Name: multimodal imaging and spectroscopy fiber-bundle microendoscopy platform for non-invasive, in vivo tissue analysis
Uploaded: 2016-10-17T14:00:00
Description: Watch this Scientific Journal Video about Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis at JoVE.com

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 mod...

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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

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		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:373px;">0.5&#34; Post Holder, L = 1.5&#34;</td>
			<td style="width:175px;">Thorlabs, Inc.</td>
			<td style="width:171px;">PH1.5</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:373px;">0.5&#34; Optical Post, L = 4.0&#34;</td>
			<td style="width:175px;">Thorlabs, Inc.</td>
			<td style="width:171px;">TR4</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:373px;">Mounting Base, 1&#34; x 2.3&#34; x 3/8&#34;</td>
			<td style="width:175px;">Thorlabs, Inc.</td>
			<td style="width:171px;">BA1S</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:373px;">Long Lifetime Tungsten-Halogen Light Source (Vis-NIR)</td>
			<td style="width:175px;">Ocean Optics</td>
			<td style="width:171px;">HL-2000-LL</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:373px;">20X Olympus Plan Objective</td>
			<td style="width:175px;">Edmund Optics, Inc.</td>
			<td style="width:171px;">PLN20X</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:373px;">Custom-Built Aluminum Motor Arm</td>
			<td style="width:175px;">N/A</td>
			<td style="width:171px;">N/A</td>
			<td>Custom designed and built</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:373px;">Custom-Built Aluminum Motor Arm Adaptor</td>
			<td style="width:175px;">N/A</td>
			<td style="width:171px;">N/A</td>
			<td>Custom designed and built</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:373px;">Custom-Built Aluminum Motor Housing</td>
			<td style="width:175px;">N/A</td>
			<td style="width:171px;">N/A</td>
			<td>Custom designed and built</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:373px;">Stepper Motor - 400 steps/revolution</td>
			<td style="width:175px;">SparkFun Electronics</td>
			<td style="width:171px;">ROB-10846</td>
			<td>Multiple suppliers</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:373px;">Custom-Built Aluminum Optical Fiber Switch</td>
			<td style="width:175px;">N/A</td>
			<td style="width:171px;">N/A</td>
			<td>Custom designed and built</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:373px;">Custom-Built Aluminum Optical Fiber Switch Face-Plate</td>
			<td style="width:175px;">N/A</td>
			<td style="width:171px;">N/A</td>
			<td>Custom designed and built</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:373px;">Arduino Uno - R3</td>
			<td style="width:175px;">SparkFun Electronics</td>
			<td style="width:171px;">DEV-11021</td>
			<td>Multiple suppliers</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:373px;">Electronic Breadboard - Self-Adhesive</td>
			<td style="width:175px;">SparkFun Electronics</td>
			<td style="width:171px;">PRT-12002</td>
			<td>Multiple suppliers</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:373px;">EasyDriver - Stepper Motor Driver</td>
			<td style="width:175px;">Sparkfun Electronics</td>
			<td style="width:171px;">ROB-12779</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:373px;">12V, 229 mA Power Supply</td>
			<td style="width:175px;">Phihong</td>
			<td style="width:171px;">PSM03A</td>
			<td>Multiple suppliers</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:373px;">Enhanced Sensitivity USB Spectrometer (Vis-NIR)</td>
			<td style="width:175px;">Ocean Optics</td>
			<td style="width:171px;">USB2000+VIS-NIR-ES</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:373px;">550 &#181;m, 0.22 NA, SMA-SMA Fiber Patch Cable</td>
			<td style="width:175px;">Thorlabs, Inc.</td>
			<td style="width:171px;">M37L01</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:373px;">Custom-Built Fiber-Optic Probe</td>
			<td style="width:175px;">Myriad Fiber Imaging</td>
			<td style="width:171px;">N/A</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:373px;">20% Spectralon Diffuse Reflectance Standard</td>
			<td style="width:175px;">Labsphere, Inc.</td>
			<td style="width:171px;">SRS-20-010</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:373px;">Standard Yellow Highlighter</td>
			<td style="width:175px;">Sharpie</td>
			<td style="width:171px;">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 ...

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.

Research

JoVE Journal

Bioengineering

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

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.  ...

Video-rate Scanning Confocal Microscopy and Microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical ...

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Viral Nanoparticles for In vivo Tumor Imaging

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by ...