This work is funded by the Cure for Cancer Foundation, Dutch Technology Foundation (STW) and The Netherlands Organisation for Health Research and Development (ZonMw).

The presented procedure takes place under a research protocol approved by the Institutional Review Board of the Academic Medical Center Amsterdam, registration number NL41985.018. Written informed consent is required from all participants.
1. System
<ol>
	<li>For this experiment, use a Fourier domain OCT system, operating at a 1,280&#8211;1,350 nm wavelength band 13. Fourier domain low-coherence interferometry allows for continuous scanning which increases the data acquisition speed when compared to the first generation time-domain OCT systems. Note: The OCT system is interfaced with a fiber optic probe, scanning helically at ~90&#176; angle. It has an outer diameter of 2.7F (0.9 mm) and an insertable length of 135 cm. The probe connects to the OCT console through a drive motor and optical controller (mounting dock) with a pullback range of 54 mm. The acquired OCT datasets consist of 541 cross-sectional images (B-scans) with an axial resolution of 15 &#181;m (Figure 1, 2).</li>
	<li>To guarantee accurate and reproducible attenuation measurements, calibrate by measuring &#956;oct for increasing concentrations based on weight percentage of a fat emulsion, (e.g., Intralipid) as described previously by Kodach et al.14, 15. 
	In short:
	<ol>
		<li>Dilute a standard batch of 20% fat emulsion with demineralized H2O to achieve concentrations of 0.125, 0.250, 0.5, 1.0, 2.0, 4.0, 10, 15 and 20 (stock) percent.
		<ol>
			<li>Place the OCT probe in 200 ml of fat emulsion mixture and acquire an OCT measurement.</li>
			<li>Cross reference extracted &#956;oct values with known values in literature.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Time Out and Patient Positioning
<ol>
	<li>Prior to starting the procedure, perform a &#8220;time out&#8221; checking name, date of birth, procedure, procedural side, anticoagulant use, and allergies.</li>
	<li>Depending on the tumor location, place the patient in either prone or lateral decubitus position. Provide the patient with adequate support and verify if he/she expects to be comfortable in this position over a 20 to 40 min period.</li>
	<li>Using ultrasound (US)16, localize the tumor and mark the needle entry point on the skin with permanent ink. 
	NOTE: When using computed tomography (CT), use a flexible needle guidance template to localize the preferred position of the access needle.</li>
</ol>
3. Disinfection and Sterile Draping
<ol>
	<li>Put on a surgical cap and mouth cover.</li>
	<li>Clean the skin around the puncture site using a chlorhexidine/alcohol solution, taking care not to remove the previously placed needle entry mark (step 2.3). Disinfecting a wide area will prevent the necessity for additional cleaning in case of unexpected access needle repositioning.</li>
	<li>With regard of the sterile content, open the percutaneous puncture set containing: a 10 ml syringe, a blunt aspiration needle, a 21 G injection needle, a scalpel, a 15 G co-axial introducer needle, a 18 G trocar needle, and a 16 G core biopsy gun.</li>
	<li>Wash hands thoroughly, applying hand disinfectant afterwards. Put on a surgical gown and sterile gloves.</li>
	<li>Cover the patient in sterile drapes.</li>
	<li>Apply a sterile cover around the ultrasound probe and fix the needle guide in place.</li>
</ol>
4. OCT Preparation
<ol>
	<li>Start the OCT console and enter the patient details in the fields labeled patient ID, last name, first name and DOB (date of birth) using the console interface.</li>
	<li>With regard of the sterile content, unpack the OCT package containing an OCT probe, a sterile mounting dock cover, and a 5 ml luer-lock syringe.</li>
	<li>Apply the sterile cover to the OCT console mounting dock. Guiding the non-sterile mounting dock requires the help of an assistant.</li>
	<li>Fill the 5 ml syringe with 0.9% NaCl and attach it to the flushing port. Flush the OCT probe until water appears in the distal part of the probe cover.</li>
	<li>Load the OCT probe into the mounting dock. After loading the probe will rotate and emit red light confirming proper functioning. Leave the probe in its protective cover during flushing and loading to minimize the risk of damage.</li>
	<li>Remove the OCT probe from its cover. Place the probe on a hard surface and use a scalpel to shorten the tip. Fix the distal part of the probe during cutting in order to minimize pressure on the optical fiber and prism. Cut 5 mm distal from prism, using the emitted (red) light for orientation.</li>
</ol>
5. Puncture
<ol>
	<li>Anesthetize the skin and deep layers using 2% lidocaine (20 mg/ml). Wait several minutes allowing for the lidocaine to take effect. Ask the patient if there is any pain.</li>
	<li>Using the needle guide, place the 15 G co-axial introducer needle verifying the position through imaging. If placement is satisfactory, remove the obturator (sharp needle core).</li>
	<li>Place the 18 G trocar needle through the introducer needle, piercing the tumor. Again verify the position of the needle with imaging. If placement is satisfactory remove the obturator.</li>
	<li>Feed the OCT probe up the trocar needle until feeling resistance.</li>
	<li>While fixing the OCT probe, retract the trocar needle, exposing the OCT probe to the tumor tissue. Keeping the tip of the trocar needle within the tumor minimizes kinking of the OCT probe during breathing cycles. This lowers the risk of probe damage.</li>
	<li>OCT Scan:
	<ol>
		<li>Perform an OCT scan, with the console set at 541 B-scans per dataset. The OCT system used here will perform an automated pullback over a length of 5.4 cm requiring no specific parameter adjustments.</li>
		<li>Check the scan for quality, artefacts and the appearance of solid tissue (Figure 1A). Artefacts most commonly appear as circular bands standing out from the normal OCT pattern (Figure 1B).</li>
		<li>Replace the probe if artefacts persists after rescanning.</li>
	</ol>
	</li>
	<li>Repeat step 5.6 until a minimum of 3 OCT datasets are acquired.</li>
	<li>Remove the OCT probe and trocar needle, leaving the introducer needle in-place.</li>
	<li>Arm the core biopsy gun and place it through the introducer needle, verifying the position on imaging.</li>
	<li>If the positioning is satisfactory, fire the biopsy gun.</li>
	<li>Place biopsy material in container according to pathology department protocol. Here, place biopsies on a petri dish with a paper inlay, sufficiently saturated with 0.9% NaCl.</li>
	<li>Check the core biopsy quality and repeat step 5.9 and 5.10 until sufficient material is obtained.</li>
</ol>

