We would like to thank our collaborators, Francois Delori, Tomas Burke, and Tobias Duncker.
Research Support: NIH/NEI R01 EY015520 (RTS, JPG), and unrestricted funds from Research to Prevent Blindness (RTB).

Ethics Statement: All patients enrolled in these studies were done so in accordance with approved institutional review board oversight at New York University School of Medicine.
1. Patient Selection and Initial Preparation for Imaging
Note: The following materials are required: 0.5% tropicamide ophthalmic solution, 2.5% phenylephrine ophthalmic solution, cSLO equipped with spectral domain optical coherence tomography (SD-OCT), and internal fluorescence reference.
<ol>
	<li>Prior to imaging, appropriately set up cSLO for data acquisition according to manufacturer&#39;s instructions.</li>
	<li>Mount an internal fluorescent reference in the cSLO. Note: The internal fluorescent reference, as purchased from the manufacturer, is housed in a metal ring and is placed directly behind the camera lens. If a cSLO from another manufacturer is used, there may be a different configuration than that which is described in this protocol.
	<ol>
		<li>In order to insert the reference into the cSLO, twist the lens to remove it, unscrew the machine&#39;s metal ring, and replace it with the new metal ring containing the reference. 
		Note: the internal fluorescent reference is essential to the qAF technique, as it allows correction for variation in laser power and detector sensitivity/gain.</li>
	</ol>
	</li>
	<li>Have patients recruited for imaging undergo a routine dilated eye examination and obtain background information regarding past ocular history and underlying medical conditions that may manifest with ocular findings.</li>
	<li>Dilate pupils with 0.5% tropicamide and 2.5% phenylephrine. CRITICAL STEP: Dilate pupils to at least 6 mm. Note: This is essential for uninterrupted passage of light, and thus for optimal visualization and measurement of the fundus.</li>
	<li>Prior to imaging, position the patient properly on the cSLO, with chin resting on the chinrest, forehead placed against the forehead rest, and lateral canthi properly aligned with the indicators.</li>
</ol>
2. Baseline Imaging of the Ocular Fundus
<ol>
	<li>First, image the fundus with near infrared reflectance (IR) light (wavelength of 820 nm) in order to centralize the camera over the macula and obtain rough focus.
	<ol>
		<li>With patient properly positioned, switch the hardware setting on the control panel to IR imaging mode, position camera manually until the fundus is in full focus, and take an image</li>
		<li>Adjust the setting on the control panel to &#34;IR-OCT&#34;, which uses spectral-domain optical coherence tomography (SD-OCT) in conjunction with IR imaging to evaluate the macula for underlying disease.</li>
		<li>Use the guides present in the imaging window to correctly orient the OCT to the IR image of the fundus. To achieve optimal SD-OCT quality, position the camera such that the OCT image is in the top third of its imaging window. Acquire at least one horizontal line scan through the fovea and spanning the entire imaging field.</li>
	</ol>
	</li>
</ol>
3. Setting up qAF Imaging
<ol>
	<li>Use &#34;High speed&#34; image acquisition. Note: This setting allows for faster image acquisition, which decreases risk of signal loss due to patient movement and resulting blockage of light by the iris or eyelids.</li>
	<li>Use &#34;Mean of 9&#34; frames. Note: This setting allows for the rapid, sequential capture of 9 image frames, which can be subsequently &#34;averaged&#34; to reduce noise and artifact (see below).</li>
	<li>Use &#34;30 x 30 degree&#34; field. Note: This refers to the degrees of retinal area captured during image acquisition.</li>
	<li>Prior to imaging, warn patient about the blue light, as this can be alarming at first.</li>
	<li>Turn on the AF mode and align camera axis such that the screen is maximally &#34;filled&#34; with fundus AF (minimal darkening of the sides and corners of the image).
	<ol>
		<li>If patients have difficulty tolerating the bright blue light, start imaging with the camera further away from the eye and then bring the camera slowly toward the patient until the fundus is in full view. 
		CRITICAL STEP: If light entering into or exiting from the eye is obstructed, decreased signal will result. Localized decrease is visible as asymmetric darkening of an image side or corner. Generalized loss of signal is seen when eye movements alone obstruct light passage.</li>
	</ol>
	</li>
	<li>Align the camera such that the AF signal is at its highest level throughout the field. Aim for maximal signal rather than sharpest image, however they roughly correlate. Adjust the camera focus by moving the cSLO to reposition the camera either manually or with the ophthalmoscope joystick.</li>
