	<li>Close the shutter on the tungsten-halogen lamp and record a spectrum, Rdark,730&#181;m, of background noise, by clicking &#34;Acquire Spectrum&#34; in the software.</li>
	<li>Open the shutter once again.</li>
</ol>
<img alt="Figure 4" src="/files/ftp_upload/54564/54564fig4.jpg" /> 
Figure 4: Calibration of the sub-diffuse reflectance spectroscopy modality. For pre-experimental calibration, the fiber-optic probe tip must be placed at different perpendicular distances from the 20% diffuse reflectance standard depending on the SDS. To consistently achieve these perpendicular distances across all experiments, a calibration standard device was designed (device cross section shown in (a)) to hold the probe at exact distances from the 20% diffuse reflectance standard. In this specific fiber-optic probe setup, light from the tungsten-halogen lamp is shown through the optical switch at source-detector separations of (b) 374 &#181;m and (c) 730 &#181;m (with motor and motor arm removed from the optical path for clarity). Distances of (d) 2.1 mm for the 374 &#181;m SDS, and (e) 3.9 mm for the 730 &#181;m SDS are required for calibration. <a href="http://cloudfront.jove.com/files/ftp_upload/54564/54564fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. In Vivo Data Acquisition and Optical Property Extraction from Human Skin
In this section, the multimodal microendoscope technique will be demonstrated on in vivo human skin.
<ol>
	<li>Open the custom data acquisition software and adjust the spectrometer integration time by clicking &#34;Integration Time&#34; and set it so that it is the same as during calibration, which was 500 msec in this case (step 3.8).</li>
	<li>Determine the area of skin in which to acquire data, which may differ on the application of the investigator. In this case, the thin skin of the forearm was chosen as a demonstration.</li>
	<li>If the skin area contains hair, remove the hair with a disposable sterile razor.</li>
	<li>Obtain a standard yellow highlighter, which contains pyranine ink, and lightly mark the chosen skin area.</li>
	<li>Turn on the 455 nm LED and close the shutter to the tungsten-halogen lamp.</li>
	<li>Place the probe in gentle contact with the skin.</li>
	<li>Move the probe around on the stained area of tissue to view a live high-resolution feed of apical keratinocyte architecture on the viewing window of the software.</li>
	<li>Select an appropriate exposure time and gain, 150 msec and 10 dB gain in this case, to avoid image saturation, by clicking &#34;Exposure Time&#34; and &#34;Gain&#34;, typing in the appropriate values, and then clicking &#34;Apply Settings&#34; in the software interface.</li>
	<li>Acquire an image by clicking &#34;Acquire Image&#34; in the software interface.</li>
	<li>While keeping the probe at the same image site, turn off the 455 nm LED and open the shutter to the tungsten-halogen lamp.</li>
	<li>Adjust the motorized optical switch to the left position such that the spectrometer is connected to the second SDS of 374 &#181;m.</li>
	<li>Acquire spectra, Rtissue,374&#181;m, by clicking &#34;Acquire Spectra&#34; in the software interface.</li>
	<li>Adjust the motorized optical switch to the middle position such that the spectrometer is connected to the second SDS of 730 &#181;m.</li>
	<li>Acquire spectra, Rtissue,730&#181;m, by clicking &#34;Acquire Spectra&#34; in the software interface.</li>
	<li>Open the custom post-processing software.</li>
	<li>Run the post-processing software by clicking &#34;Run&#34; and select the high-resolution fluorescence image, four calibration spectra, and the two in vivo spectra from the folder in which the data was saved when prompted by the software. 
	NOTE: The custom software obtains the true absolute reflectance (Rabs,374&#181;m and Rabs,730&#181;m) using the following equations. 
	<img alt="Equation 1" src="/files/ftp_upload/54564/54564eq1.jpg" /> 
	<img alt="Equation 2" src="/files/ftp_upload/54564/54564eq2.jpg" /> 
	The post-processing code, as previously described, calculates a fitted curve to the diffuse reflectance spectra (equations 1 and 2) and then determines tissue physiological parameters including ([Hb], [Mel], and SaO2).11,22,24</li>
</ol>

