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Abstract

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Acknowledgements

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Bioengineering

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

Published: October 17th, 2016

DOI:

10.3791/54564

1Department of Biomedical Engineering, 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.

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.

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, investigator....

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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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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.......

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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.......

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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.

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

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