Name: multimodal volumetric retinal imaging by oblique scanning laser ophthalmoscopy (oslo) and optical coherence tomography (oct)
Uploaded: 2018-08-04T14:30:00.000+00:00
Duration: 12 min 22 s
Description: Watch this Scientific Journal Video about Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy oSLO and Optical Coherence Tomography OCT at JoVE.com

Funding is from the Evans Medical foundation funding from Boston Medical Center as well as a sub-contract from NIH 5R01CA183101, BU-CTSI pilot grant 1UL1TR001430, BU-Joslin pilot program, and BU-CTSI KL2TR001411.

All methods described here have been approved by the Animal Care and Use Committee (ACUC) of Boston Medical Center.
1. System Setup
<ol>
	<li>oSLO System
	<ol>
		<li>Use a supercontinuum laser source as the system laser source.
		<ol>
			<li>Separate the visible light range (450-650 nm) from higher wavelength range (650-2000 nm) by a dichroic mirror (DM1). Expand the spectrum with a pair of dispersive prisms after the beam passing through a polarization beam splitter (PBS).</li>
			<li>Place a slit to select the excitation wavelength range (475-495 nm). Use a reflective mirror to reflect the filtered beam back to the prism pair and then couple the light into a single mode fiber (SMF 1).</li>
			<li>Use a spectrometer to confirm the wavelength selection at the output of the single mode fiber.</li>
		</ol>
		</li>
		<li>Connect the single mode fiber to two cascaded optical fiber couplers as shown in Figure 2. One of the fiber output port from the second fiber coupler delivers the light to the oSLO system.</li>
		<li>Collimate the laser first in the oSLO system.
		<ol>
			<li>Deflect the laser by a galvanometer mirror (GM1). Relay the laser to a second galvanometer mirror (GM2) by a 1:1 telescope system, and further relay to the pupil of the eye by a 3:1 telescope system.</li>
			<li>Install a dichroic mirror (DM2) within the 3:1 telescope system to reflect the fluorescence signals.</li>
		</ol>
		</li>
		<li>Mount the 3:1 telescope system and the dichroic mirror (DM2) on a customized dove tail slider to offset the optical axis and create the oblique scanning illumination as shown in Figure 3. Use a caliper to precisely control the offset length as desired.</li>
		<li>Fluorescence imaging optical path.
		<ol>
			<li>Reflect the fluorescence by the dichroic mirror and relay to the third galvanometer mirror to de-scan the slow scanning.</li>
			<li>Relay the light to an imaging objective lens by another 1:1 telescope system. Install the above optics on a translation stage. 
			NOTE: Two additional translation stages are installed under the third galvanometer mirror (GM3) to provide redundancy in the degrees of freedom for optimizing the imaging.</li>
		</ol>
		</li>
		<li>Mount a final imaging system on a stage that has three degrees of freedom (rotation, and two axis of translation). Use a planar camera to capture the cross-sectional fluorescence images.</li>
	</ol>
	</li>
	<li>Optical Coherence Tomography System
	<ol>
		<li>Use the same supercontinuum laser source as the system laser source.
		<ol>
			<li>Separate the near infrared (NIR) range (650-900 nm) from the remaining light (650-2000 nm) by another dichroic mirror (DM3). Use a long pass filter to further limit the bandwidth to 800-900 nm. Couple the beam into a single mode fiber (SMF 2).</li>
		</ol>
		</li>
		<li>Connect the single mode fiber to the other input port of the two cascaded optical fiber couplers to combine with the blue oSLO excitation. Direct the light from the second output port of the second fiber coupler to the OCT reference arm, which has a variable neutral density filter (VNDF), dispersion compensation plates and a reflective mirror. 
		NOTE: The light returned from the reference arm and the eye recombines at the second optical fiber coupler and is delivered to the OCT spectrometer to collect the signal.</li>
	</ol>
	</li>
	<li>Data Acquisition
	<ol>
		<li>Use a data acquisition system software written in LabVIEW and modified from the scanning protocol of OCTA7,8,9,10. For each B-scan, an 80% duty cycle saw tooth with 500 steps is output by an analog output board (AO1) to control the x&#39; fast scanning mirror, GM2.</li>
		<li>Trigger the line scan camera at each step to acquire data for the OCT only when the mirror is in the forward scanning direction. Set the exposure time for the line scan camera to be 17 &#181;s.</li>
		<li>To acquire the OCTA signal, repeat the measurement 5 times at the same B-scan location.</li>
		<li>Set the AO output rate at 100 kHz, and the OCT A-line rate at 50 kHz. Control the y&#39; slow scanning mirror, GM1, by a ramping waveform. Synchronize the de-scanning mirror, GM3, with GM1 to de-scan the slow scanning.</li>