<ol>
	<li>Jemal, A., Siegel, R., Xu, J., Ward, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics,+2010.">Cancer statistics, 2010.</a> CA Cancer J. Clin. 60, 277-300 (2010).</li><li>Mathew, A., Devesa, S. S., Fraumeni, J. F., Chow, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+increases+in+kidney+cancer+incidence,+1973-1992.">Global increases in kidney cancer incidence, 1973-1992.</a> Eur. J. Cancer Prev. 11, 171-178 (2002).</li><li>Volpe, A., 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=Contemporary+management+of+small+renal+masses.">Contemporary management of small renal masses.</a> Eur. Urol. 60, 501-515 (2011).</li><li>Ljungberg, B., 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=EAU+guidelines+on+renal+cell+carcinoma:+the+2010+update.">EAU guidelines on renal cell carcinoma: the 2010 update.</a> Eur. Urol. 58, 398-406 (2010).</li><li>Donat, S. M., 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=Follow-up+for+Clinically+Localized+Renal+Neoplasms.">Follow-up for Clinically Localized Renal Neoplasms.</a> AUA Guideline, J. Urol. 190, 407-416 (2013).</li><li>Faber, D. J., van der Meer, F. J., Aalders, M. C. G., van Leeuwen, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+measurement+of+attenuation+coefficients+of+weakly+scattering+media+using+optical+coherence+tomography.">Quantitative measurement of attenuation coefficients of weakly scattering media using optical coherence tomography.</a> Optics Express. 12, 4353-4365 (2004).</li><li>Xie, T. Q., Zeidel, M. L., Pan, Y. 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Int. 110, E415-E420 (2012).</li><li>Barwari, K., 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=Advanced+diagnostics+in+renal+mass+using+optical+coherence+tomography:+a+preliminary+report.">Advanced diagnostics in renal mass using optical coherence tomography: a preliminary report.</a> J. Endourol. 25, 311-315 (2011).</li><li>Cauberg, E. C., 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+measurement+of+attenuation+coefficients+of+bladder+biopsies+using+optical+coherence+tomography+for+grading+urothelial+carcinoma+of+the+bladder.">Quantitative measurement of attenuation coefficients of bladder biopsies using optical coherence tomography for grading urothelial carcinoma of the bladder.</a> J. Biomed. Opt. 15, 066013 (2010).</li><li>Bus, M. 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=Volumetric+in+vivo+visualization+of+upper+urinary+tract+tumors+using+optical+coherence+tomography:+a+pilot+study.">Volumetric in vivo visualization of upper urinary tract tumors using optical coherence tomography: a pilot study.</a> J. Urol. 190, 2236-2242 (2013).</li><li>Wessels, R., 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=Optical+coherence+tomography+in+vulvar+intraepithelial+neoplasia.">Optical coherence tomography in vulvar intraepithelial neoplasia.</a> Journal of Biomedical Optics. 17, (2012).</li><li>Yun, S. H., Tearney, G. J., de Boer, J. F., Iftimia, N., Bouma, B. 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The authors of this article have nothing to disclose.

