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...

<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 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.

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

Research

JoVE Journal

Bioengineering

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.  ...

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 sectioning of complex systems. Confocal microscopy is routinely used, for example, to study specific cellular targets1, monitor dynamics in living cells2-4, and visualize the three dimensional evolution of entire organisms5,6. Extensions of confocal imaging systems, such as confocal microendoscopes, allow for high-resolution imaging in vivo7 and are currently being applied to disease imaging and diagnosis in clinical settings8,9.
Confocal microscopy provides three-dimensional resolution by creating so-called "optical sections" using straightforward geometrical optics. In a standard wide-field microscope, fluorescence generated from a sample is collected by an objective lens and relayed directly to a detector. While acceptable for imaging thin samples, thick samples become blurred by fluorescence generated above and below the objective focal plane. In contrast, confocal microscopy enables virtual, optical sectioning of samples, rejecting out-of-focus light to build high resolution three-dimensional representations of samples.
Confocal microscopes achieve this feat by using a confocal aperture in the detection beam path. The fluorescence collected from a sample by the objective is relayed back through the scanning mirrors and through the primary dichroic mirror, a mirror carefully selected to reflect shorter wavelengths such as the laser excitation beam while passing the longer, Stokes-shifted fluorescence emission. This long-wavelength fluorescence signal is then passed to a pair of lenses on either side of a pinhole that is positioned at a plane exactly conjugate with the focal plane of the objective lens. Photons collected from the focal volume of the object are collimated by the objective lens and are focused by the confocal lenses through the pinhole. Fluorescence generated above or below the focal plane will therefore not be collimated properly, and will not pass through the confocal pinhole1, creating an optical section in which only light from the microscope focus is visible. (Fig 1). Thus the pinhole effectively acts as a virtual aperture in the focal plane, confining the detected emission to only one limited spatial location.
Modern commercial confocal microscopes offer users fully automated operation, making formerly complex imaging procedures relatively straightforward and accessible. Despite the flexibility and power of these systems, commercial confocal microscopes are not well suited for all confocal imaging tasks, such as many in vivo imaging applications. Without the ability to create customized imaging systems to meet their needs, important experiments can remain out of reach to many scientists.
In this article, we provide a step-by-step method for the complete construction of a custom, video-rate confocal imaging system from basic components. The upright microscope will be constructed using a resonant galvanometric mirror to provide the fast scanning axis, while a standard speed resonant galvanometric mirror will scan the slow axis. To create a precise scanned beam in the objective lens focus, these mirrors will be positioned at the so-called telecentric planes using four relay lenses. Confocal detection will be accomplished using a standard, off-the-shelf photomultiplier tube (PMT), and the images will be captured and displayed using a Matrox framegrabber card and the included software.

The complete construction of a custom, real-time confocal scanning imaging system is described. This system, which can be readily used for video-rate microscopy and microendoscopy, allows for an array of imaging geometries and applications not accessible using standard commercial confocal systems, at a fraction of the cost.

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, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

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

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence 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 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

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 contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels.

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

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, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T1 and T2-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

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 magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

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 high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal 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

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 their natural environment, root hair morphology and function are difficult to study and often excluded from plant research. In recent years, microfluidic platforms have offered a way to visualize root systems at high resolution without disturbing the roots during transfer to an imaging system. The microfluidic platform presented here builds on previous plant-on-a-chip research by incorporating a two-layer device to confine the Arabidopsis thaliana main root to the same optical plane as the root hairs. This design enables the quantification of root hairs on a cellular and organelle level and also prevents z-axis drifting during the addition of experimental treatments. We describe how to store the devices in a contained and hydrated environment, without the need for fluidic pumps, while maintaining a gnotobiotic environment for the seedling. After the optical imaging experiment, the device may be disassembled and used as a substrate for atomic force or scanning electron microscopy while keeping fine root structures intact.

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

This article demonstrates how to culture Arabidopsis thaliana seedlings in a two-layer microfluidic platform that confines the main root and root hairs to a single optical plane. This platform can be used for real-time optical imaging of fine root morphology as well as for high-resolution imaging by other means.

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 ...