	<li>Adjust the sensitivity/gain such that fundus AF is easily visible, but avoid over-saturation. During image acquisition, colored pixels visible in either the internal reference (located at the top of the image) or the fundus indicate over-saturation and thus loss of signal.</li>
</ol>
4. Image Acquisition
Note: CRITICAL STEP: The goal in image acquisition should be to obtain 2 high quality 9-frame image stacks per session to control for variability between images within a session. After repositioning the patient and camera, obtain a second session of two images to assess and control for variability. All images will ultimately be calibrated to the internal reference (described below).
<ol>
	<li>Acquire images only after at least 20 sec of &#34;bleaching&#34; (full exposure of retina to AF light) to minimize absorption of light by rhodopsin in the sensory retina 9.
	<ol>
		<li>Use this period to optimize camera alignment, focus and sensitivity.</li>
	</ol>
	</li>
	<li>Have the subjects blink before each image acquisition as a fresh tear film improves signal quality.</li>
	<li>Avoid eyelids in the plane of acquisition. 
	Note: It may be helpful to manually open eyelids for more challenging patients. The authors recommend that an assistant performs this task.</li>
	<li>Optimize alignment before each image acquisition to ensure that light is not obstructed by the iris resulting in decreased signal. 
	Note: As at least minor movement is very common, the authors recommend fine realignment as necessary before each image acquisition. Guiding and encouraging the patient while imaging helps decrease movement. The authors also recommend using the machine&#39;s foot-pedal as the &#34;acquisition button&#34; so as to minimize operator distraction.</li>
	<li>Perform post-image processing by computing the &#34;mean&#34; of the 9-frame stack to increase signal-to-noise ratio. Calculate the mean of a stack using the cSLO software by selecting the option to calculate the mean. 
	Note: There may be stacks where some frames are not of optimal quality (i.e., have localized or generalized decreased signal relative to the optimal frames), therefore carefully inspect each stack and deselect suboptimal frames before computation of the mean. 
	Note: Mean images with acceptable signal-to-noise ratios can be processed from at least 3 frames. It is normal for the sides and corners of each image to have lower signal than the central 20 degrees due to limitations within the cSLO itself.
	<ol>
		<li>If the software asks whether the operator would like to normalize the gray levels (i.e., stretch the histogram) between 0 and 255, select &#34;No&#34;. This maintains unaltered gray levels for analysis.</li>
	</ol>
	</li>
</ol>
5. Image Analysis
<ol>
	<li>Analyze AF images as previously described using image analysis software developed for the qAF technique (IGOR; 9). A brief description of a typical image analysis using this software is included below.
	<ol>
		<li>Load the qAF program in IGOR and import images (in .bmp format) exported from the cSLO software with pixel aspect ratio 768 x 768 (default output parameter).</li>
		<li>Select the image in the drop-down menu and initiate analysis.</li>
		<li>Reposition calibration indicators on the image such that the &#34;crosshairs&#34; overlie the fovea and the &#34;bracket&#34; abuts the optic disc (these indicators are used to scale and position the ROIs). When properly positioned, use software prompts then populate the image with circumferential ROIs surrounding the fovea (see Figures 1, 2).</li>
		<li>Select the option in IGOR to calibrate the overall gray levels of each image to those levels present in the internal fluorescent reference. Note: This step allows for calibration of machine-related factors, including the electronic zero level of each image and the internal reference calibration factor of each machine, as well as patient-related factors, including age, refraction and corneal curvature. 
		Note: The analysis software then displays the fixed regions on the resultant image and a qAF value is demonstrated within each region. qAF values are also automatically entered into a spreadsheet format in a separate window.&#34;</li>