In this publication we report on the feasibility of percutaneous, needle based, OCT of the kidney. This is an essential first step in the development of OCT into a clinically applicable technique for tumor differentiation, termed as an &#8220;Optical Biopsy&#8221;. Our first 25 patients have shown percutaneous OCT to be an easy and safe procedure. An optical biopsy has two advantages over conventional core biopsies. First, the real time acquisition and analysis of OCT data will provide instant diagnostic results, compare...

The past decades have shown a steady increase in the incidence of renal masses 1,2. Until now, renal mass treatment decisions have been made primarily on the basis of MRI and CT imaging characteristics, age and comorbidity. However these diagnostic methods and clinical parameters lack the finesse to truly detect the malignant potential of a renal mass. A core biopsy or fine needle aspiration with sufficient tissue for pathological evaluation (diagnostic) provides objective tumor differentiation with both sensitivity and specificity in the range of 95-100% 3. Therefore biopsy is gaining acceptance in the evaluation of suspicious renal masses 4,5. However, biopsies without sufficient tissue to establish a diagnosis or with normal renal parenchyma (non-diagnostic) occur at a rate of 10-20% overall, and even up to 30% in small renal masses (&#60;4 cm, SRMs), delaying the diagnostic process due to the frequent necessity for additional biopsy procedures3,5.
Optical coherence tomography (OCT) is a novel imaging modality that has the potential to overcome the aforementioned hurdles in renal mass differentiation. Based on the backscattering of near infrared light, OCT provides images with a 15 &#181;m axial resolution at an effective tissue penetration of 2-3 mm (Figure 1, 2). The loss of signal intensity per millimeter of tissue penetration, a resultant of tissue-specific light scattering, is expressed as the attenuation coefficient (&#181;OCT: mm-1) as described by Faber et al. 6. Histological characteristics can be correlated to &#181;OCT values providing a quantitative parameter for tissue differentiation (Figure 3).
During carcinogenesis, malignant cells display an increased number, larger and more irregularly shaped nuclei with a higher refractive index and more active mitochondria. Due to this overexpression of cell components, a change in &#956;oct is to be expected when comparing malignant tumors to benign tumors or unaffected tissue 7.
Recently we studied the ability of superficial OCT to differentiate between benign and malignant renal masses 8,9. In 16 patients, intra-operative OCT measurements of tumor tissue were obtained using an externally placed OCT probe. The control arm comprised of OCT measurements of unaffected tissue in the same patients. Normal tissue showed a significantly lower median attenuation coefficient compared to malignant tissue, confirming the potential of OCT for tumor differentiation. This quantitative analysis has been applied in a similar fashion to grade other types of malignant tissue, such as urothelial carcinoma 10,11 and vulvar epithelial neoplasia differentiation 12.
