Two-photon imaging of the mammalian retina with ultrafast pulsing laser
Grazyna Palczewska, Patrycjusz Stremplewski, Susie Suh, Nathan Alexander, David Salom, Zhiqian Dong, Daniel Ruminski, Elliot H. Choi, Avery E. Sears, Timothy S. Kern, Maciej Wojtkowski, Krzysztof Palczewski
Grazyna Palczewska, Patrycjusz Stremplewski, Susie Suh, Nathan Alexander, David Salom, Zhiqian Dong, Daniel Ruminski, Elliot H. Choi, Avery E. Sears, Timothy S. Kern, Maciej Wojtkowski, Krzysztof Palczewski
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Resource and Technical Advance Ophthalmology

Two-photon imaging of the mammalian retina with ultrafast pulsing laser

Abstract

Noninvasive imaging of visual system components in vivo is critical for understanding the causal mechanisms of retinal diseases and for developing therapies for their treatment. However, ultraviolet light needed to excite endogenous fluorophores that participate in metabolic processes of the retina is highly attenuated by the anterior segment of the human eye. In contrast, 2-photon excitation fluorescence imaging with pulsed infrared light overcomes this obstacle. Reducing retinal exposure to laser radiation remains a major barrier in advancing this technology to studies in humans. To increase fluorescence intensity and reduce the requisite laser power, we modulated ultrashort laser pulses with high-order dispersion compensation and applied sensorless adaptive optics and custom image recovery software and observed an over 300% increase in fluorescence of endogenous retinal fluorophores when laser pulses were shortened from 75 fs to 20 fs. No functional or structural changes to the retina were detected after exposure to 2-photon excitation imaging light with 20-fs pulses. Moreover, wide bandwidth associated with short pulses enables excitation of multiple fluorophores with different absorption spectra and thus can provide information about their relative changes and intracellular distribution. These data constitute a substantial advancement for safe 2-photon fluorescence imaging of the human eye.

Authors

Grazyna Palczewska, Patrycjusz Stremplewski, Susie Suh, Nathan Alexander, David Salom, Zhiqian Dong, Daniel Ruminski, Elliot H. Choi, Avery E. Sears, Timothy S. Kern, Maciej Wojtkowski, Krzysztof Palczewski

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Figure 1

Noninvasive TPEF imaging of mouse retina.

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Noninvasive TPEF imaging of mouse retina. (A) Depiction of the experimen...
(A) Depiction of the experimental setup that includes a dispersion compensation system, adaptive optics, an electro-optic modulator (EOM) and 3 lasers: (i) a tunable-wavelength and -pulse-duration laser capable of delivering 20-fs pulses; (ii) a laser with a fixed pulse duration at 75 fs and a tunable wavelength, and (iii) a laser with a fixed pulse duration at 32 fs and fixed wavelength at 740 nm. Two black, dual arrowheads indicate switching locations between the lasers. The 75-fs laser is equipped with a prism pair–based dispersion compensation unit (DC). Light beams from 20-fs and 32-fs lasers are routed through a diffraction grating and spatial light modulator–based (SLM-based), custom-built diffraction compensation system. (B) Calculated temporal profiles of 32-fs Gaussian-shaped pulses: black represents bandwidth limited, and red represents pulse impacted by third-order dispersion (calculated for the amount of NBK7 glass generating a GDD equivalent to that of the optical setup). (C) Mean TPEF from a paper target, as a function of excitation power, acquired with 75-fs and 32-fs lasers. Solid circles represent measured data points; solid lines represent the linear data fit with slopes equal to 1.9. (D) TPEF images of mouse RPE obtained with 740-nm excitation with both 75-fs and 32-fs pulses after optimization of dispersion compensation. Ex vivo (left column) and in vivo (right column); images of the RPE obtained with 75-fs laser in the upper row, images obtained with 32-fs laser are shown in the bottom row. Ex vivo images were obtained with 6.3 mW, in vivo images were obtained with 7.5 mW; image acquisition time was 2.6 seconds. Ratios of mean pixel gray value of TPEF images of the RPE in Rpe65–/– mice obtained with a 32-fs laser to that obtained with a 75-fs laser were equal to 2.9 ex vivo and 2.2 in vivo. Scale bars: 50 μm ex vivo (left column); 200 μm in vivo (right column).