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 2

Impact of pulse duration on TPEF of native retinal fluorophores.

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Impact of pulse duration on TPEF of native retinal fluorophores. (A) Spe...
(A) Spectra of laser light with different pulse durations as measured in the sample plane. (B) Two-photon excitation spectra from the RPE of Abca4−/− Rdh8−/− (Dko) and Rpe65–/– mice. Plotted are mean gray pixel values from areas equivalent to at least 10 RPE cells. (C) Images of RPE in Rpe65–/– (left panels) and in Dko (right panels) mice. Durations of laser pulses are indicated in each image. Small green-colored dots indicated with yellow arrows are retinosomes. To avoid saturation of the detector when imaging retinosomes, both the gain and laser power were kept low. Thus, retinosomes are only faintly visible in images obtained with 35-fs light as compared with images obtained with 20-fs light. Scale bars: 50 μm. Images of Rpe65–/– were obtained with 5.5 mW, and images of Dko were obtained with 4.9 mW, and image acquisition time was 2.6 seconds. (D) RPE fluorescence measured as mean gray pixel values of the raw images obtained with laser light pulses at 18 fs, 20 fs, 25 fs, and 35 fs durations. Data were analyzed as mean gray pixel values from areas equivalent to at least 10 RPE cells. Solid, filled symbols represent measured data points, dashed lines represent results of modeling. Dotted cyan line represents fit to 1/pulse duration (1/τ). Data are shown as means ± SD, n = 3.