In vivo imaging of the human retina using a two-photon excited fluorescence ophthalmoscope.

Noninvasive imaging of endogenous retinal fluorophores, including vitamin A derivatives, is vital to developing new treatments for retinal diseases. Here, we present a protocol for obtaining in vivo two-photon excited fluorescence images of the fundus in the human eye. We describe steps for laser characterization, system alignment, positioning human subjects, and data registration. We detail data processing and demonstrate analysis with example datasets. This technique allays safety concerns by allowing for the acquisition of informative images at low laser exposure. For complete details on the use and execution of this protocol, please refer to Boguslawski et al. (2022).1.

In vivo imaging of the human eye using a 2-photon-excited fluorescence scanning laser ophthalmoscope.

BackgroundNoninvasive assessment of metabolic processes that sustain regeneration of human retinal visual pigments (visual cycle) is essential to improve ophthalmic diagnostics and to accelerate development of new treatments to counter retinal diseases. Fluorescent vitamin A derivatives, which are the chemical intermediates of these processes, are highly sensitive to UV light; thus, safe analyses of these processes in humans are currently beyond the reach of even the most modern ocular imaging modalities.MethodsWe present a compact, 2-photon-excited fluorescence scanning laser ophthalmoscope and spectrally resolved images of the human retina based on 2-photon excitation (TPE) with near-infrared light. A custom Er:fiber laser with integrated pulse selection, along with intelligent postprocessing of data, enables excitation with low laser power and precise measurement of weak signals.ResultsWe demonstrate spectrally resolved TPE fundus images of human subjects. Comparison of TPE data between human and mouse models of retinal diseases revealed similarity with mouse models that rapidly accumulate bisretinoid condensation products. Thus, visual cycle intermediates and toxic byproducts of this metabolic pathway can be measured and quantified by TPE imaging.ConclusionOur work establishes a TPE instrument and measurement method for noninvasive metabolic assessment of the human retina. This approach opens the possibility for monitoring eye diseases in the earliest stages before structural damage to the retina occurs.FundingNIH, Research to Prevent Blindness, Foundation for Polish Science, European Regional Development Fund, Polish National Agency for Academic Exchange, and Polish Ministry of Science and Higher Education.

Influence of the excitation light intensity on the rate of fluorescence quenching reactions: pulsed experiments.

The effect of multiple light excitation events on bimolecular photo-induced electron transfer reactions in liquid solution is studied experimentally. It is found that the decay of fluorescence can be up to 25% faster if a second photon is absorbed after a first cycle of quenching and recombination. A theoretical model is presented which ascribes this effect to the enrichment of the concentration of quenchers in the immediate vicinity of fluorophores that have been previously excited. Despite its simplicity, the model delivers a qualitative agreement with the observed experimental trends. The original theory by Burshtein and Igoshin (J. Chem. Phys., 2000, 112, 10930-10940) was created for continuous light excitation though. A qualitative extrapolation from the here presented pulse experiments to the continuous excitation conditions lead us to conclude that in the latter the order of magnitude of the increase of the quenching efficiency upon increasing the light intensity of excitation, must also be on the order of tens of percent. These results mean that the rate constant for photo-induced bimolecular reactions depends not only on the usual known factors, such as temperature, viscosity and other properties of the medium, but also on the intensity of the excitation light.

Characterization of dimethylsulfoxide/glycerol mixtures: a binary solvent system for the study of "friction-dependent" chemical reactivity.

The properties of binary mixtures of dimethylsulfoxide and glycerol, measured using several techniques, are reported. Special attention is given to those properties contributing or affecting chemical reactions. In this respect the investigated mixture behaves as a relatively simple solvent and it is especially well suited for studies on the influence of viscosity on chemical reactivity. This is due to the relative invariance of the dielectric properties of the mixture. However, special caution must be taken with specific solvation, as the hydrogen-bonding properties of the solvent change with the molar fraction of glycerol.

Photodissociation of ClNO in the 2 1A' state: computational and experimental NO product state distributions.

Ultrafast photodissociation of the 2 (1)A' state of ClNO, which has an absorption spectrum peaking at 335 nm, is studied by computational and experimental methods. New potential-energy surfaces are calculated for the 1 and 2 (1)A' states at the multireference configuration interaction (MRCI) level. Wavepacket dynamics simulations performed both exactly and by using the multiconfiguration time-dependent Hartree method yield essentially identical results. Transition dipole moments at a range of geometries are included in these calculations to correctly model the excitation. Vibrational and rotational state distributions of the NO product are obtained both computationally by analysing the quantum flux on the 2 (1)A' surface and experimentally by use of 3D resonant multiphoton ionisation (REMPI), a variant of the velocity map imaging technique. The nascent NO is found to be only marginally vibrationally excited, with 91% formed in v=0. The calculated NO rotational distribution peaks in the j=45-55 region, which compares favourably to experiment.

Direct mid-infrared femtosecond pulse shaping with a calomel acousto-optic programmable dispersive filter.

Direct amplitude and phase shaping of mid-infrared femtosecond pulses is realized with a calomel-based acousto-optic programmable dispersive filter transparent between 0.4 and 20 µm. The shaped pulse electric field is fully characterized with high accuracy, using chirped-pulse upconversion and time-encoded arrangement spectral phase interferometry for direct electric field reconstruction techniques. Complex mid-infrared pulse shapes at a center wavelength of 4.9 µm are generated with a spectral resolution of 14 cm(-1), which exceeds by a factor of 5 the reported experimental resolutions of calomel-based filters.