Polgenix, Inc., Department of Medical Devices, Cleveland, Ohio, USA.
Department of Physical Chemistry of Biological Systems, Institute of Physical Chemistry, Polish Academy of Sciences, Warsaw, Poland.
JCI Insight. 2018 Sep 6;3(17). doi: 10.1172/jci.insight.121555.
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.
在体无创性可视化研究是了解视网膜疾病的发病机制和寻找治疗方法的关键。然而，参与视网膜代谢过程的内源性荧光团需要被紫外线激发，而人眼的前段组织会强烈吸收紫外线。相比之下，利用脉冲红外光的双光子激发荧光成像技术则克服了这一障碍。然而，将激光辐射对视网膜的暴露降至最低仍然是该技术在人类研究中取得进展的主要障碍。为了提高荧光强度并降低所需的激光功率，我们采用了高阶色散补偿调制超短激光脉冲，并应用了无传感器自适应光学和定制的图像恢复软件，结果表明，当激光脉冲从 75fs 缩短至 20fs 时，内源性视网膜荧光团的荧光强度增加了 300%以上。在暴露于 20fs 脉冲的双光子激发成像光后，未检测到视网膜的功能或结构发生变化。此外，短脉冲所具有的宽带宽使得能够激发具有不同吸收光谱的多个荧光团，从而可以提供有关它们的相对变化和细胞内分布的信息。这些数据为安全的人眼双光子荧光成像技术的发展提供了实质性的推动。
