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>
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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. 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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, compared to the 5-10 days of processing time of conventional pathology. Second, OCT has the potential to reduce the amount of non-diagnostic procedures, which is 20% for conventional biopsies. When an OCT scan reveals perirenal fat or unaffected renal tissue (non-diagnostic results) the OCT operator can reposition the OCT probe to successfully target the tumor.
Two adverse events (AE) occurred among the first 25 patients. The first AE was post procedural hypotension, in a patient with known episodes of hypotension, which resolved after rest and 0.9% NaCl infusion.
In the second AE a fragment of the OCT probe tip sheared of. Requesting the patient to hold his breath during measurements prompted deep inspiration. Excessive kidney movement caused the OCT probe to kink and subsequently shear of on the edge of the trocar needle. A probe fragment of 1-2 mm remained in situ, yet caused no problems or discomfort. This AE took place during the OCT procedure of patient number 10. In the following patients the tip of the trocar needle was kept within the tumor (protocol step 5.5) minimizing kinking of the OCT probe, on the edge of the trocar needle, during breathing cycles. This modification of the OCT procedure has shown substantially less stress on the OCT probe. However, further prospective evaluation is necessary.
The OCT probe used in this study is designed for intravascular imaging of the coronary arteries. The possibility of automated pullback scanning combined with the 2.7 F (0.9 mm) diameter makes this probe suitable for needle based OCT of renal tumors. However, the delicate nature of the optical fiber and the prism fused to the distal tip make the probe susceptible to damage. In 3 cases, probe manipulation during the procedure caused probe failure, in 1 case before an OCT dataset could be acquired. Microscopic inspection of the prism showed no abnormalities, making a break in the optical fiber the most probable cause of failure.
Quantitative data analysis requires derivation of tissue specific attenuation cut-off values. This provides the means for objective tissue differentiation. We hypothesize that OCT is able to differentiate between benign and malignant lesions and subsequently between the 3 main malignant subgroups of renal cell carcinoma. Currently, attenuation values are calculated manually from selected areas of interest, which is a time consuming process. We have developed software for automated attenuation calculation. This reduces the inter- and intra-observer variability on ROI selection, speeds up the analysis process and increases the number of measurements per dataset. The integration of this software for instant attenuation coefficient calculation into the OCT console is a necessary future step in the development of a fully functional and clinically applicable optical biopsy technique.
In addition, a qualitative analysis protocol is necessary. Intra-procedural recognition of features of unaffected tissue (i.e. recognition of perirenal fat) could prompt OCT probe repositioning, reducing the number of non-diagnostic procedures. Furthermore, qualitative analysis is needed to select a ROI for attenuation coefficient calculation. Currently, we are developing a protocol consisting of predefined visual aspects to be scored. When sufficient datasets are acquired, blinded observers will validate this protocol.
The success of renal mass treatment strategies relies on accurate demarcation and profile determination, using smart treatment planning protocols and real time identification (subtyping and grading) and follow-up of the lesion. Both follow-up strategies and real-time identification of a lesion are unmet challenges using the current diagnostic techniques. OCT in the form of an optical biopsy has the potential to fulfil these requirements, providing minimal invasive analysis of carcinogenesis related change of optical properties and changes of layered tissue architecture i.e. visual detection of the structures in the OCT image.

