Protocol - Retinal Digital Photography

Note: Example photographs A6, A7, and A8 referenced in the digital grading section of this protocol are provided and used with permission by the Atherosclerosis Risk in Communities (ARIC) Study Research Group.

Photography Protocol

1. EQUIPMENT SET-UP

The Canon Non-Mydriatic Retinal CR6-45NM camera equipped with a digital Canon D-60 camera back should be placed in a room that can be completely darkened during the photography procedure. This is because no dilating drops will be used and we rely on the natural dilation of the pupil that occurs in the dark to perform the fundus photography. The camera should be placed in the room so that the patient has easy access to the examination stool and so that the photographer has ready access to the room light switch. A small reading lamp located near the camera may be used to help the photographer see to navigate and write during the procedure. This lamp should be turned off during photography to reduce the risk of pupil constriction. Light from the computer screen may need to be adjusted to minimize pupil constriction.

The fundus camera will be set on the motorized instrument table and the laptop computer will be attached to the articulating arm located above the camera's video display monitor. The pneumatic stools are positioned on opposite sides of the fundus camera.

1.1 Daily Set-up Procedure

The camera dust cover and lens cap should be removed at the beginning of the day and the lens inspected and cleaned (see section 1.2) as necessary. Dust is the greatest enemy, producing the majority of artifacts on the photographs. When the camera is not in use, the lens cap should be in place and the special dust cover must remain on the camera.

1.2 Camera Lens and Body Care

Before each photograph, the camera lens must be inspected and, if dirty, cleaned with the brush and air bulb to remove debris. Should more extensive cleaning of the lens be required, the lens can be fogged with your breath or moistened with absolute alcohol and then cotton should be used in a circular polishing motion until no dirt or oily film is visible on the lens when it is viewed from the front with the alignment lens removed and the view lamp on and turned up to its near maximum intensity (see page 28 in the Canon Non-Mydriatic Retinal Camera CR6-45NM Operation Manual). The body of the camera should be kept clean and free of dirt with a soft cloth and water or a common spray cleaner. The forehead rest may be cleaned with alcohol.

1.3 Flash, View and Split Lamp Concerns

The Canon cameras are equipped with new lamps at the beginning of the study. It is not anticipated that the flash, view or split lamps will fail during the study. The view and split lamp should last approximately one to two years and are easily replaced as needed. The flash lamp has a life of at least 5,000 flashes. Since the view lamps are relatively inexpensive bulbs, one spare should be ordered from Canon USA, Inc. and kept at the field center. Clinic staff in the field can replace the view lamp. Remember to keep all oil from your fingers off these lamps during replacement. The flash and split lamp should be replaced by a qualified technician. Both lamps can be ordered from Canon USA, Inc. when needed for overnight delivery.

As the flash lamp ages, the light output can diminish, producing progressively darker photographs. This can temporarily be over-ridden by an adjustment of the transformer output, though ultimately the lamp should be replaced. The flash lamp requires careful handling during installation (the burnt out lamp may be hot, and the new lamp must be properly aligned. Replacement should be attempted only by staff trained to do this.

2. SUBJECT PHOTOGRAPHY OVERVIEW

Digital images will be captured into a table mounted laptop computer, archived to CDs for analysis. Before the first patient of the day is photographed, the photographer prepares the camera by first turning on the power to the Canon D-60 camera back. The power is then turned on for the Canon Non-Mydriatic Retinal CR6-45NM camera and to the Dell laptop computer. The photographer escorts the patient into the photography room, positioning them before the fundus camera, and explains the procedure. The EyeQSL software program is initiated and subject ID and photographer information is entered into the appropriate computer screens and the software is set to acquire images.

Patients will have one eye assigned as the study eye and the other eye assigned as the

fellow eye. Photography will be performed of both eyes with the study eye photographed

first. All subjects will have two 45-degree photographs taken of each eye. The first photograph will be centered on the optic nerve and this is referred to as Field 1. The second photograph will be centered on the macula and will be referred to as Field 2. A diagram of the location of these two fields can be seen below in Figure 1. The images are stored on the hard drive of the computer as the procedure for the first patient is ended and the camera is prepared for images acquisition of the next subject. At the end of each week, the images are archived to CD.

