Hybrid confocal fluorescence and photoacoustic microscopy for the label-free investigation of melanin accumulation in fish scales.

Lower vertebrates, including fish, can rapidly alter skin lightness through changes in melanin concentration and melanosomes' mobility according to various factors, which include background color, light intensity, ambient temperature, social context, husbandry practices and acute or chronic stressful stimuli. Within this framework, the determination of skin chromaticity parameters in fish species is estimated either in specific areas using colorimeters or at the whole animal level using image processing and analysis software. Nevertheless, the accurate quantification of melanin content or melanophore coverage in fish skin is quite challenging as a result of the laborious chemical analysis and the typical application of simple optical imaging methods, requiring also to euthanize the fish in order to obtain large skin samples for relevant investigations. Here we present the application of a novel hybrid confocal fluorescence and photoacoustic microscopy prototype for the label-free imaging and quantification of melanin in fish scales samples with high spatial resolution, sensitivity and detection specificity. The hybrid images are automatically processed through optimized algorithms, aiming at the accurate and rapid extraction of various melanin accumulation indices in large datasets (i.e., total melanin content, melanophores' area, density and coverage) corresponding to different fish species and groups. Furthermore, convolutional neural network-based algorithms have been trained using the recorded data towards the classification of different scales' samples with high accuracy. In this context, we demonstrate that the proposed methodology may increase substantially the precision, as well as, simplify and expedite the relevant procedures for the quantification of melanin content in marine organisms.

Full image reconstruction in frequency-domain photoacoustic microscopy by means of a low-cost I/Q demodulator.

We present a full image reconstruction methodology in frequency-domain photoacoustic (PA) microscopy using a low-cost I/Q demodulator for the recording of the amplitude and phase of the signals. By modulating the intensity of a continuous-wave diode laser at 10 MHz, we have been able to provide accurate optical absorption images and surface reconstructions of phantom samples, comparing also the extracted results with standard time-domain approaches. The findings of the study in this Letter could be utilized towards the development of inexpensive PA microscopes with multispectral capabilities for a wide range of biomedical studies, requiring the sensitive detection of endogenous or exogenous absorbers in tissues.

Hybrid autofluorescence and photoacoustic label-free microscopy for the investigation and identification of malignancies in ocular biopsies.

We demonstrate the development and application of a prototype hybrid microscopy system integrating autofluorescence (AF) and photoacoustic (PA) label-free contrast modes, for the differentiation of ocular tumors in human surgical biopsies. Hybrid imaging was performed in conjunctival nevi and uveal melanomas tissue sections to acquire quantified data for each molecular background. The AF and PA signals were spatially correlated to establish a novel malignancy indicator that could detect melanomas with high accuracy (t-test; p<0.01). The proposed methodology has the potential to simplify relevant diagnostic procedures and paves the way for the development of novel ophthalmoscopes aiming to the early diagnosis of ocular malignancies in a clinical setting.

Simple precision measurements of optical beam sizes.

We present a simple high-precision method to quickly and accurately measure the diameters of Gaussian beams, Airy spots, and central peaks of Bessel beams ranging from sub-millimeter to many centimeters without specialized equipment. By simply moving a wire through the beam and recording the relative losses using an optical power meter, one can easily measure the beam diameters with a precision of 1%. The accuracy of this method has been experimentally verified for Gaussian beams down to the limit of a commercial slit-based beam profiler (3%).