		<li>Trigger the planar camera by another analog output board (AO2) to capture one fluorescent image at each y&#39; location. Crop the imaging size or bin the neighbor pixels to increase the speed and sensitivity as desired.</li>
	</ol>
	</li>
</ol>
2. System Alignment
<ol>
	<li>Adjust the slit in the oSLO light source to select the blue excitation wavelength. Use a spectrometer to monitor the spectral range to be around 475-490 nm.</li>
	<li>Adjust the dove tail mount slider to shift the optical axis by ~5 mm. This will result in an offset at the rat pupil by ~1.7 mm, resulting in an oblique angle of ~15&#176; on the retina.</li>
	<li>Adjust the translation stage of the fluorescence detection optics by the same 5 mm.</li>
	<li>Adjust the final fluorescence imaging system to be ~30&#176;.</li>
	<li>Measure the optical power using a power meter. Make sure the blue oSLO excitation power is &#8804;0.2 mW and the OCT laser power &#8804;0.8 mW, which will not cause retina damage. 
	NOTE: Based on the ANSI standard, the maximum permissive exposure (MPE) to the retina is at the level of ~2mW7,8 in visible light range. According to the formula by Delori et al.9, the MPE for the near infrared light is about two times higher than the visible light, at about 4 mW.</li>
</ol>
3. In Vivo Animal Experiment
<ol>
	<li>Transfer a 12-weeks male Long Evans rat into the induction chamber. Anesthetize the rat with 4.5% isoflurane in oxygen for 10 minutes with a flow rate of 2 L/min by an isoflurane vaporizer.
	<ol>
		<li>Confirm depth of anesthesia as determined by a lack of withdrawal reflex during an interdigital pinch.</li>
	</ol>
	</li>
	<li>After the induction, place the rat on a 5-axis (x, y, z translations, yaw and pitch) holder. Provide supplemental heat by use of a heated stage, circulating warm water blanket or other suitable method in a prolonged experiment. Maintain the anesthesia at 1.5% of isofluorane with a flow rate of 2 liter/minutes during the remaining part of the experiment. When not using an induction chamber with active exhaust, the induction chamber should be placed on a backdraft or downdraft table or under a snorkel to scavenge isoflurane.</li>
	<li>Dilate the pupil with 1% Tropicamide ophthalmic solution for 2 minutes. Apply 0.5% Tetracaine HCl ophthalmic solution on the rat&#39;s eye for additional local anesthesia, if necessary. Keep the eye moisturized with commercial artificial tears at least once every minute during the experiment.</li>
	<li>Inject fluorescein salt (10% w/w) or FITC (10% w/w) diluted in sterile saline (0.1-0.3 mL) through the tail vein with a 1 mL syringe and a 29G needle.</li>
	<li>Turn on the laser source. Place a neutral density filter to attenuate the blue light excitation during alignment. Measure the power of the OCT light to be ~0.8 mW, and the blue light &#60;0.01 mW to avoid the formation of cataracts.</li>
	<li>Start the galvanometer scanning and alignment mode. Adjust the height of the eye ball to make a stationary laser spot on the cornea. Adjust the rat eye position to make the rim of the pupil roughly perpendicular to the laser, and offset the laser to the apical center of the eye to about ~1.5 mm.</li>
	<li>Further adjust the animal holder until OCT images reach optimal quality. In the x&#39; fast scanning direction, make sure that the cross-sectional B-scan image appears flat. When switching to the y&#39; slow scanning direction, make sure the cross-section B-scan image appears tilted, due to the oblique scanning.</li>
	<li>Remove the neutral density filter to the blue light excitation and monitor the real time feed from the camera. Cross sectional fluorescent image should appear showing blood vessels appearing in different depths.
	<ol>
		<li>Adjust the focus of the final fluorescence imaging system to reach the optimal focus. Allow fine adjustments of the eye position in the lateral plane to reach optimal oSLO image quality.</li>
	</ol>
	</li>
	<li>After the alignment, start to acquire simultaneous OCTA and volumetric fluorescein angiography (vFA).</li>
	<li>Construct the volumetric images for both OCTA and oSLO by Matlab. The algorithms are previously described in detail10. Generate depth-resolved retinal vasculatures by image segmentation.</li>
	<li>After completing the imaging, turn off the laser, release the animal and apply some ophthalmic ointment on the eyes, and then place the animal in a recovery box.</li>
	<li>Do not leave the animal unattended until it has regained sufficient consciousness to maintain sternal recumbency or as per institutional policy.</li>
</ol>