		<li>Generate qAF &#34;heat maps&#34; by selecting the option within the command prompts. All images and data can be exported from the IGOR software in an excel spreadsheet by selecting the appropriate options.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Strauss, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+retinal+pigment+epithelium+in+visual+function.">The retinal pigment epithelium in visual function.</a> Physiological reviews. 85, 845-881 (2005).</li><li>Ach, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+autofluorescence+and+cell+density+maps+of+the+human+retinal+pigment+epithelium.">Quantitative autofluorescence and cell density maps of the human retinal pigment epithelium.</a> Investigative ophthalmology &amp; visual science. 55, 4832-4841 (2014).</li><li>Ach, 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=Lipofuscin+redistribution+and+loss+accompanied+by+cytoskeletal+stress+in+retinal+pigment+epithelium+of+eyes+with+age-related+macular+degeneration.">Lipofuscin redistribution and loss accompanied by cytoskeletal stress in retinal pigment epithelium of eyes with age-related macular degeneration.</a> Investigative ophthalmology &amp; visual science. 56, 3242-3252 (2015).</li><li>Schmitz-Valckenberg, S., Jorzik, J., Unnebrink, K., Holz, F. G., Group, F. A. M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+digital+scanning+laser+ophthalmoscopy+fundus+autofluorescence+images+of+geographic+atrophy+in+advanced+age-related+macular+degeneration.">Analysis of digital scanning laser ophthalmoscopy fundus autofluorescence images of geographic atrophy in advanced age-related macular degeneration.</a> Graefe's archive for clinical and experimental ophthalmology = Albrecht von Graefes Archiv fur klinische und experimentelle Ophthalmologie. 240, 73-78 (2002).</li><li>Weng, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+the+function+of+Rim+protein+in+photoreceptors+and+etiology+of+Stargardt's+disease+from+the+phenotype+in+abcr+knockout+mice.">Insights into the function of Rim protein in photoreceptors and etiology of Stargardt's disease from the phenotype in abcr knockout mice.</a> Cell. 98, 13-23 (1999).</li><li>Holz, F. G., 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=Inhibition+of+lysosomal+degradative+functions+in+RPE+cells+by+a+retinoid+component+of+lipofuscin.">Inhibition of lysosomal degradative functions in RPE cells by a retinoid component of lipofuscin.</a> Investigative ophthalmology &amp; visual science. 40, 737-743 (1999).</li><li>Sparrow, J. R., Nakanishi, K., Parish, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lipofuscin+fluorophore+A2E+mediates+blue+light-induced+damage+to+retinal+pigmented+epithelial+cells.">The lipofuscin fluorophore A2E mediates blue light-induced damage to retinal pigmented epithelial cells.</a> Investigative ophthalmology &amp; visual science. 41, 1981-1989 (2000).</li><li>Smith, R. 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=Lipofuscin+and+autofluorescence+metrics+in+progressive+STGD.">Lipofuscin and autofluorescence metrics in progressive STGD.</a> Investigative ophthalmology &amp; visual science. 50, 3907-3914 (2009).</li><li>Delori, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+measurements+of+autofluorescence+with+the+scanning+laser+ophthalmoscope.">Quantitative measurements of autofluorescence with the scanning laser ophthalmoscope.</a> Investigative ophthalmology &amp; visual science. 52, 9379-9390 (2011).</li><li>Burke, T. 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=Quantitative+fundus+autofluorescence+in+recessive+Stargardt+disease.">Quantitative fundus autofluorescence in recessive Stargardt disease.</a> Investigative ophthalmology &amp; visual science. 55, 2841-2852 (2014).</li><li>Duncker, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+fundus+autofluorescence+and+optical+coherence+tomography+in+best+vitelliform+macular+dystrophy.">Quantitative fundus autofluorescence and optical coherence tomography in best vitelliform macular dystrophy.</a> Investigative ophthalmology &amp; visual science. 55, 1471-1482 (2014).</li><li>Duncker, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+fundus+autofluorescence+distinguishes+ABCA4-associated+and+non-ABCA4-associated+bull's-eye+maculopathy.">Quantitative fundus autofluorescence distinguishes ABCA4-associated and non-ABCA4-associated bull's-eye maculopathy.</a> Ophthalmology. 122, 345-355 (2015).</li><li>Greenberg, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+fundus+autofluorescence+in+healthy+eyes.">Quantitative fundus autofluorescence in healthy eyes.</a> Investigative ophthalmology &amp; visual science. 54, 5684-5693 (2013).</li><li>Delori, F. C., Goger, D. G., Dorey, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+accumulation+and+spatial+distribution+of+lipofuscin+in+RPE+of+normal+subjects.">Age-related accumulation and spatial distribution of lipofuscin in RPE of normal subjects.</a> Investigative ophthalmology &amp; visual science. 42, 1855-1866 (2001).</li><li>Sparrow, J. 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=Quantitative+fundus+autofluorescence+in+mice:+correlation+with+HPLC+quantitation+of+RPE+lipofuscin+and+measurement+of+retina+outer+nuclear+layer+thickness.">Quantitative fundus autofluorescence in mice: correlation with HPLC quantitation of RPE lipofuscin and measurement of retina outer nuclear layer thickness.</a> Investigative ophthalmology &amp; visual science. 54, 2812-2820 (2013).</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, 1250-1265 (2007).</li></ol>

