Name: multimodal volumetric retinal imaging by oblique scanning laser ophthalmoscopy (oslo) and optical coherence tomography (oct)
Uploaded: 2018-08-04T14:30:00
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...

<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>

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.
<p class="...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#215;2</td>
			<td></td>
		</tr>
		<tr>
			<td>3:1 Telescope System</td>
			<td>Thorlabs</td>
			<td>AC254-150-A&#215;2</td>
			<td></td>
		</tr>
		<tr>
			<td>3:1 Telescope System</td>
			<td>Thorlabs</td>
			<td>AC254-50-A&#215;2</td>
			<td></td>
		</tr>
		<tr>
			<td>1:1 Telescope System</td>
			<td>Thorlabs</td>
			<td>AC254-100-A&#215;2</td>
			<td></td>
		</tr>
		<tr>
			<td>Galvo Mirrors (GM1,GM2)</td>
			<td>Thorlabs</td>
			<td>GVS201&#215;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&#215;/0.75</td>
			<td></td>
		</tr>
		<tr>
			<td>Final imaging system</td>
			<td>Olympus</td>
			<td>UplanFL N 10&#215;/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 la...

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 ...

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