<ol>
	<li>Muldoon, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular-resolution+molecular+imaging+within+living+tissue+by+fiber+microendoscopy.">Subcellular-resolution molecular imaging within living tissue by fiber microendoscopy.</a> Opt Express. 15, 16413-16423 (2007).</li><li>Rajaram, N., Reichenberg, J. S., Migden, M. R., Nguyen, T. H., Tunnell, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pilot+clinical+study+for+quantitative+spectral+diagnosis+of+non-melanoma+skin+cancer.">Pilot clinical study for quantitative spectral diagnosis of non-melanoma skin cancer.</a> Lasers Surg Med. 42, 716-727 (2010).</li><li>Louie, J. S., Richards-Kortum, R., Anandasabapathy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+and+advancements+in+the+use+of+high-resolution+microendoscopy+for+detection+of+gastrointestinal+neoplasia.">Applications and advancements in the use of high-resolution microendoscopy for detection of gastrointestinal neoplasia.</a> Clin Gastroenterol Hepatol. 12, 1789-1792 (2014).</li><li>Chang, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+microendoscopy+for+classification+of+colorectal+polyps.">High resolution microendoscopy for classification of colorectal polyps.</a> Endoscopy. 45, 553-559 (2013).</li><li>Muldoon, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+imaging+of+oral+neoplasia+with+a+high-resolution+fiber-optic+microendoscope.">Noninvasive imaging of oral neoplasia with a high-resolution fiber-optic microendoscope.</a> Head Neck. 34, 305-312 (2011).</li><li>Muldoon, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+quantitative+image+analysis+criteria+for+the+high-resolution+microendoscopic+detection+of+neoplasia+in+Barrett's+esophagus.">Evaluation of quantitative image analysis criteria for the high-resolution microendoscopic detection of neoplasia in Barrett's esophagus.</a> J Biomed Opt. 15, 026027 (2010).</li><li>Prieto, S. P., Powless, A. J., Boice, J. W., Sharma, S. G., Muldoon, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proflavine+Hemisulfate+as+a+Fluorescent+Contrast+Agent+for+Point-of-Care+Cytology.">Proflavine Hemisulfate as a Fluorescent Contrast Agent for Point-of-Care Cytology.</a> PLoS One. 10, e0125598 (2015).</li><li>Parikh, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+diagnostic+accuracy+of+high+resolution+microendoscopy+in+differentiating+neoplastic+from+non-neoplastic+colorectal+polyps:+a+prospective+study.">In vivo diagnostic accuracy of high resolution microendoscopy in differentiating neoplastic from non-neoplastic colorectal polyps: a prospective study.</a> Am J Gastroenterol. 109, 68-75 (2014).</li><li>Shin, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+high-resolution+microendoscopic+images+for+diagnosis+of+esophageal+squamous+cell+carcinoma.">Quantitative analysis of high-resolution microendoscopic images for diagnosis of esophageal squamous cell carcinoma.</a> Clin Gastroenterol Hepatol. 13, 272-279 (2015).</li><li>Prieto, S. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Qualitative+and+quantitative+comparison+of+colonic+microendoscopy+image+features+to+histopathology.">Qualitative and quantitative comparison of colonic microendoscopy image features to histopathology.</a> Proc SPIE Int Soc Opt Eng. 9328, (2015).</li><li>Greening, G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiber-bundle+microendoscopy+with+sub-diffuse+reflectance+spectroscopy+and+intensity+mapping+for+multimodal+optical+biopsy+of+stratified+epithelium.">Fiber-bundle microendoscopy with sub-diffuse reflectance spectroscopy and intensity mapping for multimodal optical biopsy of stratified epithelium.</a> Biomed Opt Express. 6, 4934-4950 (2015).</li><li>Rajaram, N., Gopal, A., Zhang, X., Tunnell, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+validation+of+the+effects+of+microvasculature+pigment+packaging+on+in+vivo+diffuse+reflectance+spectroscopy.">Experimental validation of the effects of microvasculature pigment packaging on in vivo diffuse reflectance spectroscopy.</a> Lasers Surg Med. 42, 680-688 (2010).</li><li>Spliethoff, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+tumor+response+to+cisplatin+using+optical+spectroscopy.">Monitoring of tumor response to cisplatin using optical spectroscopy.</a> Transl Oncol. 7, 230-239 (2014).</li><li>Chang, V. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+physiology+of+the+precancerous+cervix+in+vivo+through+optical+spectroscopy.">Quantitative physiology of the precancerous cervix in vivo through optical spectroscopy.