<ol>
	<li>Webb, R. H., Hughes, G. W., Delori, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+scanning+laser+ophthalmoscope.">Confocal scanning laser ophthalmoscope.</a> Applied Optics. 26 (8), 1492-1499 (1987).</li><li>Roorda, 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=Adaptive+optics+scanning+laser+ophthalmoscopy.">Adaptive optics scanning laser ophthalmoscopy.</a> Optics Express. 10 (9), 405-412 (2002).</li><li>Huang, 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=Optical+coherence+tomography.">Optical coherence tomography.</a> Science. 254 (5035), 1178-1181 (1991).</li><li>de Carlo, T. E., Romano, A., Waheed, N. K., Duker, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+optical+coherence+tomography+angiography+(OCTA).">A review of optical coherence tomography angiography (OCTA).</a> International Journal of Retina and Vitreous. 1 (1), 5 (2015).</li><li>Jia, Y., 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+Optical+Coherence+Tomography+Angiography+of+Choroidal+Neovascularization+in+Age-Related+Macular+Degeneration.">Quantitative Optical Coherence Tomography Angiography of Choroidal Neovascularization in Age-Related Macular Degeneration.</a> Ophthalmology. 121 (7), 1435-1444 (2014).</li><li>Chen, C. -. L., Wang, R. K. <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+based+angiography+[Invited].">Optical coherence tomography based angiography [Invited].</a> Biomedical Optics Express. 8 (2), 1056-1082 (2017).</li><li>Yi, J., Chen, S. Y., Shu, X., Fawzi, A. A., Zhang, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+retinal+imaging+using+visible-light+optical+coherence+tomography+guided+by+scanning+laser+ophthalmoscopy.">Human retinal imaging using visible-light optical coherence tomography guided by scanning laser ophthalmoscopy.</a> Biomedical Optics Express. 6 (10), 3701-3713 (2015).</li><li>Zhang, X. Y., 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=Dual-band+spectral-domain+optical+coherence+tomography+for+in+vivo+imaging+the+spectral+contrasts+of+the+retinal+nerve+fiber+layer.">Dual-band spectral-domain optical coherence tomography for in vivo imaging the spectral contrasts of the retinal nerve fiber layer.</a> Optics Express. 19 (20), 19653-19667 (2011).</li><li>Delori, F. C., Webb, R. H., Sliney, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximum+permissible+exposures+for+ocular+safety+(ANSI+2000),+with+emphasis+on+ophthalmic+devices.">Maximum permissible exposures for ocular safety (ANSI 2000), with emphasis on ophthalmic devices.</a> Journal of the Optical Society of America a-Optics Image Science and Vision. 24 (5), 1250-1265 (2007).</li><li>Zhang, L., 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+fluorescence+retinal+imaging+in+vivo+over+a+30-degree+field+of+view+by+oblique+scanning+laser+ophthalmoscopy+(oSLO).">Volumetric fluorescence retinal imaging in vivo over a 30-degree field of view by oblique scanning laser ophthalmoscopy (oSLO).</a> Biomedical Optics Express. 9 (1), 25-40 (2018).</li></ol>

Ji Yi holds a pending patent for oSLO. The other author(s) declare no competing financial interests.

