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Zebrafish are powerful animal models for understanding biological processes and the molecular mechanisms involved in different human diseases. Advanced optical techniques based on fluorescence microscopy have become the main imaging method to characterize the development of these organisms at the microscopic level. However, the need for fluorescence probes and the consequent high light doses required to excite fluorophores can affect the biological process under observation including modification of metabolic function or phototoxicity. Here, without using any labels, we propose an implementation of a Mueller-matrix polarimeter into a commercial optical scanning microscope to characterize the polarimetric transformation of zebrafish preserved at different embryonic developmental stages. By combining the full polarimetric measurements with statistical analysis of the Lu and Chipman mathematical decomposition, we demonstrate that it is possible to quantify the structural changes of the biological organization of fixed zebrafish embryos and larvae at the cellular scale. This convenient implementation, with low light intensity requirement and cheap price, coupled with the quantitative nature of Mueller-matrix formalism, can pave the way for a better understanding of developmental biology, in which label-free techniques become a standard tool to study organisms.

Quantifying the imaging performances in an unbiased way is of outmost importance in super-resolution microscopy. Here, we describe an algorithm based on image correlation spectroscopy (ICS) that can be used to assess the quality of super-resolution images. The algorithm is based on the calculation of an autocorrelation function and provides three different parameters: the width of the autocorrelation function, related to the spatial resolution; the brightness, related to the image contrast; the relative noise variance, related to the signal-to-noise ratio of the image. We use this algorithm to evaluate the quality of stimulated emission depletion (STED) images of DNA replication foci in U937 cells acquired under different imaging conditions. Increasing the STED depletion power improves the resolution but may reduce the image contrast. Increasing the number of line averages improves the signal-to-noise ratio but facilitates the onset of photobleaching and subsequent reduction of the image contrast. Finally, we evaluate the performances of two different separation of photons by lifetime tuning (SPLIT) approaches: the method of tunable STED depletion power and the commercially available Leica Tau-STED. We find that SPLIT provides an efficient way to improve the resolution and contrast in STED microscopy.

Polarized light scanning microscopy is a non-invasive and contrast-enhancing technique to investigate anisotropic specimens and chiral organizations. However, such arrangements suffer from insensitivity to confined blend of structures at sub-diffraction level. Here for the first time, we present that the pixel-by-pixel polarization modulation converted to an image phasor approach issues an insightful view of cells to distinguish anomalous subcellular organizations. To this target, we propose an innovative robust way for identifying changes in the chromatin compaction and distortion of nucleus morphology induced by the activation of the lamin-A gene from Hutchinson–Gilford progeria syndrome that induces a strong polarization response. The phasor mapping is evaluated based on the modulation and phase image acquired from a scanning microscope compared to a confocal fluorescence modality of normal cell opposed to the progeria. The method is validated by characterizing polarization response of starch crystalline granules. Additionally, we show that the conversion of the polarization-resolved images into the phasor could further utilized for segmenting specific structures presenting various optical properties under the polarized light. In summary, image phasor analysis offers a distinctly sensitive fast and easy representation of the polarimetric contrast that can pave the way for remote diagnosis of pathological tissues in real-time.

Ruthenium–bipyridinetriphenylphosphine–GABA (RuBi–GABA) is a caged compound that allows studying the neuronal transmission in a specific region of a neuron. The inhibitory neurotransmitter γ-aminobutyric acid (GABA) is bound to a caged group that blocks the interaction of the neurotransmitter with its receptor site. Following linear—one-photon (1P)—and non-linear—multi-photon—absorption of light, the covalent bond of the caged molecule is broken, and GABA is released. Such a controlled release in time and space allows investigating the interaction with its receptor in four dimensions (X,Y,Z,t). Taking advantage of this strategy, we succeeded in addressing the modulation of GABAA in rat cerebellar neurons by coupling the photoactivation process, by confocal or two-photon excitation microscopy, with the electrophysiological technique of the patch-clamp in the whole-cell configuration. Key parameters have been comprehensively investigated and correlated in a temporally and spatially confined way, namely: photoactivation laser power, time of exposure, and distance of the uncaging point from the cell of interest along the X, Y, Z spatial coordinates. The goal of studying specific biological events as a function of controlled physical parameters has been achieved.

Antibacterial treatments based on photosensitized production of reactive oxygen species is a promising approach to address local microbial infections. Given the small size of bacterial cells, identification of the sites of binding of the photosensitizing molecules is a difficult issue to address with conventional microscopy. We show that the excited state properties of the naturally occurring photosensitizer hypericin can be exploited to perform STED microscopy on bacteria incubated with the complex between hypericin and apomyoglobin, a self-assembled nanostructure that confers very good bioavailability to the photosensitizer. Hypericin fluorescence is mostly localized at the bacterial wall and accumulates at the polar regions of the cell and at sites of cell wall growth. While these features are shared by Gram-negative and Gram-positive bacteria, only the latter are effectively photoinactivated by light exposure.