</a> Neoplasia. 11, 325-332 (2009).</li><li>Yu, B., Shah, A., Nagarajan, V. K., Ferris, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffuse+reflectance+spectroscopy+of+epithelial+tissue+with+a+smart+fiber-optic+probe.">Diffuse reflectance spectroscopy of epithelial tissue with a smart fiber-optic probe.</a> Biomed Opt Express. 5, 675-689 (2014).</li><li>Hennessy, R., Goth, W., Sharma, M., Markey, M. K., Tunnell, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+probe+geometry+and+optical+properties+on+the+sampling+depth+for+diffuse+reflectance+spectroscopy.">Effect of probe geometry and optical properties on the sampling depth for diffuse reflectance spectroscopy.</a> J Biomedical Opt. 19, 107002 (2014).</li><li>Ghassemi, P., Travis, T. E., Moffatt, L. T., Shupp, J. W., Ramella-Roman, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+polarized+multispectral+imaging+system+for+quantitative+assessment+of+hypertrophic+scars.">A polarized multispectral imaging system for quantitative assessment of hypertrophic scars.</a> Biomed Opt Express. 5, 3337-3354 (2014).</li><li>Vasefi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarization-sensitive+hyperspectral+imaging+in+vivo:+a+multimode+dermoscope+for+skin+analysis.">Polarization-sensitive hyperspectral imaging in vivo: a multimode dermoscope for skin analysis.</a> Sci Rep. 4, (2014).</li><li>Winkler, A. M., Rice, P. F. S., Drezek, R. A., Barton, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+tool+for+rapid+disease+mapping+using+optical+coherence+tomography+images+of+azoxymethane-treated+mouse+colon.">Quantitative tool for rapid disease mapping using optical coherence tomography images of azoxymethane-treated mouse colon.</a> J Biomedl Opt. 15, 041512 (2010).</li><li>Bish, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Handheld+Diffuse+Reflectance+Spectral+Imaging+(DRSi)+for+in-vivo+characterization+of+skin.">Handheld Diffuse Reflectance Spectral Imaging (DRSi) for in-vivo characterization of skin.</a> Biomed Opt Express. 5, 573-586 (2014).</li><li>Prahl, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Optical Absorption of Hemoglobin. , (1999).</li><li>Rajaram, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+validation+of+a+clinical+instrument+for+spectral+diagnosis+of+cutaneous+malignancy.">Design and validation of a clinical instrument for spectral diagnosis of cutaneous malignancy.</a> Appl Opt. 49, 142-152 (2010).</li><li>Hennessy, R., Markey, M. K., Tunnell, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+one-layer+assumption+on+diffuse+reflectance+spectroscopy+of+skin.">Impact of one-layer assumption on diffuse reflectance spectroscopy of skin.</a> J Biomed Opt. 20, 27001 (2015).</li><li>Rajaram, N., Nguyen, T. H., Tunnell, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lookup+table-based+inverse+model+for+determining+optical+properties+of+turbid+media.">Lookup table-based inverse model for determining optical properties of turbid media.</a> J Biomed Opt. 13, 050501 (2008).</li><li>Nichols, B. S., Rajaram, N., Tunnell, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+a+lookup+table-based+approach+for+measuring+tissue+optical+properties+with+diffuse+optical+spectroscopy.">Performance of a lookup table-based approach for measuring tissue optical properties with diffuse optical spectroscopy.</a> J Biomed Opt. 17, 057001 (2012).</li><li>Greening, G. J., James, H. M., Muldoon, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Optical Phantoms: Diffuse and Sub-diffuse Imaging and Spectroscopy Validation. , 1-37 (2015).</li><li>Karsten, A. E., Smit, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+and+verification+of+melanin+concentration+on+human+skin+type.">Modeling and verification of melanin concentration on human skin type.</a> Photochem Photobiol. 88, 469-474 (2012).</li><li>Glennie, D. L., Hayward, J. E., Farrell, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+changes+in+the+hemoglobin+concentration+of+skin+with+total+diffuse+reflectance+spectroscopy.">Modeling changes in the hemoglobin concentration of skin with total diffuse reflectance spectroscopy.</a> J Biomed Opt. 20, 035002 (2015).</li><li>Lim, L., Nichols, B., Rajaram, N., Tunnell, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probe+pressure+effects+on+human+skin+diffuse+reflectance+and+fluorescence+spectroscopy+measurements.">Probe pressure effects on human skin diffuse reflectance and fluorescence spectroscopy measurements.</a> J Biomed Opt. 16, 011012 (2011).</li></ol>