STA: no disclosures, JPG: no disclosures, RTS: consultant, Ocata Therapeutics.

Abnormal RPE lipofuscin distribution, whether increased or decreased, is a sensitive marker of retinal disease and is generally associated with loss of sensory retina function. Here, we describe application of qAF for the evaluation of RPE lipofuscin. Incorporation of an internal fluorescent reference to correct for variable laser power and detector sensitivity9 alongside our standardized imaging technique allows for reliable quantitation of AF levels. It is our goal that this method will aid in the diagnosis and monitoring of retinal disease, and eventually in assessing the efficacy of therapeutic interventions, such as drug or gene therapy. qAF may also assist in stratification of individuals at-risk for conditions such as AMD.
We have generated a large normative database of qAF data to be used as a reference tool for the interpretation of retinal pathology 13, and have also described qAF in several disease states, including Stargardt disease 10, bull&#39;s eye maculopathy 12 and Best disease 11. In healthy retina, there are different qAF levels between ethnic groups, with significantly higher qAF in whites than blacks and Asians, and demonstrated that women have higher qAF levels than men. Perhaps most strikingly, qAF increases as patients age, which corresponds with RPE lipofuscin levels, as previously measured by spectrofluorometry 14 . Although current normative data only extend up to age 60, it appears that patients have a measured decrease in RPE lipofuscin after age 70 14. Interestingly, it does not appear that changes in RPE cell number occur as patients age 2and so the observed decreases in AF may be due to a redistribution or reduction in RPE lipofuscin 3. It will be interesting to determine whether these decreases in AF in old age correlate with impaired RPE function and increased risk of AMD.
This protocol allows for reliable quantitative results that can be compared longitudinally, between patients, and between cSLO machines. One limitation is that qAF cannot accurately measure AF in older patients with lens opacities, which limits the technique in a subset of this high-risk population. One possible solution is to use a reflectometry method to individually estimate light losses in the media. Fortunately, older patients who are pseudophakic with clear posterior capsules and known intraocular lens transmission characteristics are good candidates for qAF. However, the pupils of elderly patients and those with prior cataract surgery often do not dilate to more than 6mm, which limits light passage and prevents accurate data collection. In order to image such patients, an aperture may be inserted into the cSLO by the manufacturer in order to decrease the diameter of the laser. This modification has been successfully utilized to perform qAF imaging in mice 15. Another limitation is that the technique is challenging to perform and is highly operator dependent. It is essential to consistently obtain high quality images to ensure accurate measurements of AF levels. In order to achieve this, the authors recommend strict adherence to the imaging protocol as well as adequate practice. Although some patients poorly tolerate the bright flashes of light required for AF imaging, assuring patients that exposure levels are well within the safe limits is helpful 1. To allow for optimal imaging, it is critical to dilate pupils to at least 6 mm and to obtain multiple images with unobstructed light transmission (see above). Achieving optimal focus and avoiding oversaturation of pixels are also essential. Helpful practices include communication with the patient while imaging, use of an assistant if the eyelid needs to be lifted and use the foot pedal for triggering the camera, as described above.
In summary, understanding the pathophysiology of the RPE in retinal degenerations remains an area of active research, and of potentially important therapeutic impact. As qAF allows for direct comparison of AF levels in images obtained longitudinally, between patients and between centers, it is a valuable tool that can contribute to this understanding as well as provide useful clinical information. The detailed protocol outlined here will assist others in the acquisition of reliable qAF data, and that the important clinical applications of qAF will encourage research centers and clinical retina specialists to make use of the qAF technique.

