From mouse to human: Accessing the biochemistry of vision in vivo by two-photon excitation

Two-photon excitation fluorescence in ophthalmology: safety and improved imaging for functional diagnostics

“…Safety has been a critical element in developing 2Ph imaging for application in ophthalmology, especially when imaging the retina where melanin-mediated interference can preclude detection of the signal of interest. Thus, advances in manufacturing the laser source (with spatial, temporal and spectral properties finely modulated) were key in maximizing the signal and minimizing the exposure to laser light ( 6 , 19 ); i.e., very intense but short pulses (which, if continued, would vaporize the biological samples), at a high repetition rate, that produce high instantaneous energy but low average ( 20 ). The probability of TPEF events increases with photon flux, thus requiring powerful lasers (or less powerful if the laser produces short fs pulses, making the instantaneous intensity very high).…”

“…Despite the use of these high-intensity short pulses, eye tissue interrogations remain within safe limits because of the minimal cross-section of material illuminated, and the very limited time period over which the pulses are delivered ( Figure 1M ). Technology tested in mice revealed the possibility of extracting biochemical information using TPEF imaging, within safe limits and without any perceived damage judged by several criteria, including in vivo imaging using SLO and OCT; retinal function assay by ERG; and ex vivo quantification of rhodopsin and 11-cis retinal by immune histology and TPEF imaging ( 6 , 21 ). To ensure a more significant safety margin for use in human retinas, Palczewska et al ( 21 ) further diminished the potential sources of photodamage by decreasing the pulse repetition frequency (PRF) ( 17 ), and by adding other design features in the setup ( 22 , 23 ).…”

“…Adaptive optics (AO) is a technique that at its core uses a wavefront sensor to measure the distortion in an optical wavefront, and corrects them using a deformable mirror, thus in ophthalmology AO corrects for eye aberrations ( 25 ). Additionally, one impact of reducing the pulse duration in TPEF imaging is the increased phase distortion, caused not only by the tissues but also by the optical component due to the increase in the pulse spectral bandwidth, which needs to be compensated for ( 6 , 26 ). Another impact to some extent is wavefront distortion, due to intermediate tissue layers; this problem may be overcome using AO ( 6 , 27 – 29 ).…”

Lipofuscin, Its Origin, Properties, and Contribution to Retinal Fluorescence as a Potential Biomarker of Oxidative Damage to the Retina

“…Photoreceptors, Müller cells and RPE contain considerable concentrations of retinoids, which also exhibit fluorescence properties [254,255]. In particular, retinyl palmitate, which serves as a storage form for vitamin A from which the chromophores for visual pigments are synthesized, can accumulate in particularly high concentrations in the RPE and emits broad range fluorescence between 400-650 nm with a maximum at ~490 nm when excited with ultraviolet light [254][255][256].…”

“…This can be achieved by employing short pulses of high-intensity near-infrared laser. The most recent development in clinical imaging of fundus fluorescence is spectral imaging of fluorescence emitted in the range of 400-700 nm upon intense pulse excitation with two photons of near-infrared wavelengths and the use of adaptive optics [217,254,255,295,[308][309][310][311][312][313][314][315][316][317][318][319][320][321][322]. The emitted light can be spectrally analyzed, which enables the detection of specific fluorophores.…”