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><tbody><tr><td>15 G/7.5 cm Co-Axial Introducer Needle</td><td>Angiotech, Gainesville, USA</td><td>MCXS1612SX</td><td></td></tr><tr><td>18 G/20 cm Trocar Needle</td><td>Cook medical, Bloomington, USA</td><td>DTN-18-20.0-U</td><td></td></tr><tr><td>16 G/20 cm 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 &amp;amp; FFR)</td><td>St. Jude medical, St. Paul, USA</td><td>C408650</td><td>Part of Dragonfly Kit.&lt;br/&gt; 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.&lt;br/&gt; 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.&lt;br/&gt; St. Jude medical, St. Paul, USA. (C4088643)</td></tr><tr><td>5 ml Luer-lock Syringe</td><td>Merit Med. Syst., South Jordan, USA</td><td>C408647</td><td></td></tr><tr><td>10 ml Syringe</td><td>BD, Franklin Lakes, USA</td><td>300912</td><td></td></tr><tr><td>18 G Blunt Fill Needle</td><td>BD, Franklin Lakes, USA</td><td>305180</td><td></td></tr><tr><td>21 G 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&amp;iuml;ne HCl 2% (20 mg/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>FEI Visualization Sciences Group, Hillsboro, USA</td><td>Software platform for 3D data analysis</td><td></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 attenuation measurements, the OCT data can be exported as raw data, TIFF, DICOM or AVI format. Quantitative analysis of &#181;oct of the OCT data is done using in-house developed software.
Using planimetric software, a 3D volume is rendered from the RAW data (Figure 2A). This provides a 3D overview of the scanned trajectory with the possibility of orthoslicing along 3 axes. The displayed dataset in Figure 2 shows good quality over the full pullback length. A clear visual distinction can be made between solid tissue (Figure 2B-C), perirenal fat tissue (Figure 2D) and the inside of the trocar needle. Exported TIFF files are loaded into an ImageJ based software package to be viewed in 2D by scrolling through the stacked B-scans. Combining the 2D and 3D visualization of the OCT dataset a region of interest (ROI) is selected.
Within the ROI equally spaced B-scans are selected (Figure 2, 3). Within the respective B-scans the attenuation coefficient is determined along a straight line radiating outward from the heart of the probe (Figure 3A, D). The ImageJ based software package has the option of plotting the data points along the attenuation line in a graph. The slope of the displayed graph represents the attenuation coefficient (Figure 3B, E).
By correlating attenuation measurements to histopathology results (Figure 3C, F), tissue specific cut-off values can be derived providing the means for tumor differentiation.
<img alt="Figure 1" src="/files/ftp_upload/52574/52574fig1.jpg" /> 
Figure 1: (A) OCT B-scan of solid tissue. B) OCT B-scan with circular artefact.
<img alt="Figure 2" src="/files/ftp_upload/52574/52574fig2.jpg" /> 
Figure 2: (A) 3D volume rendered from 541 stacked B-scans. (B-C) Selected B-scans showing solid tissue, indicating successful OCT probe placement. (D) Selected B-scan showing perirenal fat tissue.
<img alt="Figure 3" src="/files/ftp_upload/52574/52574fig3.jpg" /> 
Figure 3: OCT analysis and correlation of a clear cell renal cell carcinoma (A-C) and an oncocytoma (D-E). Plotting the points along the highlighted line (A, D) provides the depicted graphs (B, E). The slope of the graphs represents the attenuation coefficient. Subsequently, the attenuation coefficient is correlated to the pathology specimen from the same location (C, F) in order to derive tissue specific cut-off values.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Patient No.</td>
			<td>23</td>
		</tr>
		<tr>
			<td>Tumor No.</td>
			<td>25</td>
		</tr>
		<tr>
			<td>Age (yrs): AVG (range)</td>
			<td>63.7 (32-83)</td>
		</tr>
		<tr>
			<td>Max tumor diameter (cm): AVG (range)</td>
			<td>3.5 (1.4-7.5)</td>
		</tr>
		<tr>
			<td>Sex</td>
			<td></td>
		</tr>
		<tr>
			<td>Male (%)</td>
			<td>17 (68)</td>
		</tr>
		<tr>
			<td>Female (%)</td>
			<td>8 (32)</td>
		</tr>
		<tr>
			<td>Tumor side</td>
			<td></td>
		</tr>
		<tr>
			<td>Left (%)</td>
			<td>15 (60)</td>
		</tr>
		<tr>
			<td>Right (%)</td>
			<td>10 (40)</td>
		</tr>
		<tr>
			<td>Tumor location</td>
			<td></td>
		</tr>
		<tr>
			<td>Entirely above upper polar line or below lower polar line (%)</td>
			<td>8 (32)</td>
		</tr>
		<tr>
			<td>Crosses polar line (%)</td>
			<td>9 (36)</td>
		</tr>
		<tr>
			<td>&#62;50% across polar line or crosses axial midline or between polar lines (%)</td>
			<td>8 (32)</td>
		</tr>
		<tr>
			<td>Exophytic / endophytic properties</td>
			<td></td>
		</tr>
		<tr>
			<td>&#8805;50% exophytic (%)</td>
			<td>10 (40)</td>
		</tr>
		<tr>
			<td>&#60;50% exophytic (%)</td>
			<td>14 (56)</td>
		</tr>
		<tr>
			<td>Entirely endophytic (%)</td>
			<td>1 (4)</td>
		</tr>
	</tbody>
</table>
Table 1: Patient characteristics.