The authors declare that they have no competing financial interests.

The multimodal high-resolution imaging and sub-diffuse reflectance spectroscopy fiber-bundle microendoscope reported here can be optimized and used by investigators for a variety of applications including endoscopic or handheld use for human or animal studies. It thus provides a flexible method for visualizing in vivo apical tissue micro-architecture alongside measurements of hemoglobin concentration, melanin concentration, and tissue oxygen saturation from two different tissue depths. This article describes the...

Fiber-bundle microendoscopy techniques typically analyze in vivo tissue using either imaging techniques or a combination of spectroscopy techniques.1-3 One such imaging technique, high-resolution fluorescence microendoscopy, can image apical tissue micro-architecture with sub-cellular resolution in a small, microscale field-of-view, using a topical contrast agent such as proflavine, fluorescein, or pyranine ink.1,3-11 This imaging modality has shown promising clinical performance in qualitatively differentiating diseased and healthy epithelial tissue in real-time with low inter-observer variability.8 Occasionally, investigators will use high-resolution fluorescence microscopy data to extract quantitative features such as cell and nuclear size or gland area, but this remains a primarily qualitative technique targeted towards visualizing tissue morphology.1,3,8-10 On the other hand, spectroscopy techniques, such as diffuse reflectance spectroscopy, are targeted towards providing functional tissue information and have shown promising clinical performance in quantitatively identifying cancerous lesions in multiple organs.2,12-15
Therefore, there is a need for a device incorporating both types of modalities to potentially further reduce inter-observer variability, maintain real-time visualization of tissue micro-architecture, and provide a more complete analysis of tissue health. To accomplish this goal, a multimodal probe-based instrument was constructed that combines two modalities in a single fiber-optic probe: high-resolution fluorescence microendoscopy and sub-diffuse reflectance spectroscopy.11 This method co-registers qualitative high-resolution images of apical tissue morphology (structural properties) with quantitative spectral information (functional properties) from two distinct tissue depths including local hemoglobin concentration ([Hb]), melanin concentration ([Mel]), and oxygen saturation (SaO2).11,12,16 This specific sub-diffuse reflectance spectroscopy modality uses two source-detector separations (SDSs) to sample two unique tissue depths to provide a more comprehensive picture of tissue health by sampling down to the basement membrane and underlying tissue stroma.11
The fiber-probe consists of a central 1 mm-diameter image fiber with approximately 50,000 4.5 &#181;m diameter fiber elements, a cladding diameter of 1.1 mm and an overall coating diameter of 1.2 mm. The image fiber is surrounded by five 200 &#181;m diameter fibers with cladding diameters of 220 &#181;m. Each 200 &#181;m multimode fiber is located a center-to-center distance of 864 &#181;m away from the center of the image fiber. Each of the 200 &#181;m multimode fibers are 25&#176; apart. Using the leftmost 200 &#181;m multimode fiber as the &#34;source&#34; fiber, and the additional three 200 &#181;m multimode fibers as the &#34;collection&#34; fibers, this geometry necessarily creates three center-to-center SDSs of 374 &#181;m, 730 &#181;m, 1,051 &#181;m, and 1,323 &#181;m. The fiber tips are enclosed in a cylindrical metal casing that keeps the distances between fibers constant. The diameter of the cylindrical metal casing is 3 mm. The distal end (towards the fiber-optic probe tip) of the fiber-optic probe is 2 feet long. The probe then separates into the six respective individual fibers at the proximal end (towards the instrumentation) which is an additional 2 feet long, for a total length of 4 feet. Figure 1 shows a representation of the fiber-optic probe.
<img alt="Figure 1" src="/files/ftp_upload/54564/54564fig1.jpg" /> 
Figure 1: Fiber-optic probe design. The fiber-optic probe consists of one 1 mm-diameter image fiber and four 200 &#181;m multimode fibers. This figure shows representations of (a) the metal end cap which constrains the geometry of the fibers at the probe tip to yield SDSs of 374, 730, and 1,051 &#181;m with respect to the leftmost 200 &#181;m multimode fiber (Scale bar &#8776; 1 mm), (b) the fibers being constrained within the metal cap, showing the fiber cores, fiber cladding, and fiber coating (Scale bar &#8776; 1 mm), (c) the protective polyamide sheathing around fibers (Scale bar &#8776; 1 mm), (d) the finished distal tip of the probe, with the metal finger grip and single black cable containing all fibers (Scale bar &#8776; 4 mm), and (e) a picture of the distal tip of the probe (Scale bar &#8776; 4 mm). <a href="http://cloudflare.jove.com/files/ftp_upload/54564/54564fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
This multimodal instrumentation and associated technique is the first combination of these modalities within a single probe, although other combined structural/functional techniques do exist that combine different modalities. For example, hyperspectral imaging combines wide-field imaging with quantitative hemoglobin and melanin properties,17,18 and other techniques have been developed that combine optical coherence tomography (OCT) with analysis of tissue protein expression,19 to name a few. This article reports on a compact and easy-to-implement instrumentation setup that uses a general fiber-optic probe which can be optimized for various purposes including endoscopic use in the lower gastrointestinal tract and esophagus or as a handheld probe for use in the oral cavity and external skin placement.11,20
The hardware for this instrumentation requires both custom data acquisition and post-processing code to acquire diffuse reflectance spectra and then extract the resulting volume-averaged tissue physiological parameters including [Hb], [Mel], and SaO2. The custom data acquisition code was built to allow the simultaneous acquisition from a camera (for high-resolution fluorescence microscopy) and a spectrometer (for diffuse reflectance spectroscopy). Drivers are often available from the manufacturers&#39; websites to allow integration with a variety of programming languages. The custom post-processing code imports a priori absorption values of in vivo [Hb] and [Mel]21 and then utilizes a previously developed nonlinear optimization fitting process that creates a fitted curve of the spectra.22 The fitted curve is built by minimizing the &#967;2 value between itself and the raw spectra and determining the tissue physiological parameters ([Hb], [Mel], and SaO2) from the fitted curve and with the lowest &#967;2 value.22 The code can be modified to include absorption from other chromophores as well, such as the exogenous pyranine ink used here, so that target physiological parameters are unaffected.
Physiological indicators of tissue health, such as [Hb], [Mel], and SaO2, can be used as reports of tumor response to therapy or as indicators of local vascularization and angiogenesis.14,23 Including a high-resolution fluorescence microendoscopy modality helps guide probe placement and provides investigators with a more complete picture of the relationship between epithelial tissue structure and function. In this article, construction and application of the multimodal microendoscope is described.11