The retinal pigment epithelium (RPE) supports the function of the sensory retina through numerous processes 1. Age-related macular degeneration (AMD) is the most important cause of untreatable blindness in industrialized countries and is characterized by changes in the RPE, including loss of pigment, loss of function and atrophy. In AMD and in normal aging, the RPE accumulates fluorescent, lysosome-derived organelles containing phagocytosed photoreceptor fragments, referred to as lipofuscin granules. The accumulation of RPE lipofuscin has been thought to indicate oxidative dysfunction 1, but recent studies have shown that the RPE morphology remains normal in aged eyes with high lipofuscin levels 2. However, abnormal patterns of lipofuscin distribution, in particular loss of lipofuscin, are documented markers for AMD and AMD progression, both histologically and clinically 3,4
Defective processing of RPE lipofuscin has also been shown to occur in certain inherited retinal degenerations. Patients suffering from Stargardt disease (STGD) accumulate lipofuscin in the RPE at a young age, eventually developing vision loss similar to that seen in AMD 5. These findings suggested that lipofuscin accumulation may itself be toxic and drive RPE dysfunction 6,7. However, a detailed imaging study of subjects with STGD over time did not confirm that focal lipofuscin accumulation led to subsequent RPE loss 8. Hence, although lipofuscin abnormalities are markers for retinal degenerations, a role for direct toxicity of lipofuscin remains unproven.
The RPE is the most posterior cell layer of the retina, but generates the majority of fluorescent signal from the ocular fundus. Generation and detection of autofluorescence (AF) derived from the RPE can be performed using confocal scanning laser ophthalmoscopy (cSLO), which allows for visualization of the spatial distribution of fundus AF. Certain retinal degenerations demonstrate distinctive patterns of fundus AF, and AF imaging aids in the diagnosis and monitoring of these conditions. Although standard AF imaging is clinically important, quantitative AF (qAF) has become an important means of assessing RPE health. We and others have developed a standardized approach that can reliably determine qAF levels at specific retinal locations 9. qAF has potential applications in the diagnosis and monitoring of retinal conditions, and may also have utility in prognosis and risk stratification. In addition, the diagnostic capabilities of qAF have also been described for certain retinal disorders 10-12. Here, we provide step-wise details for performing our technique accompanied by a visual demonstration of its application in the evaluation of healthy and diseased eyes.

<table border="0" cellpadding="0" cellspacing="0" width="671">
	<tbody>
		<tr height="19">
			<td height="19" style="height:19px;width:243px;">Spectralis HRA + OCT</td>
			<td style="width:143px;">Heidelberg Engineering</td>
			<td style="width:119px;">n/a</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">0.5% tropicamide ophthalmic solution</td>
			<td style="width:143px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>Any brand can be used</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">2.5% phenylephrine ophthalmic solution</td>
			<td style="width:143px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>Any brand can be used</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:243px;">Internal fluorescent reference</td>
			<td style="width:143px;">Heidelberg Engineering</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">IGOR Pro software</td>
			<td style="width:143px;">WaveMetrics</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitative fundus autofluorescence for the evaluation of retinal diseases

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks performed by the RPE are phagocytosis and processing of outer photoreceptor segments through lysosome-derived organelles. These degradation products, stored and referred to as lipofuscin granules, are composed partially of bisretinoids, which have broad fluorescence absorption and emission spectra that can be detected clinically as fundus autofluorescence with confocal scanning laser ophthalmoscopy (cSLO). Lipofuscin accumulation is associated with increasing age, but is also found in various patterns in both acquired and inherited degenerative diseases of the retina. Thus, studying its pattern of accumulation and correlating such patterns with changes in the overlying sensory retina are essential to understanding the pathophysiology and progression of retinal disease. Here, we describe a technique employed by our lab and others that uses cSLO in order to quantify the level of RPE lipofuscin in both healthy and diseased eyes.

