Fluorescence lifetime imaging for retinal function assessment

This invention uses a laser-based imaging system to study the eye’s retina. By measuring light responses, it provides insights into retinal health and potential diseases like age-related macular degeneration.

Background

Two-photon excited fluorescence (TPEF) is a powerful technique used to study the eye. TPEF allows researchers to excite intrinsic retinal fluorophores, which are molecules that emit light when excited by a specific wavelength. These fluorophores are involved in cellular metabolism and the visual cycle, making them valuable markers for understanding retinal function. By analyzing the fluorescence signals, researchers can gain insights into the health and function of retinal cells, particularly photoreceptors and the retinal pigment epithelium (RPE), which are crucial for vision.

While intensity-based TPEF has been successful in visualizing retinal structures, it has limitations in providing detailed information about the cellular processes within the retina. Specifically, intensity-based measurements can be influenced by factors like excitation power and probe concentration, making it difficult to isolate the specific contributions of different fluorophores. In addition, this approach may not fully capture the dynamic changes in retinal physiology that occur during the light-dark visual cycle, which is essential for understanding how the retina adapts to different light conditions.

Technology description

This technology utilizes a two-photon fluorescence lifetime imaging (FLIM) assay to study the autofluorescence of rabbit photoreceptors and retinal pigment epithelium (RPE) cells during light-dark visual cycles. The system uses a femtosecond laser to excite intrinsic retinal fluorophores, allowing for the measurement of fluorescence lifetimes at a cellular level. The fluorescence lifetime of these cells varies with light and dark exposure, reflecting changes in retinoid levels crucial for the visual cycle.

The setup includes a specialized ophthalmoscopy system with advanced scanners and a cooled photomultiplier tube for detecting emitted fluorescence. Data analysis using Gaussian Mixture Models helps differentiate fluorescence signals from multiple fluorophores, providing insights into retinal physiology and potential disease states.

This technology differentiates itself by offering a detailed, cellular-level understanding of retinal function and the impact of visual cycles on cellular metabolism. Unlike intensity-based methods, FLIM is insensitive to excitation power and probe concentration, making it more reliable. Additionally, the use of two-photon excitation enhances depth resolution and minimizes photobleaching, which is particularly beneficial for imaging delicate retinal layers.

By analyzing the distinct fluorescence lifetimes of retinoids like all-trans-retinol and all-trans-retinal, this approach provides valuable insights into the dynamics of the visual cycle and its potential disruption in diseases like age-related macular degeneration.

Features

The invention is a two-photon fluorescence lifetime imaging (FLIM) assay that studies the autofluorescence of rabbit photoreceptors and retinal pigment epithelium (RPE) during light-dark visual cycles.
It measures fluorescence lifetimes at a cellular scale, providing insights into retinal physiology and potential disease states.
The fluorescence lifetime of photoreceptors and RPE cells varies with light and dark exposure, reflecting changes in retinoid levels crucial for phototransduction and the visual cycle.
The system uses a fiber-based femtosecond laser to excite intrinsic retinal fluorophores and a two-photon fluorescence lifetime ophthalmoscopy (2P-FLIO) system for detection.
Data analysis using Gaussian Mixture Models unmixes fluorescence signals from multiple fluorophores, offering a detailed understanding of retinal function and the impact of visual cycles on cellular metabolism.
The invention has implications for studying diseases like age-related macular degeneration.