We aim to develop OCT into an optical biopsy, providing real-time imaging combined with on-the-spot tumor differentiation. The goal of the current study is to describe a percutaneous, needle based, OCT approach in patients diagnosed with a solid enhancing renal mass. This method description is, to our knowledge, the first to assess the possibility of needle based OCT of renal tumors.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>15G / 7.5cm Co-Axial Introducer Needle</td>
			<td>Angiotech, Gainesville, USA</td>
			<td>MCXS1612SX</td>
			<td></td>
		</tr>
		<tr>
			<td>18G / 20cm Trocar Needle</td>
			<td>Cook medical, Bloomington, USA</td>
			<td>DTN-18-20.0-U</td>
			<td></td>
		</tr>
		<tr>
			<td>16G / 20cm Quick-Core Biopsy Gun</td>
			<td>Cook Medical, Bloomington, USA</td>
			<td>G07827</td>
			<td></td>
		</tr>
		<tr>
			<td>Ilumien Optis PCI Optimization System (OCT &#38; FFR)</td>
			<td>St. Jude medical, St. Paul, USA</td>
			<td>C408650</td>
			<td>Part of Dragonfly Kit. 
			St. Jude medical, St. Paul, USA. (C4088643)</td>
		</tr>
		<tr>
			<td>Dragonfly Duo Imaging Catheter</td>
			<td>LightLab Imaging, Westford, USA</td>
			<td>C408644</td>
			<td>Part of Dragonfly Kit. 
			St. Jude medical, St. Paul, USA. (C4088643)</td>
		</tr>
		<tr>
			<td>Sterile Dock Cover</td>
			<td>CFI Med. Solutions, Fenton, USA</td>
			<td>200-700-00</td>
			<td>Part of Dragonfly Kit. 
			St. Jude medical, St. Paul, USA. (C4088643)</td>
		</tr>
		<tr>
			<td>5ml Luer-lock Syringe</td>
			<td>Merit Med. Syst., South Jordan, USA</td>
			<td>C408647</td>
			<td></td>
		</tr>
		<tr>
			<td>10ml Syringe</td>
			<td>BD, Franklin Lakes, USA</td>
			<td>300912</td>
			<td></td>
		</tr>
		<tr>
			<td>18G Blunt Fill Needle</td>
			<td>BD, Franklin Lakes, USA</td>
			<td>305180</td>
			<td></td>
		</tr>
		<tr>
			<td>21G Injection Needle</td>
			<td>BD, Franklin Lakes, USA</td>
			<td>301155</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile scalpel</td>
			<td>BD, Franklin Lakes, USA</td>
			<td>372611</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl 0,9% solution</td>
			<td>Braun, Melsungen AG, Germany</td>
			<td>222434</td>
			<td></td>
		</tr>
		<tr>
			<td>Lidoca&#239;ne HCl 2% (20mg/ml) solution</td>
			<td>Braun, Melsungen AG, Germany</td>
			<td>3624480</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Ultrasound Gel, Aquasonic 100</td>
			<td>Parker Lab. Inc., Fairfield, USA</td>
			<td>GE424609</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Ultrasound Cover</td>
			<td>Microtek Med., Alpharetta, USA</td>
			<td>PC1289EU</td>
			<td></td>
		</tr>
		<tr>
			<td>Pathology Container</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AMIRA software package</td>
			<td colspan="2">FEI Visualization Sciences Group, Hillsboro, USA</td>
			<td>Software platform for 3D data analysis</td>
		</tr>
		<tr>
			<td>FIJI software package (open source)</td>
			<td>Open source, http://fiji.sc/Fiji</td>
			<td></td>
			<td>Open source image processing software</td>
		</tr>
	</tbody>
</table>

in vivo, percutaneous, needle based, optical coherence tomography of renal masses