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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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Nanyang Technological University

Research

JoVE Journal

Medicine

8.8K Views.  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.

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

Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation

Lung cancer is the leading cause of cancer-related deaths1. Squamous cell and small cell cancers typically arise in association with the conducting airways, whereas adenocarcinomas are typically more peripheral in location. Lung malignancy detection early in the disease process may be difficult due to several limitations: radiological resolution, bronchoscopic limitations in evaluating tissue underlying the airway mucosa and identifying early pathologic changes, and small sample size and/or incomplete sampling in histology biopsies. High resolution imaging modalities, such as optical frequency domain imaging (OFDI), provide non-destructive, large area 3-dimensional views of tissue microstructure to depths approaching 2 mm in real time (Figure 1)2-6. OFDI has been utilized in a variety of applications, including evaluation of coronary artery atherosclerosis6,7 and esophageal intestinal metaplasia and dysplasia6,8-10.
Bronchoscopic OCT/OFDI has been demonstrated as a safe in vivo imaging tool for evaluating the pulmonary airways11-23 (Animation). OCT has been assessed in pulmonary airways16,23 and parenchyma17,22 of animal models and in vivo human airway14,15. OCT imaging of normal airway has demonstrated visualization of airway layering and alveolar attachments, and evaluation of dysplastic lesions has been found useful in distinguishing grades of dysplasia in the bronchial mucosa11,12,20,21. OFDI imaging of bronchial mucosa has been demonstrated in a short bronchial segment (0.8 cm)18. Additionally, volumetric OFDI spanning multiple airway generations in swine and human pulmonary airways in vivo has been described19. Endobronchial OCT/OFDI is typically performed using thin, flexible catheters, which are compatible with standard bronchoscopic access ports. Additionally, OCT and OFDI needle-based probes have recently been developed, which may be used to image regions of the lung beyond the airway wall or pleural surface17.
While OCT/OFDI has been utilized and demonstrated as feasible for in vivo pulmonary imaging, no studies with precisely matched one-to-one OFDI:histology have been performed. Therefore, specific imaging criteria for various pulmonary pathologies have yet to be developed. Histopathological counterparts obtained in vivo consist of only small biopsy fragments, which are difficult to correlate with large OFDI datasets. Additionally, they do not provide the comprehensive histology needed for registration with large volume OFDI. As a result, specific imaging features of pulmonary pathology cannot be developed in the in vivo setting. Precisely matched, one-to-one OFDI and histology correlation is vital to accurately evaluate features seen in OFDI against histology as a gold standard in order to derive specific image interpretation criteria for pulmonary neoplasms and other pulmonary pathologies. Once specific imaging criteria have been developed and validated ex vivo with matched one-to-one histology, the criteria may then be applied to in vivo imaging studies. Here, we present a method for precise, one to one correlation between high resolution optical imaging and histology in ex vivo lung resection specimens. Throughout this manuscript, we describe the techniques used to match OFDI images to histology. However, this method is not specific to OFDI and can be used to obtain histology-registered images for any optical imaging technique. We performed airway centered OFDI with a specialized custom built bronchoscopic 2.4 French (0.8 mm diameter) catheter. Tissue samples were marked with tissue dye, visible in both OFDI and histology. Careful orientation procedures were used to precisely correlate imaging and histological sampling locations. The techniques outlined in this manuscript were used to conduct the first demonstration of volumetric OFDI with precise correlation to tissue-based diagnosis for evaluating pulmonary pathology24. This straightforward, effective technique may be extended to other tissue types to provide precise imaging to histology correlation needed to determine fine imaging features of both normal and diseased tissues.

Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation

A method to image ex vivo pulmonary resection specimens with optical frequency domain imaging (OFDI) and obtain precise correlation to histology is described, which is essential to developing specific OFDI interpretation criteria for pulmonary pathology. This method is applicable to other tissue types and imaging techniques to obtain precise imaging to histology correlation for accurate image interpretation and assessment. Imaging criteria established with this technique would then be applicable to image assessment in future in vivo studies.