Here, we have described oSLO, an in vivo volumetric fluorescent retinal imaging technique with a FOV over 30&#176;. Compared to OCT, a current standard of care imaging method in ophthalmology, oSLO offers a similar 3D imaging capability yet allows fluorescence contrast that OCT is not sensitive to. The advantage of oSLO is that it requires only one raster scan, and thus allows the seamless combination of OCT, providing two complementary techniques for structural and fluorescent volumetric imaging.
In this protocol, the key point to obtain good image quality is the clarity of the rat eye. If the oSLO image is obscured, examine whether there is cataract formation. Several factors such as ketamine/xylazine anesthesia, cornea drying, and over-exposure to blue light will cause formation of cataracts, which would significantly deteriorate the image quality. To prevent the cataracts, avoid continuous exposure to blue light for more than 2 minutes; apply artificial eye tear at least once every minute to prevent cornea drying; and allow the eye to rest at least 30 seconds between imaging sections by blocking the light.
We envision that oSLO can significantly impact the clinical practice of fluorescence imaging. We have shown that the depth resolving power can effectively eliminate the signal from the outer retinal, yielding high-contrast volumetric FA images down to the single capillary level, which is unobtainable with conventional SLO. The dramatically improved image clarity would allow for more sensitive detection and quantification of blood-retina barrier disruption and retinal capillary leakage, the hallmark of vision-threatening macular edema in DR and other chorioretinal vascular diseases.
In the current system, the speed of the CCD camera is 20 frames per second, which leads to &#62;25 second acquisition times. A scientific CMOS camera will dramatically improve the system speed. The fluorescence detection has a different optical path separated from the illumination. It will simplify the system if using the same optical path for the illumination and detection in the future design. In summary, a novel multimodal platform for volumetric retinal imaging, namely oblique scanning laser ophthalmoscopy (oSLO), was presented. The large FOV in vivo imaging over a 30&#176; viewing angle of rat retina using oSLO and simultaneous optical coherence tomography (OCT) was demonstrated.

Ophthalmology and vision science greatly benefit from the modern optical imaging techniques, since the retina can be easily accessed with light. Fluorescence retinal imaging is an essential tool in the diagnosis and management of chorioretinal vascular diseases such as diabetic retinopathy (DR) and age-related macular degeneration (AMD), both of which are leading causes of blindness in the United States.
However, it is still challenging to acquire a large field of view (FOV), three-dimensional (3D) retinal imaging by using fluorescence imaging. Fundus photography does not have the depth-resolving capability and does not reject diffuse light. As a result, the mixing of signals from different depth reduces the image quality. Scanning laser ophthalmoscopy (SLO) and confocal SLO (cSLO) can reduce the effect of diffused light by using confocal gating1. However, it is difficult for SLO or cSLO to acquire a 3D human retinal image due to the limit of their depth of focus. Adaptive optics SLO (AOSLO) can provide superb resolution and contrast by correcting for the wavefront aberrations introduced by the human eye. However, AOSLO would still need z-stacking for volumetric imaging2. Optical coherence tomography (OCT)3 and OCT angiography (OCTA) systems overcome these restrictions to provide three-dimensional (3D) anatomical and vascular images4,5,6, but the dye-free nature of OCT cannot visualize leakage indicative of vascular dysfunction.
This protocol describes a novel multimodal platform for 3D volumetric fluorescence retinal imaging, namely oblique scanning laser ophthalmoscopy (oSLO). In this imaging system, an oblique scanning is generated by a dove tail slider, and a final imaging system is aligned in an angle to detect fluorescence cross sectional images. The system uses laser scanning methods, and these techniques allow easy incorporation with OCT as a complementary volumetric structural imaging modality. The current depth resolution is about 25 &#181;m in the rat retina and the field of view is 30&#176;. Essentially, the oSLO allows a fluorescent version of OCT and can be simultaneously combined with OCT and OCTA over a large FOV.
In this protocol, we will describe the setup of the oSLO, the method of alignment and construction, the method of in vivo imaging of rat retina, and the representative results.