Figure 1 - Retinal Digital Photography

The study eye will be selected based on the subject's ID number. When the sixth digit of the ID number is even, the right eye will be photographed first, and when it is odd; the left eye will be photographed first. If the eye specified by this algorithm is considered too difficult to photograph with adequate photographic quality, only the opposite eye should be photographed, and an explanatory note entered in the MESA Photography Completion form. Conditions falling into this category are (based upon the technician's judgment): eye missing, inability to dilate at least 4 mm, inability to fixate adequately for proper photographic fields, and opacities of the media preventing a reasonably clear view of the retinal vasculature and resulting in unreadable digital images.

2.1 Subject Exclusion

The photographer will attempt photography on subjects with poor visual acuity who may be unable to direct their gaze so that their optic nerve or macula is properly positioned on the camera monitor (as may be the case where both eyes are blind or when the subject is deaf and communication with them is impossible). In these cases, the photographer should get the best field definition possible. If, in the photographer's judgment, no acceptable photograph can be taken of either eye, the subject will be excused from photography.

The photographer should attempt photography on those subjects who are physically disabled, to the point that they can be comfortably positioned at the camera. To facilitate this, the subject may remain in a wheel chair positioned before the motorized camera table lowered to the appropriate height. Care should be taken when lowering the camera table to avoid pressing against the subject's legs. If, in the photographer's estimation, the subject cannot be comfortably positioned, no photography will be performed.

2.2 Subject Explanation

Photography begins with a complete explanation of the procedure to the participant by the photographer. A color illustration may be useful to show what the retina looks like. It is important to reassure the subject that no retinal damage is caused by this procedure. The subject should know to expect several flashes. The pictures will include the macula (area of central vision) and it is normal to experience a blue or red tint to vision immediately following the flash. This disappears within five to seven minutes. No dilation drops will be used for this examination, and the eyes will not be touched. A sample script of a typical retinal photography explanation (suitable for use as written material for deaf or interested subjects) follows:

We will be taking several photographs of the inside of the back of both of your eyes (the retina) so we can study the blood vessels and look for any unusual changes. We will not be touching your eyes or be giving you any eye drops to take the pictures. Instead, you will be asked to sit in a darkened room before a special camera with your chin in a chin rest. We darken the room so that your pupils will dilate and we can align and focus the camera on your retina. While your pupils are dilating, we may ask you some questions about your vision and the health of your eyes. During the aligning process you will only be aware of some small red lights and a blinking green box and red bars visible in the camera lens. We will ask you to follow the blinking green box as we move it. Just before we take the picture, we will ask you to blink your eyes and then open them real wide. The camera will flash a light from within the camera lens as each picture is taken.

Just after the picture is taken, you may see a blue or red circular spot before the eye photographed. This will disappear within 5-7 minutes and causes no damage to the eye. Please remember that we are only taking pictures (not an x-ray) of a small portion of the back of your eyes and that this picture will not substitute as an eye examination. You will certainly be notified should we notice anything requiring immediate attention. Please continue to see your eye doctor on a regular basis for your complete eye examinations.

2.3 The Photography Completion Form

The Photography Completion Form records the circumstances of the photographic session, and can only be completed as the session begins. The participant's ID# and acrostic will appear at the top right hand corner of the form. The sixth digit of this number will determine the study eye (right eye if even, left eye if odd); this eye should be photographed first. Before beginning the photography session, the photographer records the site number, photographer ID#, and the photography date. If either eye cannot be photographed for a reason gathered during the ophthalmic history (e.g., that eye has been enucleated) or for a reason that emerges during the first part of the session (one eye does not dilate sufficiently well to be photographed), the photographer should record this on the form. The flash setting (2 unless re-adjusted by the photographer) and the estimated diameter of the pupil at the time of the first photograph of each eye should be recorded. The photographer is encouraged to comment on anything unusual such as artifacts, pathology or other problems encountered during the session. When the photography session is over and the photographer is reviewing the images in the contact strip, the eye and fields photographed should be marked. If extra images were taken, mark other and record the reason for extra images in the comments section. When the final images for each have been decided upon by the photographer, he/she can record the total number of images taken of each eye. Photocopies of this form will be mailed along with the images for analysis.