Lung cancer is the leading cause of cancer-related deaths1. Squamous cell and small cell cancers typically arise in association with the conducting ...

Bioengineering

Longitudinal Morphological and Physiological Monitoring of Three-dimensional Tumor Spheroids Using Optical Coherence Tomography

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, high-throughput imaging modalities utilizing bright field or fluorescence detection, are unable to resolve the overall 3D structure of the tumor spheroid due to limited light penetration, diffusion of fluorescent dyes and depth-resolvability. Recently, our lab demonstrated the use of optical coherence tomography (OCT), a label-free and non-destructive 3D imaging modality, to perform longitudinal characterization of multicellular tumor spheroids in a 96-well plate. OCT was capable of obtaining 3D morphological and physiological information of tumor spheroids growing up to about 600 &#181;m in height. In this article, we demonstrate a high-throughput OCT (HT-OCT) imaging system that scans the whole multi-well plate and obtains 3D OCT data of tumor spheroids automatically. We describe the details of the HT-OCT system and construction guidelines in the protocol. From the 3D OCT data, one can visualize the overall structure of the spheroid with 3D rendered and orthogonal slices, characterize the longitudinal growth curve of the tumor spheroid based on the morphological information of size and volume, and monitor the growth of the dead-cell regions in the tumor spheroid based on optical intrinsic attenuation contrast. We show that HT-OCT can be used as a high-throughput imaging modality for drug screening as well as characterizing biofabricated samples.

Optical coherence tomography (OCT), a three-dimensional imaging technology, was used to monitor and characterize the growth kinetics of multicellular tumor spheroids. Precise volumetric quantification of tumor spheroids using a voxel counting approach, and label-free dead tissue detection in the spheroids based on intrinsic optical attenuation contrast, were demonstrated.

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, ...

Cancer Research

Novel Photoacoustic Microscopy and Optical Coherence Tomography Dual-modality Chorioretinal Imaging in Living Rabbit Eyes

Photoacoustic ocular imaging is an emerging ophthalmic imaging technology that can noninvasively visualize ocular tissue by converting light energy into sound waves and is currently under intensive investigation. However, most reported work to date is focused on the imaging of the posterior segment of the eyes of small animals, such as rats and mice, which poses challenges for clinical human translation due to small eyeball sizes. This manuscript describes a novel photoacoustic microscopy (PAM) and optical coherence tomography (OCT) dual-modality system for posterior segment imaging of the eyes of larger animals, such as rabbits. The system configuration, system alignment, animal preparation, and dual-modality experimental protocols for in vivo, noninvasive, label-free chorioretinal imaging in rabbits are detailed. The effectiveness of the method is demonstrated through representative experimental results, including retinal and choroidal vasculature obtained by the PAM and OCT. This manuscript provides a practical guide to reproducing the imaging results in rabbits and advancing photoacoustic ocular imaging in larger animals.

This manuscript describes the novel setup and operating procedure of a photoacoustic microscopy and optical coherence tomography dual-modality system for noninvasive, label-free chorioretinal imaging of larger animals, such as rabbits.

Photoacoustic ocular imaging is an emerging ophthalmic imaging technology that can noninvasively visualize ocular tissue by converting light energy into ...

Engineering

Evaluating Ocular Surface Squamous Neoplasia Using Anterior Segment HR-OCT

Anterior High-Resolution Optical Coherence Tomography in the Diagnosis and Therapeutic Monitoring of Ocular Surface Squamous Neoplasia

Ocular surface squamous neoplasia (OSSN) is the most common tumor of the ocular surface, ranging from mild dysplasia to invasive squamous carcinoma. Traditionally, the diagnosis of OSSN relies on histopathological confirmation followed by a full-thickness biopsy. However, in the past two decades, the therapeutic approach to OSSN has shifted from surgical intervention to topical chemotherapy regimens in clinical settings. This shift emphasizes the need for less invasive or non-invasive methods to diagnose ocular surface pathologies. Among various imaging devices, commercially available high-resolution optical coherence tomography (HR-OCT) has emerged as a powerful tool for the characterization of OSSN. HR-OCT provides an in vivo, cross-sectional view of ocular surface lesions, offering an "optical biopsy" for OSSN with high sensitivity and specificity. It provides valuable information in differentiating intraepithelial or invasive OSSN from other benign lesions. Additionally, HR-OCT can be used to monitor the response to topical chemotherapy and to detect subclinical OSSN during follow-up visits. In this article, the scanning protocol for image acquisition is presented, and image interpretation for OSSN is outlined. This standardized, practical, and reproducible approach is recommended in clinical workflows and is expected to assist clinicians in the management of OSSN.