<table><tbody><tr><td>Supercontinuum Laser Source</td><td>NKT Photonics</td><td>SuperK EXTREME EXU-OCT6</td><td></td></tr><tr><td>Dichroic Mirror (DM1)</td><td>Thorlabs</td><td>DMLP650R</td><td></td></tr><tr><td>Dichroic Mirror (DM2)</td><td>Chroma</td><td>ZT514/1064rpc</td><td></td></tr><tr><td>Dichroic Mirror (DM3)</td><td>Thorlabs</td><td>DMLP900R</td><td></td></tr><tr><td>Single Mode Fiber (SMF 1)</td><td>Thorlabs</td><td>P3-460B-FC-2</td><td></td></tr><tr><td>Single Mode Fiber (SMF 2)</td><td>Thorlabs</td><td>P3-780A-FC-2</td><td></td></tr><tr><td>Optic Fiber Coupler</td><td>Thorlabs</td><td>TW850R5A2</td><td></td></tr><tr><td>1:1 Telescope System</td><td>Thorlabs</td><td>AC254-100-A&amp;times;2</td><td></td></tr><tr><td>3:1 Telescope System</td><td>Thorlabs</td><td>AC254-150-A&amp;times;2</td><td></td></tr><tr><td>3:1 Telescope System</td><td>Thorlabs</td><td>AC254-50-A&amp;times;2</td><td></td></tr><tr><td>Galvo Mirrors (GM1,GM2)</td><td>Thorlabs</td><td>GVS201&amp;times;2</td><td></td></tr><tr><td>De-sacn Galvo Mirrors (GM3)</td><td>Thorlabs</td><td>GVS011</td><td></td></tr><tr><td>Objective Lens</td><td>Olympus</td><td>UplanSApo 20&amp;times;/0.75</td><td></td></tr><tr><td>Final imaging system</td><td>Olympus</td><td>UplanFL N 10&amp;times;/0.3</td><td></td></tr><tr><td>Final imaging system</td><td>Computar</td><td>12-36mm/1:2.8</td><td></td></tr><tr><td>Camera</td><td>PCO</td><td>Pco.pixelfly usb</td><td></td></tr><tr><td>Filter</td><td>Thorlabs</td><td>FEL0800</td><td></td></tr><tr><td>Mounted Continuously Variable ND Filter</td><td>Thorlabs</td><td>NDC-50C-4M-A</td><td></td></tr><tr><td>Line Scan Camera</td><td>Thorlabs</td><td>SPL2048-140K</td><td></td></tr><tr><td>Analog Output Board (AO1)</td><td>National Instrument</td><td>PCI-6731</td><td></td></tr><tr><td>Analog Output Board (AO2)</td><td>National Instrument</td><td>PCIe-6351</td><td></td></tr><tr><td>Long pass filter</td><td>Thorlabs</td><td>FEL0800</td><td></td></tr></tbody></table>

multimodal volumetric retinal imaging by oblique scanning laser ophthalmoscopy (oslo) and optical coherence tomography (oct)

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big challenge with the state-of-the-art retinal imaging modalities because they would require z-stacking to compile a volumetric dataset. Newer optical coherence tomography (OCT) and OCT angiography (OCTA) systems overcome these restrictions to provide three-dimensional (3D) anatomical and vascular images, but the dye-free nature of OCT cannot visualize leakage indicative of vascular dysfunction. This protocol describes a novel oblique scanning laser ophthalmoscopy (oSLO) technique that provides 3D volumetric fluorescence retinal imaging. The setup of the imaging system generates the oblique scanning by a dove tail slider and aligns the final imaging system at an angle to detect fluorescent cross-sectional images. The system uses the laser scanning method, and therefore, allows an easy incorporation of OCT as a complementary volumetric structural imaging modality. In vivo imaging on rat retina is demonstrated here. Fluorescein solution is intravenously injected to produce volumetric fluorescein angiography (vFA).

