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

Abstract

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.

Hybrid Fluorescence and Frequency-Domain Photoacoustic Microscopy for Imaging Development of Parhyale hawaiensis Embryos

“…Aiming at the delivery of complementary contrast in biological tissues, several studies have demonstrated the development of multimodal instruments, effectively combining time-domain (TD) PA microscopy with well-established optical diagnostic techniques such as confocal fluorescence microscopy, multiphoton imaging and optical coherence tomography (OCT) [34]. It is worth mentioning that these hybrid imaging developments have found various significant applications, including the sensitive detection of capillary blood vessels in mice [35][36][37][38], the delineation of ocular anatomy [39][40][41], the investigation of zebrafish larvae [42,43], the measurement of melanin content in fish scales [44] as well as the spatial mapping of pigments in vegetative tissues [45]. Nevertheless, all the aforementioned experimental configurations have integrated expensive nanosecond Q-switched laser sources for the efficient excitation of PA signals, restricting significantly the wider accessibility of the hybrid imaging methods by many biological laboratories.…”

Hybrid Autofluorescence and Optoacoustic Microscopy for the Label-Free, Early and Rapid Detection of Pathogenic Infections in Vegetative Tissues

“…A detailed technical description of the hybrid confocal fluorescence and optoacoustic microscopy setup ( Figure 1 ) has been provided in a previous study [ 26 ]. In brief, autofluorescence (AF) excitation is achieved by employing a low-power continuous wave (CW) diode laser at 450 nm (CPS450, Thorlabs, Newton, NJ, USA; output power 4.5 mW).…”

“…This custom-developed hybrid system integrates state-of the-art confocal fluorescence and optoacoustic microscopy modalities to provide complementary label-free imaging contrast at high spatial resolution. While such multimodal instruments have been previously employed for several imaging applications, including the sensitive detection of pigments in plant leaves [ 22 ], the examination of various ocular tissues [ 23 , 24 ], the investigation of emerging models organisms [ 25 ], as well as the quantification of melanin in fish [ 26 ], this is the first time to our knowledge that they have been utilized for the early and rapid diagnosis of pathogenic infections in plants, paving the way for new and significant developments in the field.…”

Polydopamine-Based Nanoprobes Application in Optical Biosensing

“…Also, as already mentioned in paragraph 2, fluorescence is generated by melanin-PDA synthesis intermediates and oligomers, which, by in situ polymerization, can be more easily monitored. In other cases, melanin or its precursors are simply not only the probe but also the target, as in the work of Tserevelakis and co-workers, who monitored melanin accumulation in fish scales, exploiting melanin PAI [98]. They implemented a hybrid confocal fluorescence and photoacoustic microscopy that could detect, by the combination of photoacoustic signal and tissue autofluorescence, the melanin content along the fish scales with high accuracy.…”