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real time images with a 15 &#181;m axial resolution at an effective tissue penetration of 2-3 mm. Within the OCT images the loss of signal intensity per millimeter of tissue penetration, the attenuation coefficient, is calculated. The attenuation coefficient is a tissue specific property, providing a quantitative parameter for tissue differentiation. 
Until now, renal mass treatment decisions have been made primarily on the basis of MRI and CT imaging characteristics, age and comorbidity. However these parameters and diagnostic methods lack the finesse to truly detect the malignant potential of a renal mass. A successful core biopsy or fine needle aspiration provides objective tumor differentiation with both sensitivity and specificity in the range of 95-100%. However, a non-diagnostic rate of 10-20% overall, and even up to 30% in SRMs, is to be expected, delaying the diagnostic process due to the frequent necessity for additional biopsy procedures. 
We aim to develop OCT into an optical biopsy, providing real-time imaging combined with on-the-spot tumor differentiation. This publication provides a detailed step-by-step approach for percutaneous, needle based, OCT of renal masses.

Among the first 25 tumors (23 patients), a total of 24 successful OCT procedures were performed. In one case a probe malfunction led to the inability to acquire an OCT scan. Two adverse events (AE) occurred, which are described in detail in the discussion section. General patient characteristics are found in Table 1.
The OCT console has pre-installed software providing real-time OCT images for immediate qualitative analysis of acquired datasets. For further analysis and attenu...

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In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Optical coherence tomography (OCT) is a high resolution imaging technique that allows analysis of tissue specific optical properties providing the means for tissue differentiation. We developed needle based OCT, providing real-time imaging combined with on-the-spot tumor differentiation. This publication describes a method for percutaneous, needle based OCT of renal masses.

In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real ...

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8.8K Aufrufe.  Academic Medical Center. Optical coherence tomography (OCT) is a high resolution imaging technique that allows analysis of tissue specific optical properties providing the means for tissue differentiation. We developed needle based OCT, providing real-time imaging combined with on-the-spot tumor differentiation. This publication describes a method for percutaneous, needle based OCT of renal masses.

Serie: In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Doppler Optical Coherence Tomography of Retinal Circulation

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double circular scan (CDCS) that scans around the optic nerve head at 3.40 mm and 3.75 mm diameters. The double concentric circles are performed 6 times consecutively over 2 sec. The CDCS scan is saved with Doppler shift information from which flow can be calculated. The standard clinical protocol calls for 3 CDCS scans made with the OCT beam passing through the superonasal edge of the pupil and 3 CDCS scan through the inferonal pupil. This double-angle protocol ensures that acceptable Doppler angle is obtained on each retinal branch vessel in at least 1 scan. The CDCS scan data, a 3-dimensional volumetric OCT scan of the optic disc scan, and a color photograph of the optic disc are used together to obtain retinal blood flow measurement on an eye. We have developed a blood flow measurement software called "Doppler optical coherence tomography of retinal circulation" (DOCTORC). This semi-automated software is used to measure total retinal blood flow, vessel cross section area, and average blood velocity. The flow of each vessel is calculated from the Doppler shift in the vessel cross-sectional area and the Doppler angle between the vessel and the OCT beam. Total retinal blood flow measurement is summed from the veins around the optic disc. The results obtained at our Doppler OCT reading center showed good reproducibility between graders and methods (&lt;10%). Total retinal blood flow could be useful in the management of glaucoma, other retinal diseases, and retinal diseases. In glaucoma patients, OCT retinal blood flow measurement was highly correlated with visual field loss (R2&gt;0.57 with visual field pattern deviation). Doppler OCT is a new method to perform rapid, noncontact, and repeatable measurement of total retinal blood flow using widely available Fourier-domain OCT instrumentation. This new technology may improve the practicality of making these measurements in clinical studies and routine clinical practice.

Total retinal blood flow is measured by Doppler optical coherence tomography and semi-automated grading software.

