>Simultaneous imaging of rat retina using optical coherence tomography and autofluorescence
Optical coherence tomography (OCT) and autofluorescence (AF) microimaging are two important biomedical optical imaging methods. In ophthalmic applications, OCT in the spectral domain is based on scattering contrast and provides high-resolution three-dimensional imaging capabilities, which has become an indispensable imaging method for diagnosing retinal diseases. AF imaging is based on detecting AF emissions to map the distribution of endogenous molecules when stimulated with photons of the appropriate wavelength. AF imaging has been shown to detect lipofuscin accumulation in retinal pigment epithelial cells (RPE), which is related to the pathophysiology of retinal aging and age-related macular degeneration (AMD). Dual-mode ophthalmic imaging combining OCT and AF may be a valuable tool for AMD research and clinical diagnosis.
This paper introduces the use of a single broadband light source with a center wavelength of 480nm to achieve OCT and AF dual-mode imaging with good image quality.
Figure 1 (a) shows the schematic diagram of the experimental system. Among them, C1-C3: collimator; L1-L3: lens; DM: dichroic mirror; LPF: long-pass filter; PC: polarization controller; PMT: photomultiplier tube. A broadband super-continuous laser (SuperK EXTREME, NKT Photonics) is used as the light source, and a band selection module (SuperK VARIA) is added. Figure 1 (b) shows the measured spectrum of the light source used in the imaging system at the selected output.
The output light source is coupled to a light source arm of a single-mode fiber (fused 2 × 2 fiber coupler, with a center wavelength of 514 nm, OZ Optics, Ottawa, Canada), that is, a Michelson interferometer. After leaving the sample arm, the light is collimated, reflected by a dichroic mirror (DMLP505, cut-off wavelength: 505nm, Thorlabs), scanned by an XY galvanometer scanner, and then transmitted to the retina through a combination of relay lenses and eyepieces. The system has a lateral resolution of 7.3 μm in the retina. In the reference arm, a glass plate is used to compensate for the group velocity dispersion mismatch between the reference arm and the sample arm. In the detection arm, a 1800 line / mm transmission grating, a multi-element imaging lens (f = 150 mm), and a line scan CCD camera (Aviiva-SM2-CL-2010, 2048 pixels, 10 μm pixel size, 12 bits Mode, e2V) spectrometer collimates and detects the mixed reflected light from the sample arm and the reference arm. The image acquisition board (IMAQ PCI-1428, National Instruments) acquires the interference spectrum collected by the camera and transmits it to the workstation (DELL Precision T7500, 4GB memory) for signal processing and image display. The linear CCD camera in the OCT spectrometer operates at a linear rate of 24 kHz.
In order to test the imaging quality of the dual-mode imaging technique, this experiment images the retina of an albinism rat. The rats were anesthetized by intraperitoneal injection of a mixture containing ketamine (54 mg / kg body weight) and xylazine (6 mg / kg body weight), the pupils were dilated with a 10% phenylephrine solution, and the rats were restrained in an animal stent.
Figure 2 shows a typical visible optical coherence tomography (VIS-OCT) cross-sectional image of a rat retina obtained with this system. The image consists of 2048 A-lines (depth scan). The cross-sectional image shown in Figure 2 has a better depth resolution (improved from 12 μm to 5.8 μm) than the image obtained at 415 nm using the system to better visualize the retinal layer. In addition, stronger signals from deeper layers such as the choroid can be observed. Compared to the OCT image in the near infrared (NIR), the blood vessels of the VIS-OCT image cast a sharper shadow on the retinal layer behind it, due to the high hemoglobin absorption in the visible spectrum.
Figure 3 shows images of the retinas of VIS-OCT [Figure 3 (a) and 3 (c)] and AF [Figure 3 (b)] rats acquired at the same time. Figure 3 (a) is an OCT fundus image generated from a 3D VIS-OCT dataset. Figure 3 (c) is a VIS-OCT B-scan image, and its position is marked as a white line on Figures 3 (a) and 3 (b). The data set consists of 512 (horizontal) x 128 (vertical) A-scans. Since the OCT and AF images are generated from the same set of photons, it can be seen that Fig. 3 (a) and Fig. 3 (b) are exactly matched. At 480nm, the main fluorophore in the eye is lipofuscin. Lipofuscin is a product of photoreceptor phagocytosis and accumulates in the RPE layer. The black appearance of retinal blood vessels means that the fluorescent signal comes from the retinal layer behind the retinal blood vessels, so Figure 3 (b) represents the distribution of lipofuscin concentration in RPE cells. To test the ability of the imaging system to monitor lipofuscin accumulation longitudinally in RPE cells, three 10-week-old normal rats were followed in the experiment for 4 weeks. During this period, the average rat weight increased from 206g to 251g.
Figure 4 shows the OCT fundus image (a, d, g), AF image (b, e, h) of the same eye of a rat, and the AF intensity distribution (c, f, i) Histogram: (a)-(c) 10 weeks, (d)-(f) 12 weeks, (g)-(i) 14 weeks. The corresponding blood vessels at different time points are marked with the numbers 1, 2 and 3. The pattern of retinal blood vessels does not look exactly the same because the mouse body is soft and it is not possible to place the head in the same position to image at different times. It can be seen from FIG. 4 that the intensity of the AF signal increases strongly with aging within four weeks, while the intensity of the OCT fundus image remains relatively constant.
Time point (week)
OCT average intensity (number)
AF average intensity (number)
AF / OCT
Table 1 shows the average intensities of the OCT and AF images of the rat retina shown in FIG. 4. As can be seen from the table, the average intensity of the OCT image changed between different time points, but the percentage change from the previous measurement point was very small (less than 5%). In contrast, the average intensity of AF images increased strongly (36% and 37%).
From the histogram in Figure 4, we can clearly see that more and more pixels have higher intensity numbers as they age, although the maximum at each age point is still at a lower intensity value. The reason that the maximum value of each histogram is at a lower intensity value is that most of the dark pixels are located in the optic disc and retinal blood vessels. The intensity of the OCT fundus image depends on the detection light intensity and fundus reflectance. The relatively constant intensity of the OCT fundus image shows that the detection light intensity is constant during the imaging process. The intensity of the AF signal is proportional to the intensity of the detection light and the fluorophore concentration. Since the detection light intensity is constant during imaging, an increase in the AF signal intensity means an increase in lipofuscin concentration. It was concluded that Figure 4 shows that lipofuscin concentration in rat retina increases with aging.
Figure 5 shows the average AF intensity counts and standard deviations calculated over the entire imaging area of 3 rats of different ages. Although there were individual differences in average AF intensity, all three rats showed the same lipofuscin accumulation trend. Interestingly, all 3 rats had similar average AF intensity at the beginning of the study. The increase in mean AF intensity differences in the three rats in the following weeks may be due to exposure differences caused by different cage positions. The rapid accumulation of lipofuscin in relatively young animals may be due to the lack of protective effects on melanin in RPE cells of albino rats.
The system described herein is suitable for simultaneous OCT and AF imaging using a single broadband visible light source to provide more comprehensive retinal imaging. This technology may become a powerful tool for research and clinical diagnosis of age-related macular degeneration.
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