<table><tbody><tr><td>30 mm Cage Cube with Dichroic Filter Mount</td><td>Thorlabs, Inc.</td><td>CM1-DCH</td><td></td></tr><tr><td>470 nm Dichroic Mirror (Beam Splitter)</td><td>Chroma Corporation</td><td>T470lpxr</td><td></td></tr><tr><td>Cage Assembly Rod, 1.5&quot;, 4-Pack</td><td>Thorlabs, Inc.</td><td>ER1.5-P4</td><td></td></tr><tr><td>Cage Assembly Rod, 3.0&quot;, 4-Pack</td><td>Thorlabs, Inc.</td><td>ER3-P4</td><td></td></tr><tr><td>Cage Assembly Rod, 2.0&quot;, 4-Pack</td><td>Thorlabs, Inc.</td><td>ER2-P4</td><td></td></tr><tr><td>SM1-Threaded 30 mm Cage Plate</td><td>Thorlabs, Inc.</td><td>CP02</td><td></td></tr><tr><td>SM1 Series Stress-Free Retaining Ring</td><td>Thorlabs, Inc.</td><td>SM1PRR</td><td></td></tr><tr><td>SM1 Lens Tube, 1.00&quot; Thread Depth</td><td>Thorlabs, Inc.</td><td>SM1L10</td><td></td></tr><tr><td>Right-Angle Kinematic Mirror Mount</td><td>Thorlabs, Inc.</td><td>KCB1</td><td></td></tr><tr><td>1&quot; UV Enhanced Aluminum Mirror</td><td>Thorlabs, Inc.</td><td>PF10-03-F01</td><td></td></tr><tr><td>Z-Axis Translation Mount</td><td>Thorlabs, Inc.</td><td>SM1Z</td><td></td></tr><tr><td>10X Olympus Plan Achromatic Objective</td><td>Thorlabs, Inc.</td><td>RMS10X</td><td></td></tr><tr><td>XY Translating Lens Mount</td><td>Thorlabs, Inc.</td><td>CXY1</td><td></td></tr><tr><td>SMA Fiber Adapter Plate with SM1 Thread</td><td>Thorlabs, Inc.</td><td>SM1SMA</td><td></td></tr><tr><td>SM1 Lens Tube, 0.50&quot; Thread Depth</td><td>Thorlabs, Inc.</td><td>SM1L05</td><td></td></tr><tr><td>440/40 Bandpass Filter (Excitation)</td><td>Chroma Corporation</td><td>ET440/40x</td><td></td></tr><tr><td>525/36 Bandpass Filter (Emission)</td><td>Chroma Corporation</td><td>ET525/36m</td><td></td></tr><tr><td>Quick Set Epoxy</td><td>Loctite</td><td>1395391</td><td></td></tr><tr><td>455 nm LED Light Housing Kit - 3-Watt</td><td>LED Supply</td><td>ALK-LH-3W-KIT</td><td></td></tr><tr><td>1&quot; Achromatic Doublet, f = 50 mm</td><td>Thorlabs, Inc.</td><td>AC254-050-A</td><td></td></tr><tr><td>Flea 3 USB Monochrome Camera</td><td>Point Grey, Inc.</td><td>FL3-U3-32S2M-CS</td><td></td></tr><tr><td>0.5&quot; Post Holder, L = 1.5&quot;</td><td>Thorlabs, Inc.</td><td>PH1.5</td><td></td></tr><tr><td>0.5&quot; Optical Post, L = 4.0&quot;</td><td>Thorlabs, Inc.</td><td>TR4</td><td></td></tr><tr><td>Mounting Base, 1&quot; x 2.3&quot; x 3/8&quot;</td><td>Thorlabs, Inc.</td><td>BA1S</td><td></td></tr><tr><td>Long Lifetime Tungsten-Halogen Light Source (Vis-NIR)</td><td>Ocean Optics</td><td>HL-2000-LL</td><td></td></tr><tr><td>20X Olympus Plan Objective</td><td>Edmund Optics, Inc.</td><td>PLN20X</td><td></td></tr><tr><td>Custom-Built Aluminum Motor Arm</td><td>N/A</td><td>N/A</td><td>Custom designed and built</td></tr><tr><td>Custom-Built Aluminum Motor Arm Adaptor</td><td>N/A</td><td>N/A</td><td>Custom designed and built</td></tr><tr><td>Custom-Built Aluminum Motor Housing</td><td>N/A</td><td>N/A</td><td>Custom designed and built</td></tr><tr><td>Stepper Motor - 400 steps/revolution</td><td>SparkFun Electronics</td><td>ROB-10846</td><td>Multiple suppliers</td></tr><tr><td>Custom-Built Aluminum Optical Fiber Switch</td><td>N/A</td><td>N/A</td><td>Custom designed and built</td></tr><tr><td>Custom-Built Aluminum Optical Fiber Switch Face-Plate</td><td>N/A</td><td>N/A</td><td>Custom designed and built</td></tr><tr><td>Arduino Uno - R3</td><td>SparkFun Electronics</td><td>DEV-11021</td><td>Multiple suppliers</td></tr><tr><td>Electronic Breadboard - Self-Adhesive</td><td>SparkFun Electronics</td><td>PRT-12002</td><td>Multiple suppliers</td></tr><tr><td>EasyDriver - Stepper Motor Driver</td><td>Sparkfun Electronics</td><td>ROB-12779</td><td></td></tr><tr><td>12 V, 229 mA Power Supply</td><td>Phihong</td><td>PSM03A</td><td>Multiple suppliers</td></tr><tr><td>Enhanced Sensitivity USB Spectrometer (Vis-NIR)</td><td>Ocean Optics</td><td>USB2000+VIS-NIR-ES</td><td></td></tr><tr><td>550 &amp;micro;m, 0.22 NA, SMA-SMA Fiber Patch Cable</td><td>Thorlabs, Inc.</td><td>M37L01</td><td></td></tr><tr><td>Custom-Built Fiber-Optic Probe</td><td>Myriad Fiber Imaging</td><td>N/A</td><td></td></tr><tr><td>20% Spectralon Diffuse Reflectance Standard</td><td>Labsphere, Inc.</td><td>SRS-20-010</td><td></td></tr><tr><td>Standard Yellow Highlighter</td><td>Sharpie</td><td>25005</td><td>Multiple suppliers, proflavine or fluorescein can be substituted</td></tr></tbody></table>

