Multimodal nonlinear optical imaging of unstained retinas in the epi-direction with a sub-40 fs Yb-fiber laser

Abstract

Ultrafast lasers have potential use in ophthalmology for diagnoses through non-invasive imaging as well as for surgical therapies or for evaluating pharmacological therapies. New ultrafast laser sources, operating at 1.07 μm and sub-40 fs pulse durations, offer exciting possibilities in multiphoton imagining of the retina as the bulk of the eye is relatively transparent to this wavelength, three-photon excitation is not absorbed by DNA, and this wavelength has a greater penetration depth compared to the commonly used 800 nm Ti:Sapphire laser. In this work, we present the first epi-direction detected cross-section and depth-resolved images of unstained isolated retinas obtained using multiphoton microscopy with an ultrafast fiber laser centered at 1.07 μm and a ~38 fs pulse duration. Spectral and temporal characterization of the autofluorescence signals show two distinct regions; the first one from the nerve fiber layer to the inner receptor layer, and the second being the retinal pigmented epithelium and choroid.

Keywords: (140.3510) Lasers, fiber; (170.3880) Medical and biological imaging; (180.4315) Nonlinear microscopy; (320.7090) Ultrafast lasers.

Fig. 1

The experimental apparatus consisted of an ultrafast fiber laser operating at 1.07 μm, a MIIPS pulse shaper, a laser scanning inverted microscope, and a photon detector, mounted in the epi-direction. The TCSPC was used to collect fluorescence spectra and lifetime, whereas the single PMT was used to acquire the depth resolved images.

Fig. 2

Three colored (red, green, blue) composite multimodal images of the retinal layers from a 7 µm slice of a mouse retina taken with the TCSPC at 6.9 mW of power depths using a 1.07 µm Yb-fiber laser with 35.0 fs pulse durations. Image acquisition was done at 30-second intervals for a total of 4.5 minutes. (a) Here the blue, green, and red channels represent emission centered at 535 nm, 575 nm, and 629 nm, respectively. (b) The blue, green, and red channels represent emission centered at 355 nm, 535 nm, and 629 nm, respectively. The bandwidth of each channel is ~37.5 nm.

Fig. 3

Spectral emission from 480 to 680 nm detected from a mouse retina. a) The non-normalized emission spectra reveal that the receptor layers (ORL and IRL) have the strongest fluorescence emission. b) The normalized spectra more readily compare the different spectral shapes across retinal layers. Layers from the NFL thru the OPL all have nearly identical spectral shapes. The sclera has a unique spectral shape, where the peak at 535 nm is attributed to SHG (see Discussion).

Fig. 4

Fluorescence lifetimes across the retinal layers from a mouse over a spectral range of a) 556-594 nm, b) 610-648 nm, and c) short: 556-594 nm and long: 610-648 nm. The plots reveal that for a given spectral band, all the layers from the NFL through the ORL have nearly identical lifetimes, whereas the choroid and RPE have nearly identical lifetimes. Additionally, the lifetimes in the choroid and RPE become shorter for longer detection wavelengths (i.e. from panel a to panel b). This trend is shown directly in panel c, where the lifetimes for the choroid, RPE, IRL, and IPL are fitted for both the short wavelength range and the long wavelength range. Also in panel c is the lifetime measured at 535 nm from the sclera. SHG from collagen in the sclera is the source of this emission and coincides with the IRF of our system.

Fig. 5

Depth-resolved imaging of an unstained, fixed, Cynomolgus monkey retina flat mount. The total extent of the Cynomolgus monkey retina was 220 μm. The depth of each image is indicated in yellow. The scale bar is 15 μm and can be seen in the NFL panel. Each 2D image was an average of 5 scans, averaged for 5 seconds. Each layer, beginning with the NFL and ending with the ORL and RPE retinosomes, from left to right, was an average of 182 images (9.1 µm), 190 images (9.5 µm), 182 images (9.1 µm), 139 images (7.0 µm), 51 images (2.63 µm), 233 images (11.7 µm), 211 images (10.6 µm), and 33 images (1.7 µm), respectively. The depth resolved stack was obtained with 7 mW of average power at all depths using a 1.07 µm Yb-fiber laser with 34.8 fs pulse durations. In the IRL, inner segments of cone photoreceptors (large white features) and rod photoreceptor inner segments (smaller round features between the cone inner segments) are easily distinguishable. We believe that the bright particles in the bottom right panel are perhaps melanin or RPE retinosomes attached to the tips of photoreceptors. At each depth, the characteristic morphology of the retina layers is clear, indicating that the Yb-fiber laser is effective at achieving the cellular resolution needed for depth resolved imaging. Video of depth resolved imaging of retina used to obtain the images presented in this figure (Visualization 1).

Fig. 6

(Left). Lifetime decay fits and the corresponding residual plots for the RPE and choroid for the wavelength range of 556-594 nm. This figure shows the RPE and choroid fit poorly to a mono-exponential function. (Right). Lifetime decay fits and the corresponding residual plots for the ORL through NFL for the wavelength range of 610-648 nm. This figure demonstrates that the ORL-NFL layers fit poorly to a tri-exponential function

Fig. 7

The spectra (Left) and lifetime (Right) from a 1 mM solution of A2E. The spectra peaks near 625 nm, which is the same peak seen in the choroid, RPE, and receptor layers. The lifetime of ~173 ps agrees with the literature values [29].