This technique was used to study qAF in both healthy 13 and disease states 10-12. In healthy eyes (Figure 1), AF emitted from the RPE is distributed relatively uniformly throughout the fundus (Figure 1A). Reduced intensity is seen in the central macular region due to blocking of light by macular pigment, and at the sides and corners of the image due to the optics of the eye and camera. Vessels appear dark and should be in clear focus in well-acquired images. Figure 1B demonstrates a corresponding heat map representation of the qAF levels from Figure 1A. Cooler colors correspond to areas of lower intensity while warmer colors correspond to areas of higher intensity. Maximum intensity is generally seen in the second concentric 8-segment ring (indicated in Figure 1B). This region is also less subject to imaging related variability than regions closer to the image borders, and is outside of the central region where macula pigment has a large impact on qAF levels. Thus, the mean intensities of this ring are used for most data analyses 13. Figure 2 presents a representative analysis in an eye with AMD demonstrating geographic atrophy (GA), an advanced form of AMD. This form of AMD results in localized areas of RPE loss, evidenced by markedly reduced or absent AF, and causes progressive central vision loss.
<img alt="Figure 1" src="/files/ftp_upload/53577/53577fig1.jpg" /> 
Figure 1. Autofluorescence in healthy eye. (A) Autofluorescence (AF) image of the right eye of a normal patient. OD: optic disk, Fo: fovea, Ma: macula, Ref: internal reference. (B) Post-processing qAF map of AF image from (A). Warmer colors correlate to higher AF intensity. Fixed regions are shown, and qAF values of each region are indicated. 8-segment perifoveal ring used in data analysis is indicated by the dashed line. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53577/53577fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53577/53577fig2.jpg" /> 
Figure 2. Autofluorescence in eye displaying geographic atrophy due to AMD. (A) Autofluorescence (AF) image of the left eye of a patient with advanced AMD showing geographic atrophy (GA) of the RPE (representative region circumscribed by dashed line). OD: optic disk, Fo: fovea, Ma: macula, Ref: internal reference. Note markedly reduced and absent AF in regions corresponding to GA in the macula. (B) Post-processing qAF map of AF image from (A). Fixed regions are shown, and qAF values of each region are indicated. 8-segment perifoveal ring used in data analysis is indicated by the dashed line. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53577/53577fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Quantitative Fundus Autofluorescence for the Evaluation of Retinal Diseases at JoVE.com

Quantitative Fundus Autofluorescence for the Evaluation of Retinal Diseases

The retinal pigment epithelium (RPE) supports the sensory retina through recycling visual cycle byproducts, which accumulate as lipofuscin. These products are autofluorescent and can be qualitatively imaged in vivo. Here, we describe a method to quantitatively image RPE lipofuscin using confocal scanning laser ophthalmoscopy.

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks ...

quantitative-fundus-autofluorescence-for-evaluation-retinal

Nanyang Technological University

Research

JoVE Journal

Medicine

11.3K Views.  NYU School of Medicine. The retinal pigment epithelium (RPE) supports the sensory retina through recycling visual cycle byproducts, which accumulate as lipofuscin. These products are autofluorescent and can be qualitatively imaged in vivo. Here, we describe a method to quantitatively image RPE lipofuscin using confocal scanning laser ophthalmoscopy. 

Video: Quantitative Fundus Autofluorescence for the Evaluation of Retinal Diseases

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

Techniques for Processing Eyes Implanted with a Retinal Prosthesis for Localized Histopathological Analysis: Part 2 Epiretinal Implants with Retinal Tacks

Retinal prostheses for the treatment of certain forms of blindness are gaining traction in clinical trials around the world with commercial devices currently entering the market. In order to evaluate the safety of these devices, in preclinical studies, reliable techniques are needed. However, the hard metal components utilised in some retinal implants are not compatible with traditional histological processes, particularly in consideration for the delicate nature of the surrounding tissue. Here we describe techniques for assessing the health of the eye directly adjacent to a retinal implant secured epiretinally with a metal tack.
Retinal prostheses feature electrode arrays in contact with eye tissue. The most commonly used location for implantation is the epiretinal location (posterior chamber of the eye), where the implant is secured to the retina with a metal tack that penetrates all the layers of the eye. Previous methods have not been able to assess the proximal ocular tissue with the tack in situ, due to the inability of traditional histological techniques to cut metal objects. Consequently, it has been difficult to assess localized damage, if present, caused by tack insertion.
Therefore, we developed a technique for visualizing the tissue around a retinal tack and implant. We have modified an established technique, used for processing and visualizing hard bony tissue around a cochlear implant, for the soft delicate tissues of the eye. We orientated and embedded the fixed eye tissue, including the implant and retinal tack, in epoxy resin, to stabilise and protect the structure of the sample. Embedded samples were then ground, polished, stained, and imaged under various magnifications at incremental depths through the sample. This technique allowed the reliable assessment of eye tissue integrity and cytoarchitecture adjacent to the metal tack.

Here we describe histological techniques for visualising ocular tissue directly adjacent to a metal epiretinal tack and retinal prosthesis.

Retinal prostheses for the treatment of certain forms of blindness are gaining traction in clinical trials around the world with commercial devices ...

Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

3-[18F]fluoro-4-aminopyridine, [18F]3F4AP, is a radiofluorinated analog of the FDA-approved drug for multiple sclerosis 4-aminopyridine (4AP). This compound is currently under investigation as a PET tracer for demyelination. We recently described a novel chemical reaction to produce metafluorinated pyridines consisting of direct fluorination of a pyridine N-oxide and the utilization of this reaction for the radiochemical synthesis of [18F]3F4AP. In this article, we demonstrate how to produce this tracer using an automated synthesizer and an in-house made flow hydrogenation reactor. We also show the standard quality control procedures performed before releasing the radiotracer for preclinical animal imaging studies. This semi-automated procedure may serve as the basis for future production of [18F]3F4AP for clinical studies.

Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

We demonstrate the semi-automated radiochemical synthesis of [18F]3F4AP and quality control procedures.

3-[18F]fluoro-4-aminopyridine, [18F]3F4AP, is a radiofluorinated analog of the FDA-approved drug for multiple sclerosis 4-aminopyridine (4AP). This ...

Smartphone Fundus Photography

Smartphone fundus photography is a simple technique to obtain ocular fundus pictures using a smartphone camera and a conventional handheld indirect ophthalmoscopy lens. This technique is indispensable when picture documentation of optic nerve, retina, and retinal vessels is necessary but a fundus camera is not available. The main advantage of this technique is the widespread&#160;availability of smartphones that allows documentation of macula and optic nerve changes in many settings that was not previously possible. Following the well-defined steps detailed here, such as proper alignment of the phone camera, handheld lens, and the patient&#39;s pupil, is the key for obtaining a clear retina picture with no interfering light reflections and aberrations. In this paper, the optical principles of indirect ophthalmoscopy and fundus photography will be reviewed first. Then, the step-by-step method to record a good quality retinal image using a smartphone will be explained.

Fundus photography normally requires specialized fundus cameras that are not always available in all clinical settings.&#160;Here, a simple method to record ocular fundus images using a smartphone camera and a conventional high-plus handheld indirect ophthalmoscopy lens is described.

Smartphone fundus photography is a simple technique to obtain ocular fundus pictures using a smartphone camera and a conventional handheld indirect ...

Using Retinal Imaging to Study Dementia

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central nervous system (CNS) and shares prominent similarities with the brain in terms of embryological origin, anatomical features and physiological properties.&#160; The vascular and neuronal structure in the retina can now be visualized easily and non-invasively using retinal imaging techniques, including fundus photography and optical coherence tomography (OCT), and quantified semi-automatically using computer-assisted analysis programs. Studying the associations between vascular and neuronal changes in the retina and dementia could improve our understanding of dementia and, potentially, aid in diagnosis and risk assessment.&#160; This protocol aims to describe a method of quantifying and analyzing retinal vasculature and neuronal structure, which are potentially associated with dementia. This protocol also provides examples of retinal changes in subjects with dementia, and discusses technical issues and current limitations of retinal imaging.

The retina shares prominent similarities with the brain and thus represents a unique window to study vasculature and neuronal structure in the brain non-invasively. This protocol describes a method to study dementia using retinal imaging techniques. This method can potentially aid in diagnosis and risk assessment of dementia.

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central ...

Invasive Hemodynamic Assessment for the Right Ventricular System and Hypoxia-Induced Pulmonary Arterial Hypertension in Mice

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. However, the evaluation of right ventricular pressure (RVP) and pulmonary artery pressure (PAP) remains technically challenging in mice. RVP and PAP are more difficult to measure than left ventricular pressure because of the anatomical differences between the left and right heart systems. In this paper, we describe a stable right heart hemodynamic measurement method and its validation using healthy and PAH mice. This method is based on open-chest surgery and mechanical ventilation support. It is a complicated procedure compared to closed chest procedures. While a well-trained surgeon is required for this surgery, the advantage of this procedure is that it can generate both RVP and PAP parameters at the same time, so it is a preferable procedure for the evaluation of PAH models.

Here, we present a protocol to perform an invasive hemodynamic assessment of the right ventricle and pulmonary artery in mice using an open-chest surgery approach.

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. ...

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR model induction and its readouts in both mice and rats. Retinal neovascularization is induced in OIR by exposing rodent pups either to hyperoxia (mice) or alternating levels of hyperoxia and hypoxia (rats). The primary readouts of these models are the size of neovascular (NV) and avascular (AVA) areas in the retina. This preclinical in vivo model can be used to evaluate the efficacy of potential anti-angiogenic drugs or to address the role of specific genes in the retinal angiogenesis by using genetically manipulated animals. The model has some strain and vendor specific variation in the OIR induction which should be taken into consideration when designing the experiments.

