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 ...

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 ...