Figure 4a shows a cross-sectional OCT image of a rat retina. Figure 4b-4c show the same retinal cross-sectional images of OCTA and oSLO vFA acquired at the same time. The oSLO enables cross-sectional FA analogous to the OCT B-scan. In comparison to OCTA, the oSLO vFA cross-sectional image clearly identifies the vessels in nerve fiber layer (NFL) and ganglion cell layer (GCL), and capillaries in outer plexiform layer (OPL). Figure 4d and 4g show the superficial layer of OCTA and oSLO vFA image. Unlike OCTA, the oSLO vFA image (Figure 4g) avoids the motion artifacts (vertical stripes in Figure 4d) by utilizing fluorescence emission contrast. By comparing the oSLO vFA (Figure 4e) and OCTA (Figure 4h) images within the retinal intermediate layer, the vertically diving vessels are clearly shown in the oSLO FA image but not apparent in OCTA. This is presumably because the blood flow speed or the vessel orientation will affect the OCTA signal but not the oSLO fluorescence contrast.
Figure 4f and 4i show the images within the deep capillary plexus layer. The regions pointed out by blue arrows in oSLO vFA have a better contrast than OCTA in the vasculature. The size of the venule pointed out by white arrows in oSLO is larger than that in OCTA. Overall, the oSLO vFA image resembles the actual vascular morphology more accurately than OCTA, since it is not dependent upon blood flow speed or the vessel orientation. An en face fly-through from two simultaneously acquired volumetric data set from oSLO and OCTA are shown in Video 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57814/57814fig1.jpg" /> 
Figure 1. The system schematic. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57814/57814fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57814/57814fig2.jpg" /> 
Figure 2. The photograph of the two cascaded fiber couplers that direct oSLO and OCT light. The routes of the light pass are labeled. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57814/57814fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57814/57814fig3.jpg" /> 
Figure 3. The setup of the dove tail mount for oblique illumination. (a) The solid work model for the oblique illumination part. (b-c) The zoomed-in view, and the separate view of the dove tail mount. (d) The photography of the actual setup for oblique illumination part. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57814/57814fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57814/57814fig4v2.jpg" /> 
Figure 4. Rat retinal image acquired by oSLO and OCTA at the same time. The example of (a) the cross-sectional image of OCT (b) OCTA and (c) oSLO FA. Panels (d) and (g) show en face OCT and oSLO FA images from the superficial layer. The motion artifacts in OCTA image was pointed out by red arrows. Panel (e) and (h) show the results from the intermediate layer. The locations where the vessels dive down into next layer were pointed out by yellow arrows, which are clearer on oSLO FA than OCTA. Panels (f) and (i) show the results from the deep capillary plexuses layer. The contrast of oSLO is better than OCTA in the region pointed out by blue arrows. The size of the venule pointed out by white arrows in oSLO is larger than that in OCTA. Bars in the figure is 200 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57814/57814fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy oSLO and Optical Coherence Tomography OCT at JoVE.com

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy oSLO and Optical Coherence Tomography OCT

Here, we present a protocol to get a large field of view (FOV) three-dimensional (3D) fluorescence and OCT retinal image by using a novel imaging multimodal platform. We will introduce the system setup, the method of alignment, and the operational protocols. In vivo imaging will be demonstrated, and representative results will be provided.

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy (oSLO) and Optical Coherence Tomography (OCT)

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big ...

multimodal-volumetric-retinal-imaging-oblique-scanning-laser

Nanyang Technological University

Research

JoVE Journal

Bioengineering

8.5K Views.  Boston University School of Medicine. Hier presenteren we een protocol om een groot beeldveld (FOV) driedimensionale (3D) fluorescentie en OCT retinale beeld met behulp van een roman imaging multimodale platform. We introduceren de systeeminstellingen, de methode van de aanpassing en de operationele protocollen. In vivo imaging zal worden aangetoond, en representatieve resultaten zal worden verstrekt.