Oxygen-induced retinopathy (OIR) can be used to model ischemic retinal diseases such as retinopathy of prematurity and proliferative diabetic retinopathy and to serve as a model for proof-of-concept studies in evaluating antiangiogenic drugs for neovascular diseases. OIR induces robust and reproducible neovascularization in the retina that can be quantified.

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR ...

Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These conditions include retinitis pigmentosa (RP) and advanced forms of age-related macular degeneration (AMD), such as geographic atrophy (GA). Together, these disorders represent a significant proportion of currently untreatable blindness globally. These unmet medical needs have generated heightened academic interest in developing methods of RPE replacement. Among the animal models commonly utilized for preclinical testing of therapeutics, the non-human primate (NHP) is the only animal model that has a macula. As it shares this anatomical similarity with the human eye, the NHP eye is an important and appropriate preclinical animal model for the development of advanced therapy medicinal products (ATMPs) such as RPE cell therapy.
This manuscript describes a method for the submacular transplantation of an RPE monolayer, cultured on a polyethylene terephthalate (PET) cell carrier, underneath the macula onto a surgically created RPE wound in immunosuppressed NHPs. The fovea-the central avascular portion of the macula-is the site of the greatest mechanical weakness during the transplantation. Foveal trauma will occur if the initial subretinal fluid injection generates an excessive force on the retina. Hence, slow injection under perfluorocarbon liquid (PFCL) vitreous tamponade is recommended with a dual-bore subretinal injection cannula at low intraocular pressure (IOP) settings to create a retinal bleb. 
Pretreatment with an intravitreal plasminogen injection to release parafoveal RPE-photoreceptor adhesions is also advised. These combined strategies can reduce the likelihood of foveal tears when compared to conventional techniques. The NHP is a key animal model in the preclinical phase of RPE cell therapy development. This protocol addresses the technical challenges associated with the delivery of RPE cellular therapy in the NHP eye.

The non-human primate (NHP) is an ideal model for studying human retinal cellular therapeutics due to the anatomical and genetic similarities. This manuscript describes a method for submacular transplantation of retinal pigment epithelial cells in the NHP eye and strategies to prevent intraoperative complications associated with macular manipulation.

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These ...

Retinal Pathophysiological Evaluation in a Rat Model

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal detachment, breakdown of the blood-retinal barrier (BRB), and retinal angiogenesis. An in vivo rat model is a valuable experimental tool to examine the changes in the structure and function of the retina. We propose three different experimental techniques in the rat model to identify morphological changes of retinal cells, retinal vasculature, and compromised BRB. Retinal histology is used to study the morphology of various retinal cells. Also, quantitative measurement is performed by retinal cell count and thickness measurement of different retinal layers. A BRB breakdown assay is used to determine the leakage of extraocular proteins from the plasma to vitreous tissue due to the breakdown of BRB. Fluorescence angiography is used to study angiogenesis and leakage of blood vessels by visualizing retinal vasculature using FITC-dextran dye.

Diabetic retinopathy is one of the leading causes of blindness. Histology, blood-retinal barrier breakdown assay, and fluorescence angiography are valuable techniques to understand the pathophysiology of the retina, which could further enhance the efficient drug screening against diabetic retinopathy.

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal ...

Repetitive Blood Sampling from the Subclavian Vein of Conscious Rat

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to detect drug exposure. The rat blood sampling method determines the quality of the plasma and further affects the precision of the test results. The subclavian vein blood collection method described in this protocol collects blood samples repeatedly in the conscious state of animals to meet the needs of PK and TK tests. The skills of restraint handling and appropriate procedure of needle incision ensure the success rate of blood collection. It is easy to operate while ensuring the quality of plasma and at the same time catering to animal welfare. However, this method requires skilled operation, and an improper one may cause animal weakness, pain, lameness, and even mortality. The current method has been used in the testing facility for a 4-week oral toxicity study in Sprague Dawley (SD) rats with TK. The maximum amount of blood collected within 24 h did not exceed 20% of the animal's total blood. The animals' body weight was more than 200 g for males and females. The data showed that the animals' bodyweight increased steadily every week, and the clinical observation was normal after repetitive sample collection.

The present protocol describes a simple and efficient method for collecting blood from the subclavian vein in rats. It enables rapid, timely, and easily identifiable sampling without anesthesia and obtains high-quality blood through repetitive sample collection.

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to ...