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.

Following this protocol, the investigator will obtain an in-focus high-resolution image of the tissue site with the full field of view (Figure 5). Outlines of cells can be seen if stained with pyranine ink from a standard yellow highlighter, whereas individual cell nuclei can be seen if stained with a dye such as proflavine. Following spectral acquisition, the post-processing software uses a priori knowledge of in vivo hemoglobin concentration ([Hb]) and...

Watch this Scientific Journal Video about Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis at JoVE.com

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.

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 ...

multimodal-imaging-spectroscopy-fiber-bundle-microendoscopy-platform

Research

JoVE Journal

Bioengineering

7.8K Views.  University of Arkansas. 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.

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

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

Dual-mode Imaging of Cutaneous Tissue Oxygenation and Vascular Function

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health disorders such as chronic wounds. We report the development of a dual-mode imaging system for non-invasive and non-contact imaging of cutaneous tissue oxygenation and vascular function. The imaging system integrated an infrared camera, a CCD camera, a liquid crystal tunable filter and a high intensity fiber light source. A Labview interface was programmed for equipment control, synchronization, image acquisition, processing, and visualization. Multispectral images captured by the CCD camera were used to reconstruct the tissue oxygenation map. Dynamic thermographic images captured by the infrared camera were used to reconstruct the vascular function map. Cutaneous tissue oxygenation and vascular function images were co-registered through fiduciary markers. The performance characteristics of the dual-mode image system were tested in humans.

A dual-mode imaging system was developed for non-contact assessment of cutaneous tissue oxygenation and vascular function.

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health ...

Medicine

Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously attaining anatomical, functional and molecular contrast in deep optically opaque tissues. While enormous potential has been recently demonstrated in the application of optoacoustics for small animal research, vast efforts have also been undertaken in translating this imaging technology into clinical practice. We present here a newly developed optoacoustic tomography approach capable of delivering high resolution and spectrally enriched volumetric images of tissue morphology and function in real time. A detailed description of the experimental protocol for operating with the imaging system in both hand-held and stationary modes is provided and showcased for different potential scenarios involving functional and molecular studies in murine models and humans. The possibility for real time visualization in three dimensions along with the versatile handheld design of the imaging probe make the newly developed approach unique among the pantheon of imaging modalities used in today&#8217;s preclinical research and clinical practice.