Please see Photography Completion Form

2.4 Preparing the Camera

The Canon D-60 digital camera back must be turned on first. This is done using the small dial located on the back left side of the camera body. This must precede turning on the Canon fundus camera or computer since the camera "looks" for the D-60 camera body during start up. The Canon CR6-45NM video display is activated when the power switch on the side of the main unit is turned on. If no photography or switch operations are performed for 10 minutes, a power saving mode is activated, turning the lamps and display off to prevent unnecessary wear. During this power saving mode a green "ready" lamp blinks on the monitor. Pressing any button below the monitor will reactivate the system.

Notice that the flash power setting (located on the lower right-hand corner of the monitor) blinks when the main unit is switched on. This indicates the system is charging up. Do not attempt to take photographs until the blinking stops, indicating a fully charged flash.

The camera contains an internal clock and the date will automatically change each day. The photographer must manually change the date if this clock should fail or if the camera is left unplugged for a long period of time. The date is displayed on the fundus camera monitor and is changed through the menu located in the "Set 3" screen (see pp. 18-20 in the Canon Non-Mydriatic Retinal Camera CR6-45NM Operation Manual). The date format will read Month-Day-Year. The display form "Disp Form" screen ("Date" subscreen) is used to adjust the current date.

The Canon D-60 camera body should be attached to the main unit and set with the following settings:

Shutter speed: 60
Mode Dial: Manual
AF-WB: Flash
Quality: First Level Large
Red Eye: Off
AEB: 0-
ISO: 400
Beep: Off
Auto power off: Off
Review: On
Review Time: 8 seconds
LCD: Normal
* The camera should remain ON until the end of the day

3. EXAMINATION PROCEDURE

3.1 Subject Positioning and ID Entry

The subject and photographer are seated on the appropriate sides of the retinal camera. The subject is positioned so that he/she is comfortable with chin and forehead in the headrest. Chin height should be adjusted so that the eyes are approximately level with the height adjustment mark on the face rest pole. The room is darkened to the level where a newspaper can barely be read (equal to about 5 lux) and the camera room door is closed. The only light in the room should come from the display monitors. If a lamp is used to aid the examiner during administration of the questionnaire, it must be turned off when photography is performed.

While the subject begins to dilate, the photographer begins the image capture procedure by double clicking the desktop Digital Healthcare Client icon and selecting the EyeQSL program. A "Login" screen will appear in which the name and password that has been setup for that computer are entered. An "Unarchived Visits" screen will appear and will contain images from any patients who have not yet been archived. Select the "Next" button on this screen to open the "Identify a Patient" screen. The photographer then enters the patient's subject ID number in the Patient Number. The subject's last name and first name are not used to preserve subject anonymity. The first six letters of the acrostic (name code) and photographer ID# should be filled in the appropriate boxes. These fields must be filled in before the photographer is able to begin capturing images. Additional information may be entered into fields as indicated in the study protocol. The "Next" button is clicked until the "Confirm Patient number" box appears, at this point, the patient number is re-entered and the "OK" button is selected. A "Visual Acuity" box will appear, click "Cancel".

The photographer is ready to begin taking photographs when the 4-panel split screen and timer box appear. The timer box displays the time the photograph was taken and also the current photo number.

When the photographer takes a picture, the timer stops and the image will briefly appear on a screen located on the digital camera back. It will then take a few seconds for the image to transfer from the camera to the computer screen. The timer will restart after the image appears on the screen. The photographer will review the image for quality and may take another image if they believe a better quality image can be obtained or they may accept the image by simply taking the next photograph when the patient is ready and the pupil is adequately dilated.

