Background. Noninvasive 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.Methods. We present a compact two-photon excited fluorescence scanning laser ophthalmoscope and spectrally resolved images of the human retina based on two-photon excitation (TPE) with near-infrared (IR) light. A custom Er:fiber laser with integrated pulse selection, along with intelligent post-processing of data, enables excitation with low laser power and precise measurement of weak signals.Results. We 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. Conclusion.Our 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.

Two-photon vision is a phenomenon associated with the perception of short pulses of near-infrared radiation (900-1200 nm) as a visible light. It is caused by the nonlinear process of two-photon absorption by visual pigments. Here we present results showing the influence of pulse duration and repetition rate of short pulsed lasers on the visual threshold. We compared two-photon sensitivity maps of the retina obtained for subjects with normal vision using a cost-effective fiber laser (λc = 1028.4 nm, τp = 12.2 ps, Frep = 19.17 MHz) and a solid-state laser (λc = 1043.3 nm, τp = 0.253 ps, Frep = 62.65 MHz). We have shown that in accordance with the description of two-photon absorption, the average optical power required for two-photon vision for a fiber laser is 4 times greater than that for a solid-state laser. Mean sensitivity measured for the first one is 5.9 ± 2.8 dB lower than for the second but still 17 dB away from the safety limit, confirming that picosecond light sources can be successfully applied in microperimetry. This development would dramatically reduce the cost and complexity of future clinical devices.

We report the development of a widely-tunable femtosecond fiber laser system and its application for two-photon vision studies. The source is based on an Er-doped fiber laser with spectral shift up to 2150 nm, followed by a second harmonic generation module to generate a frequency-doubled beam tunable from 872 to 1075 nm. The source delivers sub-230 fs pulses with nearly-constant duration over the entire tuning range, with output powers between 0.68–1.24 mW, which corresponds to a pulse energy of 13.2–24.1 pJ. Such pulse energy is sufficient for employing a system for measurements of two-photon scotopic spectral sensitivity of two-photon vision in humans. The laser parameters allow for very efficient and safe two-photon stimulation of the human visual system, as proved by a good separation between one- and two-photon thresholds for wavelengths below 950 nm, which we have confirmed for 3 healthy subjects.

In this paper, a spectroscopic method for determination of cyclosporine concentrations in biological fluids is presented. Blood plasma and hemoglobin solutions are chosen for the experiment. For various cyclosporine concentrations in blood plasma and hemoglobin, absorbance measurements in spectra range from 600 to 1100 nm are performed. The measurement results are analyzed by the use of a dedicated algorithm. The obtained data are characterized by a high coefficient of correlation R 2 , which is equal to 0.9461 and 0.9808 for blood plasma and hemoglobin, respectively. The proposed method enables the selective detection of cyclosporine level and could be applied in medicine and laboratory diagnostics. The obtained result can be the base to build the point-of-care CsA level detection optical sensor.

In this paper, an investigation into detecting immunosuppressive medicine in aqueous solutions using a spectrometry-based technique is described. Using optical transmissive spectrometry, absorbance measurements in the spectra range from 250 nm to 1000 nm were carried out for different cyclosporine A (CsA) concentrations in aqueous solutions. The experiment was conducted for samples both with and without interferent substances—glucose and sodium chloride. Using a dedicated algorithm, the measured data was analyzed and a high correlation coefficient R2 = 0.8647 was achieved. The experiment showed that the described technique allowed for the detection of various CsA concentration levels in a selective, label-free and simple way. This method could be used in medicine, veterinary medicine and laboratory diagnostics.