Video: Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy oSLO and Optical Coherence Tomography OCT

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 ...

Medicine

Integrated Photoacoustic Ophthalmoscopy and Spectral-domain Optical Coherence Tomography

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging technologies. Existing retinal imaging modalities, such as fundus photography1, confocal scanning laser ophthalmoscopy (cSLO)2, and optical coherence tomography (OCT)3, have significant contributions in monitoring disease onsets and progressions, and developing new therapeutic strategies. However, they predominantly rely on the back-reflected photons from the retina. As a consequence, the optical absorption properties of the retina, which are usually strongly associated with retinal pathophysiology status, are inaccessible by the traditional imaging technologies.
Photoacoustic ophthalmoscopy (PAOM) is an emerging retinal imaging modality that permits the detection of the optical absorption contrasts in the eye with a high sensitivity4-7 . In PAOM nanosecond laser pulses are delivered through the pupil and scanned across the posterior eye to induce photoacoustic (PA) signals, which are detected by an unfocused ultrasonic transducer attached to the eyelid. Because of the strong optical absorption of hemoglobin and melanin, PAOM is capable of non-invasively imaging the retinal and choroidal vasculatures, and the retinal pigment epithelium (RPE) melanin at high contrasts 6,7. More importantly, based on the well-developed spectroscopic photoacoustic imaging5,8 , PAOM has the potential to map the hemoglobin oxygen saturation in retinal vessels, which can be critical in studying the physiology and pathology of several blinding diseases 9 such as diabetic retinopathy and neovascular age-related macular degeneration.
Moreover, being the only existing optical-absorption-based ophthalmic imaging modality, PAOM can be integrated with well-established clinical ophthalmic imaging techniques to achieve more comprehensive anatomic and functional evaluations of the eye based on multiple optical contrasts6,10 . In this work, we integrate PAOM and spectral-domain OCT (SD-OCT) for simultaneously in vivo retinal imaging of rat, where both optical absorption and scattering properties of the retina are revealed. The system configuration, system alignment and imaging acquisition are presented.

Photoacoustic ophthalmology (PAOM), an optical-absorption-based imaging modality, provides the complementary evaluation of the retina to the currently available ophthalmic imaging technologies. We report the using of PAOM integrated with spectral-domain optical coherence tomography (SD-OCT) for simultaneous multimodal retinal imaging in rats.

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging ...

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 ...

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 ...

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 ...

Mouse Retina OCT Fibergram Alignment with Confocal Images

Alignment of Visible-Light Optical Coherence Tomography Fibergrams with Confocal Images of the Same Mouse Retina

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has been increasingly applied to obtain an objective assessment of neural damage in eye diseases. Ex vivo confocal imaging of the same retina is often necessary to validate the in vivo findings especially in animal research. In this study, we demonstrated a method for aligning an ex vivo confocal image of the mouse retina with its in vivo images. A new clinical-ready imaging technology called visible light optical coherence tomography fibergraphy (vis-OCTF) was applied to acquire in vivo images of the mouse retina. We then performed the confocal imaging of the same retina as the &#34;gold standard&#34; to validate the in vivo vis-OCTF images. This study not only enables further investigation of the molecular and cellular mechanisms but also establishes a foundation for a sensitive and objective evaluation of neural damage in vivo.

Author Spotlight: Advancements in In Vivo and Ex Vivo Retinal Imaging for Improved Glaucoma Diagnosis and Treatment 

The present protocol outlines the steps for aligning in vivo visible-light optical coherence tomography fibergraphy (vis-OCTF) images with ex vivo confocal images of the same mouse retina for the purpose of verifying the observed retinal ganglion cell axon bundle morphology in the in vivo images.

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has ...

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 ...

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy (oSLO) and Optical Coherence Tomography (OCT)

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