Author Spotlight: Anterior HR-OCT as a Non-Invasive Tool for Characterizing Ocular Surface Squamous Neoplasia

Anterior segment high-resolution optical coherence tomography (HR-OCT) is a promising non-invasive modality for the diagnosis and therapeutic evaluation of ocular surface squamous neoplasia (OSSN). Here, the system setup, scanning technique, and representative diagnostic results are presented.

Ocular surface squamous neoplasia (OSSN) is the most common tumor of the ocular surface, ranging from mild dysplasia to invasive squamous carcinoma. ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

Neuroscience

In vivo Structural Assessments of Ocular Disease in Rodent Models using Optical Coherence Tomography

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable tool to evaluate and characterize changes in a variety of retinal and ocular disease and injury models. In light induced retinal degeneration models, SD-OCT can be used to track thinning of the photoreceptor layer over time. In glaucoma models, SD-OCT can be used to monitor decreased retinal nerve fiber layer and total retinal thickness and to observe optic nerve cupping after inducing ocular hypertension. In diabetic rodents, SD-OCT has helped researchers observe decreased total retinal thickness as well as decreased thickness of specific retinal layers, particularly the retinal nerve fiber layer with disease progression. In mouse models of myopia, SD-OCT can be used to evaluate axial parameters, such as axial length changes. Advantages of SD-OCT include in vivo imaging of ocular structures, the ability to quantitatively track changes in ocular dimensions over time, and its rapid scanning speed and high resolution. Here, we detail the methods of SD-OCT and show examples of its use in our laboratory in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia. Methods include anesthesia, SD-OCT imaging, and processing of the images for thickness measurements.

Here, we describe the use of spectral-domain optical coherence tomography (SD-OCT) to visualize retinal and ocular structures in vivo in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia.

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable ...

Using Optical Coherence Tomography and Optokinetic Response As Structural and Functional Visual System Readouts in Mice and Rats

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for the investigation of processes of neurodegeneration, neuroprotection and neuro-repair involving the visual system, as these often correlate well with retinal changes. As a functional readout, visually evoked compensatory eye and head movements are commonly used in experimental models involving the visual function. Combining both techniques allows a quantitative in vivo investigation of structure and function, which can be used to investigate the pathological conditions or to evaluate the potential of novel therapeutics. A great benefit of the presented techniques is the possibility to perform longitudinal analyses allowing the investigation of dynamic processes, reducing variability and cuts down the number of animals needed for the experiments. The protocol described aims to provide a manual for acquisition and analysis of high quality retinal scans of mice and rats using a low cost customized holder with an option to deliver inhalational anesthesia. Additionally, the proposed guide is intended as an instructional manual for researchers using optokinetic response (OKR) analysis in rodents, which can be adapted to their specific needs and interests.

A detailed protocol for the assessment of structural and visual readouts in rodents by optical coherence tomography and optokinetic response is presented. The results provide valuable insights for ophthalmologic as well as neurologic research.

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for ...

Retinal Vascular Reactivity as Assessed by Optical Coherence Tomography Angiography

The vascular supply to the retina has been shown to dynamically adapt through vasoconstriction and vasodilation to accommodate the metabolic demands of the retina. This process, referred to as retinal vascular reactivity (RVR), is mediated by neurovascular coupling, which is impaired very early in retinal vascular diseases such as diabetic retinopathy. Therefore, a clinically feasible method of assessing vascular function may be of significant interest in both research and clinical settings. Recently, in vivo imaging of the retinal vasculature at the capillary level has been made possible by the FDA approval of optical coherence tomography angiography (OCTA), a noninvasive, minimal risk and dyeless angiography method with capillary level resolution. Concurrently, physiological and pathological changes in RVR have been shown by several investigators. The method shown in this manuscript is designed to investigate RVR using OCTA with no need for alterations to the clinical imaging procedures or device. It demonstrates real time imaging of the retina and retinal vasculature during exposure to hypercapnic or hyperoxic conditions. The exam is easily performed with two personnel in under 30 min with minimal subject discomfort or risk. This method is adaptable to other ophthalmic imaging devices and the applications may vary based on the composition of the gas mixture and patient population. A strength of this method is that it allows for an investigation of retinal vascular function at the capillary level in human subjects in vivo. Limitations of this method are largely those of OCTA and other retinal imaging methods including imaging artifacts and a restricted dynamic range. The results obtained from the method are OCT and OCTA images of the retina. These images are amenable to any analysis that is possible on commercially available OCT or OCTA devices. The general method, however, can be adapted to any form of ophthalmic imaging.