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double ...

Near Infrared Optical Projection Tomography for Assessments of &beta;-cell Mass Distribution in Diabetes Research

By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of pancreatic tissue, such as the rat pancreas, and to increase the number of channels (cell types) that may be studied in a single specimen. We further describe the implementation of a number of computational tools that provide: 1/ accurate positioning of a specimen's (in our case the pancreas) centre of mass (COM) at the axis of rotation (AR)2; 2/ improved algorithms for post-alignment tuning which prevents geometric distortions during the tomographic reconstruction2 and 3/ a protocol for intensity equalization to increase signal to noise ratios in OPT-based BCM determinations3. In addition, we describe a sample holder that minimizes the risk for unintentional movements of the specimen during image acquisition. Together, these protocols enable assessments of BCM distribution and other features, to be performed throughout the volume of intact pancreata or other organs (e.g. in studies of islet transplantation), with a resolution down to the level of individual islets of Langerhans.

Near Infrared Optical Projection Tomography for Assessments of &amp;#946;-cell Mass Distribution in Diabetes Research

We describe the adaptation of optical projection tomography (OPT)1 to imaging in the near infrared spectrum, and the implementation of a number of computational tools. These protocols enable assessments of pancreatic &beta;-cell mass (BCM) in larger specimens, increase the multichannel capacity of the technique and increase the quality of OPT data.

By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Transcutaneous Assessment of Renal Function in Conscious Rodents

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and time-consuming. In addition, serial blood or urine samples are required, with the associated stress for the experimental animals. A recent technique significantly reduces the investment in time and resources, minimizing the invasiveness and the animal stress, but being equally valid as the traditional approaches. The method measures transcutaneously renal function. Using an optical device and the exogenous renal marker fluorescein isothiocyanate (FITC)-sinistrin, this technique is capable of measuring the elimination kinetics of the marker through the skin. With neither blood nor urine samples nor the associated laboratory assays needed, the results of the transcutaneous measurement are almost instantaneously available. The method has been already validated in different species and successfully applied in several models of renal pathology. Moreover, due to its minimally invasive characteristics, it is suitable for sequential measurements within the same animal. Here is provided a detailed protocol to carry out the transcutaneous assessment of renal function in rodents.

Determination of glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional procedures to measure this parameter are cumbersome and require a large investment of time. Here we describe a faster and minimally invasive method to determinate GFR transcutaneously.

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression. Pulled glass microneedles were used to deliver sub retinal injections of adeno-associated virus (AAV) or nanoparticles (NP) via a trans-scleral and trans-choroidal approach. Careful positioning of the needle into the subretinal space was required prior to a calibrated pressure injection, which delivers fluid into the sub retinal space. Real time subretinal surgery was conducted on our retinal imaging system (RIS). HR-SD-OCT demonstrated progressive uniform retinal degeneration due to expression of a toxic mutant human mutant rhodopsin (P347S) (RHOP347S) transgene in mice. HR-SD-OCT allows rigorous quantification of all the retinal layers. Outer nuclear layer (ONL) thickness and photoreceptor outer segment length (OSL) measurements correlate with photoreceptor vitality, degeneration, or rescue. The RIS delivery system allows real-time visualization of subretinal injections in neonatal (~P10-14) or adult mice, and HR-SD-OCT immediately determines success of delivery and maps areal extent. HR-SD-OCT is a powerful tool that can evaluate the success of subretinal surgery in mice, in addition to measuring vitality of photoreceptors in vivo. HR-SD-OCT can also be used to identify uniform animal cohorts to evaluate the extent of retinal degeneration, toxicity, and therapeutic rescue in preclinical gene therapy research studies.

Here we demonstrate a novel approach to using high resolution spectral-domain optical coherence tomography (HR-SD-OCT) to assist delivery of gene therapy agents into the subretinal space, assess its areal coverage, and characterize photoreceptor vitality.

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the ...

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 &#181;m and 6 &#181;m, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies7, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate8.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...