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.

This method can help answer key questions in the field of ophthalmology and retinal imaging. Such as imaging and quantification of blood-retina barrier disruption and retinal capillary functions. The main advantage of this technique is that it can obtain a large field of view, three-dimensional, multi-contrast retinal imaging by using an oblique scanning laser in one single raster scan.

The implications of this technique extend toward diagnosis of diabetic retinopathy and other pre-retinal diseases. Because oSLO can obtain high-contrast images of retinal microvasculature, down to single capillaries in 3D. Though this method can provide insight into retinal imaging, it can also be applied to other imaging systems using conventional objective lens.

Such as in vivo imaging of the mouse cortex. A super-continuum laser source is used as the system laser source for the oblique scanning laser ophthalmoscopy, or oSLO setup. The visible light range is separated from higher-wavelength range by the first dichroic mirror.

The light spectrum is expanded with a pair of dispersive prisms, after the beam passes through a polarization beam splitter. A slit is used to select the excitation wavelength range. And a reflective mirror reflects the filtered beam back to the prism pair to couple the light into a single-mode fiber.

A spectrometer is used to confirm the wavelength selection at the output of the single-mode fiber. The single-mode fiber is connected to two cascaded optical fiber couplers. One of the fiber output port from the second fiber coupler delivers the light to the oSLO system.

To collimate the laser in the oSLO system, the laser is deflected by a galvanometer mirror. A one-to-one telescope system relays the laser to a second galvanometer mirror, and a three-to-one telescope system further relays the laser to the pupil of the eye. A dichroic mirror within the three-to-one telescope system reflects the fluorescent signals.

The three-to-one telescope system and the dichroic mirror is mounted on a customized dovetail slider to offset the optical axis and create the oblique scanning illumination. The oblique illumination allows the volumetric fluorescence imaging without a need to have sectioning. By offsetting the laser, an oblique beam is focused on retina, and then the oblique detection can capture a tomographic fluorescence image along the oblique beam path.

To create the fluorescence imaging optical path, the fluorescence is reflected by the dichroic mirror and relayed to the third galvanometer mirror. The fluorescent light is then relayed to an imaging objective lens by another one-to-one telescope system. Two additional translation stages are installed under the third galvanometer mirror to provide redundancy in the degrees of freedom for optimizing the image.

The final imaging system is mounted on a stage that has three degrees of freedom. Rotation, and two axes of translation. A planar camera is used to capture the cross-sectional fluorescence images.

Another dichroic mirror separates the rear infrared range from the remaining light. A long pass filter is used to further limit the bandwidth to 800 to 900 nanometers. Couple the beam into a single-mode fiber.

The single-mode fiber is connected to the other input port of the two cascaded optical fiber couplers to combine with the blue oSLO excitation. The light from the second output port of the second fiber coupler is directed to the OCT reference arm. Which has dispersion compensation plates, variable neutral density filter, and a reflective mirror.

The light return from the reference arm and the eye recombines at the second optical fiber coupler, and is delivered to the OCT spectrometer to collect the signal. Use a data acquisition system software written in Labview and modified from the scanning OCTA scanning protocol. For each b-scan, an 80%duty cycle sawtooth with 500 steps is output by an analog output board to control the x-prime fast scanning mirror.

Trigger the line scan camera at each step to acquire data for the OCT, only when the mirror is in the forward scanning direction. Set the exposure time for the line scan camera to be 17 microseconds. To acquire the OCTA signal, repeat the measurement five times at the same b-scan location.

Set the AO output rate at 100 kilohertz, and the OCT A-line rate at 50 kilohertz. Control the y-prime slow scanning mirror, GM1, by a ramping waveform. Synchronize the descanning mirror, GM3, with GM1 to descan the slow scanning.

Trigger the planar camera by another analog output board to capture one fluorescent image at each y-prime location. Crop the imaging size or bin the neighbor pixels to increase the speed and sensitivity as desired. Begin by confirming an appropriate level of anesthesia in the rat by a lack of a withdrawal reflex during an intradigital pinch.

After anesthesia induction, place the rat on a holder. Fit a nose cone to maintain the anesthesia during the remainder of the experiment. Apply 5 tetracaine hydrochloride ophthalmic solution on the rat's eye for local anesthesia.

Then dilate the pupil with 1%tropicamide ophthalmic solution. After two minutes of dilation, use a one-milliliter syringe and a 29-gauge needle to inject 10%fluorescein or 10%FITC diluted in saline through the tail vein. Then turn on the laser source, and place a neutral density filter to attenuate the blue light excitation during alignment.

Measure the power of blue light, ensuring that it is less than 10 microwatts. Then switch to the optical coherence tomography light, ensuring that it is close to 8 milliwatts. Turn on the power supply to the galvanometer mirror, which is used to control the direction of the laser.

Adjust the height of the eyeball to make a stationary laser spot on the cornea. Adjust the eye position to make the rim of the pupil roughly perpendicular to the laser. And offset the laser to about 1.5 millimeters from apical center of the eye.

Further adjust the animal holder until the optical coherence tomography images reach optimal quality. In the x-prime fast scanning direction, make sure that the cross-sectional b-scan image appears flat. When switching to the y-prime slow scanning direction, make sure the cross-section b-scan image appears tilted due to the oblique scanning.

Remove the neutral density filter to the blue light excitation. And monitor the real-time feed from the camera. A cross-sectional fluorescent image should appear showing blood vessels at different depths.

Adjust the focus of the final fluorescence imaging system to reach the optimal focus. And perform fine adjustments of the eye position in the lateral plane to reach optimal oblique scanning laser ophthalmoscopy image quality. After the alignment, begin to acquire simultaneous optical coherence tomography angiography and volumetric fluorescein angiography.

This image shows a cross-sectional optical coherence tomography image of a rat retina. This is an optical coherence tomography angiography, or OCTA image, of the same region. And an oblique scanning laser ophthalmoscopy and volumetric fluorescein angiography image cross-sectional fluorescein angiography, or oSLO-VFA.

Analogous to the optical coherence tomography b-scan. In comparison to OCTA, the oblique scanning laser ophthalmoscopy and volumetric fluorescein angiography cross-sectional image clearly identifies the capillaries in the outer plexiform layer. The superficial layer of the retina is shown here in an OCTA image.

Artefacts in the form of vertical stripes are visible in the image. oSLO-VFA avoids the motion artefacts by utilizing fluorescence emission contrast. Within the retinal intermediate layer, the vertically-diving vessels are clearly shown in the oSLO FA image.

But not apparent in OCTA. While attempting this procedure, it's important to avoid the continuous laser exposure to the eye for more than two minutes. Avoid corneal drying, and allow the eye to rest at least 30 seconds between imaging sections by blocking the light.

Following this procedure, other methods like imaging genetically-modified mice to express fluorescence proteins can be performed in order to answer additional questions. Like how specific retinal cell types can change, and the pulling past seen variables with known diseases.