We provide herein a detailed description of the experimental protocol for imaging with a newly developed hand-held optoacoustic (photoacoustic) system for three-dimensional functional and molecular imaging in real time. The demonstrated powerful performance and versatility may define new application areas of the optoacoustic technology in preclinical research and clinical practice.

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously ...

Three-Dimensional Ultrasonic Needle Tip Tracking with a Fiber-Optic Ultrasound Receiver

Ultrasound is frequently used for guiding minimally invasive procedures, but visualizing medical devices is often challenging with this imaging modality. When visualization is lost, the medical device can cause trauma to critical tissue structures. Here, a method to track the needle tip during ultrasound image-guided procedures is presented. This method involves the use of a fiber-optic ultrasound receiver that is affixed within the cannula of a medical needle to communicate ultrasonically with the external ultrasound probe. This custom probe comprises a central transducer element array and side element arrays. In addition to conventional two-dimensional (2D) B-mode ultrasound imaging provided by the central array, three-dimensional (3D) needle tip tracking is provided by the side arrays. For B-mode ultrasound imaging, a standard transmit-receive sequence with electronic beamforming is performed. For ultrasonic tracking, Golay-coded ultrasound transmissions from the 4 side arrays are received by the hydrophone sensor, and subsequently the received signals are decoded to identify the needle tip&#39;s spatial location with respect to the ultrasound imaging probe. As a preliminary validation of this method, insertions of the needle/hydrophone pair were performed in clinically realistic contexts. This novel ultrasound imaging/tracking method is compatible with current clinical workflow, and it provides reliable device tracking during in-plane and out-of-plane needle insertions.

Accurate and efficient visualization of invasive medical devices is extremely important in many ultrasound-guided minimally invasive procedures. Here, a method for localizing the spatial position of a needle tip relative to the ultrasound imaging probe is presented.

Ultrasound is frequently used for guiding minimally invasive procedures, but visualizing medical devices is often challenging with this imaging modality. ...

Engineering

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo, necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo imaging and may significantly impact on preclinical research in various fields.

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Small animal imaging techniques allow serial diagnostic examinations and therapeutic interventions in vivo. Recently, the scope of applications has significantly widened and currently includes assessment of colonic tumor development, wound healing and monitoring of inflammation. This protocol illustrates these diverse potential applications of murine endoscopy.

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

Using a VSFG Microscope for Multimodal, Rapid, Hyperspectral Chemical Analysis

Multimodal Nonlinear Hyperspectral Chemical Imaging Using Line-Scanning Vibrational Sum-Frequency Generation Microscopy

Vibrational sum-frequency generation (VSFG), a second-order nonlinear optical signal, has traditionally been used to study molecules at interfaces as a spectroscopy technique with a spatial resolution of ~100 &#181;m. However, the spectroscopy is not sensitive to the heterogeneity of a sample. To study mesoscopically heterogeneous samples, we, along with others, pushed the resolution limit of VSFG spectroscopy down to ~1 &#181;m level and constructed the VSFG microscope. This imaging technique not only can resolve sample morphologies through imaging, but also record a broadband VSFG spectrum at every pixel of the images. Being a second-order nonlinear optical technique, its selection rule enables the visualization of non-centrosymmetric or chiral self-assembled structures commonly found in biology, materials science, and bioengineering, among others. In this article, the audience will be guided through an inverted transmission design that allows for imaging unfixed samples. This work also showcases that VSFG microscopy can resolve chemical-specific geometric information of individual self-assembled sheets by combining it with a neural network function solver. Lastly, the images obtained under brightfield, SHG, and VSFG configurations of various samples briefly discuss the unique information revealed by VSFG imaging.

Author Spotlight: Unveiling the Potential of VSFG Microscopy in Studying Mesoscopically Heterogeneous Self-Assembled Structures 

A multimodal, rapid hyperspectral imaging framework was developed to obtain broadband vibrational sum-frequency generation (VSFG) images, along with brightfield, second harmonic generation (SHG) imaging modalities. Due to the infrared frequency being resonant with molecular vibrations, microscopic structural and mesoscopic morphology knowledge is revealed of symmetry-allowed samples.

Vibrational sum-frequency generation (VSFG), a second-order nonlinear optical signal, has traditionally been used to study molecules at interfaces as a ...