At the time the photographer has completed the patient's photographs, he/she will select the "End" button located on the timer box. The photographs that have been taken will then appear on the computer screen in a "Contact Strip" box. The photographer reviews all of the images in the contact strip and may delete any unwanted images at this time. This is done by left clicking on the image to be deleted, making the gray border around the image turn purple. Select "Delete from..." on the menu that appears. Select "Delete from Visit." The photographer may then elect to continue the photography session, if they wish to retake a particular field. Once the technician is satisfied with the photographs, he or she can select "Quit" to end the photography session. At this point, a new subject may begin being photographed by filling in the new patient information and repeating the steps stated above. If no other participants are to be seen, the photographer can "Quit" the program.

3.2 Pupil Size and External Camera Alignment

The camera stage holding knob is unlocked, the alignment switch is turned on and the stage is moved to center the eye to be photographed horizontally and the height adjustment ring is used to position the eye vertically. The pupil should appear on the TV screen coincident with the central circle on the monitor. The camera joystick is moved forwards or backwards until the pupil appears perfectly round. At this point, proper external alignment has been achieved. A pupil larger than the central 4mm circle on the monitor is required for adequate photography. If the eye does not dilate to at least 4mm after a 5-minute waiting period, the fellow eye should be examined for pupillary dilation as well. If dilation of the fellow eye is larger, the photographer will photograph the fellow eye first. At this point, the pupil size is measured using the alignment circle on the monitor as a gauge. This measurement is estimated to the nearest 1mm and it is recorded on the Photography Completion form.

3.3 Internal Eye Alignment

Once proper external pupil alignment is achieved, the alignment switch is pressed to provide a view of the fundus, split focusing lines, corneal reflection dots, and the fixation light. If no split lines are seen, the height or left/right adjustment is improper, the "Split" (split lines) setting is set to "off" (Set 1, Split/FCD), or the diopter compensating slider is pulled out. The split lines may fade in and out if the pupil is too small, the alignment of the camera is not centered on the pupil, or if the eyelashes or lids eclipse the light. If no corneal reflection dots are seen, the forward/backward adjustment is improper. The best photographs are obtained when the eye is well dilated, fixation is on the target; and lids and lashes are held wide open.

3.4 Alignment, Focus and Proper Fixation of Fundus Photographs

While viewing the fundus image on the screen, the photographer carefully adjusts the fixation target control button to locate Fields 1 and 2 correctly on the screen. Field 1 is always photographed first.

Any fine adjustment of fixation is made by moving the fixation lever and instructing the subject to look into the lens of the camera at the green target box. In the event that the subject sees no fixation light with the eye being photographed, the photographer must carefully instruct the subject to make micro movements (fine movements up, down, left or right) until the protocol fields are located.

Once the fixation is confirmed, the photographer must constantly adjust and position the camera to maintain the correct position of the corneal reflection dots. It is important that these dots be properly positioned at the three and nine o'clock positions before the picture is taken. This will ensure the correct distance from the eye and will allow capture of a sharp digital image. Focus is done manually by dialing either of the focus knobs located on the side of the fundus camera. Proper focus is obtained by aligning the two horizontal focus bars visible on the fundus camera monitor (see p. 12 of the Canon Non-Mydriatic Retinal Camera CR6-45NM Operation Manual). Good focus is critical for grading photographs.

The photographer will instruct the subject to blink once or twice just before the picture is taken. This blinking will insure a moist (and subsequently clearer) cornea and will safeguard against unwanted blinks at the moment of exposure. Once alignment is satisfactory, the shutter release, located in the tip of the joystick, is depressed and the exposure is made. The quality of the digital image is evaluated by the examiner and ungradable images, which in the opinion of the photographer may be improved, may be retaken. Once the best quality image is obtained, the second field is photographed and the photographer conducts the same image evaluation procedure.

4. PHOTOGRAPHIC CHALLENGES

4.1 Photography Through Small Pupils

The photographer will experience much more difficulty attempting photography through small (less than 4mm) pupils because all of the camera light doesn't enter through the smaller pupil. This usually results in uneven illumination (seen as dark shadows) on the monitor. In this situation, the photographer must make careful camera adjustments to position the shadows as far away from the optic nerve in the Field 1 photo and away from the macula on the photo of Field 2.