This article describes a method for measuring retinal vasculature reactivity in vivo with human subjects using a gas breathing provocation technique to deliver vasoactive stimuli while acquiring retinal images.

The vascular supply to the retina has been shown to dynamically adapt through vasoconstriction and vasodilation to accommodate the metabolic demands of ...

Application of Optical Coherence Tomography to a Mouse Model of Retinopathy

Optical coherence tomography (OCT) offers a noninvasive method for the diagnosis of retinopathy. The OCT machine can capture retinal crosssectional images from which the retinal thickness can be calculated. Although OCT is widely used in clinical practice, its application in basic research is not as prevalent, especially in small animals such as mice. Because of the small size of their eyeballs, it is challenging to conduct fundus imaging examinations in mice. Therefore, a specialized retinal imaging system is required to accommodate OCT imaging on small animals. This article demonstrates a small-animal-specific system for OCT examination procedures and a detailed method for image analysis. The results of retinal OCT examination of very-low-density lipoprotein receptor (Vldlr) knockout mice and C57BL/6J mice are presented. The OCT images of C57BL/6J mice showed retinal layers, while those of Vldlr knockout mice showed subretinal neovascularization and retinal thinning. In summary, OCT examination could facilitate the noninvasive detection and measurement of retinopathy in mouse models.

Here, we describe an in vivo imaging technique using optical coherence tomography to facilitate the diagnosis and quantitative measurement of retinopathy in mice.

Optical coherence tomography (OCT) offers a noninvasive method for the diagnosis of retinopathy. The OCT machine can capture retinal crosssectional images ...

Image-Guided Optical Coherence Tomography to Assess Structural Changes in Rodent Retinas

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural changes. These diseases affecting the fundus always exhibit typical abnormalities in certain cell types in the retina, including photoreceptor cells, retinal ganglion cells, cells in the retinal blood vessels, and cells in the choroidal vascular cells. Noninvasive, highly efficient, and adaptable imaging techniques are required for both clinical practice and basic research. Image-guided optical coherence tomography (OCT) satisfies these requirements because it combines fundus photography and high-resolution OCT, providing an accurate diagnosis of tiny lesions as well as important changes in the retinal architecture. This study details the procedures of data collection and data analysis for image-guided OCT and demonstrates its application in rodent models of choroidal neovascularization (CNV), optic nerve crush (ONC), light-induced retinal degeneration, and experimental autoimmune uveitis (EAU). This technique helps researchers in the eye field to identify rodent retinal structural changes conveniently, reliably, and tractably.

The protocol presented here details the procedures of data collection and data analysis for image-guided optical coherence tomography (OCT) and demonstrates its application in multiple rodent models of ocular diseases.

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural ...

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

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Chapters in this video

Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation

Longitudinal Morphological and Physiological Monitoring of Three-dimensional Tumor Spheroids Using Optical Coherence Tomography

Novel Photoacoustic Microscopy and Optical Coherence Tomography Dual-modality Chorioretinal Imaging in Living Rabbit Eyes

Anterior High-Resolution Optical Coherence Tomography in the Diagnosis and Therapeutic Monitoring of Ocular Surface Squamous Neoplasia

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

In vivo Structural Assessments of Ocular Disease in Rodent Models using Optical Coherence Tomography

Using Optical Coherence Tomography and Optokinetic Response As Structural and Functional Visual System Readouts in Mice and Rats

Retinal Vascular Reactivity as Assessed by Optical Coherence Tomography Angiography

Application of Optical Coherence Tomography to a Mouse Model of Retinopathy

Image-Guided Optical Coherence Tomography to Assess Structural Changes in Rodent Retinas