Chemistry

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and geometric&#160;deformation. Our understanding of the internal relationships between these events has been limited because we can neither &#39;see&#39; inside the muscle nor precisely track the spatio-temporal nature of excitation-contraction dynamics. To resolve these problems, we have constructed a device that combines a suite of imaging modalities. Specifically, it integrates a brightfield microscope to measure local changes of sarcomere length and tissue strain, a fluorescence microscope to visualize the Ca2+ transient, and an optical coherence tomograph to capture the tissue&#39;s geometric&#160;changes throughout the time-course of a cardiac cycle. We present here the imaging infrastructure and associated data collection framework. Data are collected from isolated rod-like tissue structures known as trabeculae carneae. In our instrument, a pair of position-controlled platinum hooks hold each end of an ex vivo muscle sample while it is continuously superfused with nutrient-rich saline solution. The hooks are under independent control, permitting real-time control of muscle length and force. Lengthwise translation enables the piecewise scanning of the sample, overcoming limitations associated with the relative size of the microscope imaging window (540 &#181;m by 540 &#181;m) and the length of a typical trabecula (&#62;2000 &#181;m). Platinum electrodes at either end of the muscle chamber stimulate the trabecula at a user-defined rate. We exploit the stimulation signal as a trigger for synchronizing the data from each imaging window to reconstruct the entire sample twitching under steady-state conditions. Applying image-processing techniques to these brightfield imaging data provides tissue displacement and sarcomere length maps. Such a collection of data, when incorporated into an experiment-modeling pipeline, will provide a deeper understanding of muscle contractile homogeneity and heterogeneity in physiology and pathophysiology.

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

This protocol presents a collection of sarcomere, calcium, and macroscopic geometric&#160;data from an actively contracting cardiac trabecula ex vivo. These simultaneous measurements are made possible by the integration of three imaging modalities.

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and ...

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 ...

Biomolecular Imaging of Cellular Uptake of Nanoparticles using Multimodal Nonlinear Optical Microscopy

Probing gold nanoparticles (AuNPs) in living systems is essential to reveal the interaction between AuNPs and biological tissues. Moreover, by integrating nonlinear optical signals such as stimulated Raman scattering (SRS), two-photon excited fluorescence (TPEF), and transient absorption (TA) into an imaging platform, it can be used to reveal biomolecular contrast of cellular structures and AuNPs in a multimodal manner. This article presents a multimodal nonlinear optical microscopy and applies it to perform chemically specific imaging of AuNPs in cancer cells. This imaging platform provides a novel approach for developing more efficient functionalized AuNPs and determining whether they are within vasculatures surrounding the tumor, pericellular, or cellular spaces.

This article presents the integration of a spectral-focusing module and a dual-output pulse laser, enabling rapid hyperspectral imaging of gold nanoparticles and cancer cells. This work aims to demonstrate the details of multimodal nonlinear optical techniques on a standard laser scanning microscope.

Probing gold nanoparticles (AuNPs) in living systems is essential to reveal the interaction between AuNPs and biological tissues. Moreover, by integrating ...

Multispectral Optoacoustic Tomography for Functional Imaging in Vascular Research

Microcirculatory impairment has been recognized in various disease processes, underlying this growing theme within vascular research. In recent years, the development of live imaging systems has set the (analytical) pace in both basic and clinical research, with the objective of creating new instruments capable of providing real-time, quantifiable endpoints with clinical interest and application. Near-infrared spectroscopy (NIRS), positron emission tomography (PET), computed tomography (CT), and magnetic resonance imaging (MRI) are available, among other techniques, but cost, image resolution, and reduced contrast are recognized as common challenges. Optoacoustic tomography (OT) offers a new perspective on vascular functional imaging, combining state-of-the-art optical absorption and spatial resolution capacities (from micrometer optical to millimeter acoustic resolution) with tissue depth. In this study, we tested the applicability of multispectral optoacoustic tomography (MSOT) for functional imaging. The system uses a tunable optical parametric oscillator (OPO) pumped by an Nd: YAG laser, providing excitation pulses sensed by a 3D probe at wavelengths from 680 nm to 980 nm. Images obtained from the human forearm were reconstructed through a specific algorithm (supplied within the manufacturer&#39;s software) based on the response of specific chromophores. Maximal Oxygenated Hemoglobin (Max HbO2) and Deoxygenated Hemoglobin (Max Hb), Total Hemoglobin (HbT), and mean Oxygen Saturation (mSO2) to vascular density (&#181;Vu), inter-unit average distances (&#950;Ad), and capillary blood volume (mm3) may be measured using this system. The applicability potential found with this OT system is relevant. Ongoing software developments will surely improve the utility of this imaging system.

The present protocol describes the acquisition of multispectral optoacoustic images of in vivo human skin vasculature. These include the quantification of hemoglobin and melanin, regarded as chromophores of interest for functional analysis.

Microcirculatory impairment has been recognized in various disease processes, underlying this growing theme within vascular research. In recent years, the ...

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

Samenvatting

Meer video's verkennen