A small percentage of subject's eyes will not dilate to the minimum 4mm required for adequate photography. Certain medication may prevent any dilation and the pupil size observed on the monitor may be 2-3mm, inadequate for the photographer to appreciate all retinal landmarks on the viewing monitor. In this case it is recommended that the photographer introduce the small pupil aperture located on the left side of the fundus camera body (see p.10 of the Canon Non-Mydriatic Retinal Camera CR6-45NM Operation Manual). This function adjusts the illumination to concentrate more of the retinal illumination into a smaller area providing more even illumination to the center of the field while sacrificing illumination at the field perimeter. If after introducing the small pupil function only one focus line is visible, it might be necessary to adjust the camera position either vertically or horizontally to bring the second line into view so that accurate focus can be set. However, there may be circumstances that prevent the second line from appearing, even when the camera adjustments are made. In these cases it may be helpful to remove the small pupil function momentarily while searching for the second line and adjusting focus, reintroducing the small pupil function before taking the photograph.

If no retinal landmarks are visible, often the case with ≤2mm diameter pupils, the photographer should adjust the camera slightly to position the corneal reflection dots slightly above or below their optimum position. This technique allows a portion of the illumination light (which falls on the iris when the pupil is small) to enter the eye. If any retinal landmarks become visible with this technique, a picture should be taken. However, if no retinal landmarks are visible, no picture is taken. The photographer notes the problem on the Photography Completion form.

4.2 Exposure Compensations for Dark or Light Retinas

The normal flash output used in this study will be flash number 2. The flash output can be increased or decreased by first pressing the "DA" button followed by the +/- buttons located under the main video display screen of the fundus camera monitor. Higher numbers increase flash output and produce brighter images while lower numbers decrease flash output and produce darker images. A one number increase or decrease in flash intensity is usually all that is needed. Photography through small pupils, media opacities or of darkly pigmented retinas (Black or Asian) will require increased flash output (to flash 3) to avoid underexposed pictures. Photography of lightly pigmented retinas (blond, albino or Scandinavian) will require decreased flash output (to flash 1) to avoid overexposed pictures. The photographer should check the color saturation of the first image taken of each participant, and adjust the flash if necessary before proceeding.

4.3 Focus with High Myopia or Hyperopia

The diopter compensation slide should be set to the "0" position for most eyes. This is the only setting in which the focus bars are active and allows photography of eyes with refractions between -12 and +15 diopters. In the event that the eye photographed falls outside this range and focus cannot be achieved, as in the case of aphakia or high myopia, the diopter compensation slider must be adjusted for the clearest focus to the "+" or "-" position and the focusing knob is then turned manually to provide the sharpest image as seen on the monitor. Obtaining a brighter retinal image by increasing the view light intensity can facilitate this. The normal setting for the view light intensity adjustment is approximately 4.

Standard TV monitor functions can be adjusted for the photographer's viewing comfort (including contrast and brightness) by opening the access door below the TV monitor. These are standard controls similar to those found on a home TV set and only effect viewing; they do not affect final photo quality.

DIGITAL GRADING PROTOCOL

The photograph grader views each retinal image with a high resolution monitor using the EyeQ Lite image processing software and database, and references the written protocol and the digital photographic standards and examples to evaluate retinal abnormalities. The photograph grader directly enters his/her evaluations in a micro computer database.

1. USING THE IMAGE PROCESSING SOFTWARE/DATABASE

1.1 LOGGING IN

To access the image processing database the grader will double click on the Digital Healthcare EyeQ grading icon on the desktop. The grader will then be asked to log into the database. The login is the same for all workstations with the both the user name and password case sensitive). Then the grader selects OK from the login screen.

Login: Name:
Password:
Select OK

1.2 SELECTING A PATIENT VISIT FOR VIEWING

The first screen to appear is the Identify a Patient screen. The grader may type in the Study ID # of the patient to be reviewed and then click on Next. The database will find any patient visits with that study ID and display a list of visits with their corresponding study dates in the Patient Found in Database menu. Double click on the patient visit to be viewed. A final menu called Select Visit/Study will appear. Again double click on the visit to be viewed. A "contact strip" of 4 or more images will appear. This contact strip should contain field 1 and 2 of both eyes. (Additional images may be present.)

To select an image from the Contact Strip for further viewing or measurements either, right click on that image and the image will enlarge to full size (left clicking on the image (a purple frame will appear around the selected image) and selecting view image from the side bar menu will accomplish the same thing). Note: The title of the sidebar menu is helpful to remember in negotiating through the software.

1.3 IMAGE MANIPULATION TOOLS

There are many image manipulations available using the EyeQ software. Change in magnification, various manipulations of contrast, and splitting the image into the separate red, green and blue color channels are among the most commonly used. Further descriptions of the EyeQ software can be found in the user's manual.

The manipulations that will be performed on all images before measurements are performed are listed below in order. After each manipulation the image should be returned to its normal state before starting the next manipulation. If at any time the grader notes a suspicious area in the image the grader may zoom in/out and/or lighten/darken to enhance the area of concern

Zoom In (or Zoom Out): This will magnify the entire image for viewing. Magnifying the image two times at most is usually adequate (available on the View Image Menu). To magnify a localized area of the retina (to closely examine retinal vessels or specific lesions or color changes) the grader can left click on mouse when the magnifying glass is displayed. This can be done at any time during image manipulation even after a complete zoom has been performed. Another method for zooming in is to use the scrolling wheel on the mouse. This can be done at any time.

Lighten (or darken): (This option is available in the contrast menu only.) When an image is particularly light or dark this function is very helpful. One or two clicks is adequate to allow better viewing of poorly illuminated areas of the retina (similarly for overly exposed retinal images and darkening).

Image Manipulation Steps for all images:

1. Zoom in (lighten/darken/stretch as needed)

2. Red-Free (or 3 RGB components) (stretch lighten/darken as needed)

Optional steps if above steps demand more manipulations

3. Equalize Histogram

4. Invert image

5. Median ≥ 128

View Image Menu

1. Red-free or RGB color option: This provides the grader with a full screen view of the green channel. The grader should select Zoom In (or Zoom Out) and left click on the mouse to magnify a given area, and scan the entire photo. After magnification the grader should select the Contrast submenu, and Lighten (or darken), and/or Stretch the image for another view. (Again, suggest manipulating the image two times at most). To return the image to its normal state, click Undo to negate the lightening (or darkening) of the image and return grader to the original image. (Instead of choosing the Red-free option, the grader can view all of the color channels (red, green and blue) by selecting RGB. This will display a montage of the color image and each of its color components. The 3 color channels can be split and saved back to the contact strip for additional manipulations or measurements (lighten/darken, zoom in/out, stretch, etc) or the entire montage can be save as one image to the contact strip. It should be noted that saving these images is only temporary, remaining intact only while the grader has that patient file open. This montage is helpful in gauging areas of overexposure in the red channel, among other things.)

Contrast Menu

This menu takes the grader to a submenu of options for increasing contrast of an image. All manipulations are temporary and will never overwrite the study images. If the grader wishes to restore the image to its original state they should select Undo. If the grader wishes to perform additional manipulations or measurements on the image that are not available in the Contrast Menu then the grader should select Quit and Store. This will return the manipulated image to the contact strip. When the grader quits this patient visit, the contact strip will return to the original study images only.

2. Equalize histogram: This function examines the distribution of levels and redistributes them as evenly as possible across the range from 0 to 255 in all three channels. Values of pixels are changed so that an equal number of pixels have each of the possible levels. Thus, details in areas that are particularly bright, where there are many pixels at a similar brightness, are seen clearer. While this function dramatically shifts the colors of the image when combined with Lighten/Darken, it is often quite helpful in detecting small color changes, drusen or microaneurysms.

3.