The study of brain tissue development requires imaging approaches that ideally provide micron resolution, millimeter imaging depth, and multi-contrast capability.

We will discuss recent approaches for large-scale imaging of uncleared tissues, namely color serial two-photon imaging [1] and three-photon (3P) microscopy. We will also present a novel label-free modality that combines some of these concepts, based on third-order sum frequency generation (TSFG). TSFG microscopy provides label-free imaging of red blood cells while being compatible with deep tissue 3P imaging [2].

References:

[1] Abdeladim et al, Nat Commun (2019), https://doi.org/10.1038/s41467-019-09552-9.

[2] Ferrer Ortas et al, Light Sci App (2023), https://doi.org/10.1038/s41377-022-01064-4.

Fast and sensitive detector arrays make Image Scanning Microscopy (ISM) the natural successor of confocal microscopy. Indeed, ISM enables super-resolution at an excellent signal-to-noise ratio. Optimizing photon collection requires large detectors and so more out-of-focus light is collected. Nonetheless, the ISM dataset inherently contains information on the axial position of the fluorescence emitters. We exploit such information to directly invert the corresponding physical model with a maximum-likelihood approach and reassign the signal in the three dimensions, improving the signal-to-background ratio and resolution. We validated our method on synthetic and experimental images; these latter acquired with a custom setup equipped with a single photon avalanche diode array detector. Moreover, our method is compatible with recent developments in ISM data processing and requires minimal knowledge of physical parameters.

Confocal microscopes have been the workhorses of 3-D biological imaging, but they are slow, offer limited depth penetration and collect only ballistic photons. With their inefficient use of excitation photons they expose biological samples to an often intolerably high light burden. The speed limitation and photo-bleaching risk can be somewhat relaxed in a spinning-disk geometry, due to shorter pixel dwell times and rapid re-scans during image capture. Alternatively, light-sheet microscopes rapidly image large volumes of transparent or chemically cleared samples. Finally, with infrared excitation and efficient scattered-light collection, 2-photon microscopy allows deep-tissue imaging, but it remains slow. Here, we describe a new optical scheme that borrows the best from three different worlds: the speed and direct-view from a spinning-disk confocal, deep tissue-penetration and intrinsic optical sectioning from 2-photon excitation, and a large field of view and a low invasiveness of a selective-plane illumination microscope – all with a single objective lens. We validate the performance of our 2-photon spinning-disk microscope in various applications that have in common to simultaneously require a large depth penetration, high speed and larger fields of view. Beyond biological fluorescence, we demonstrate an application in material science, imaging coherent non-linear scattering from a 3-D nano-porous network

The ability to observe traces of biological material on buildings and stone artworks is of particular importance in understanding how to best deal with them and maybe, in the future, even make use of biofilms for conservation science. We have identified hyperspectral imaging as a viable method for the efficient analysis of such biological materials. Thanks to the high throughput of our approach based on an interferometric method based on Fourier Transform, we were not only able to detect traces of biofilms on stone samples, but also to map in which areas these were found to have higher biological activity.

The study of the interaction of biological tissue with polarized light leads to relevant information of physical properties (dichroism, retardance and depolarization) of samples. Polarimetric analysis of different characteristics in tissues is useful for applications such us tissue classification, contrast enhancement or pathology detection. By means of polarimetric imaging techniques we can characterize the polarimetric signature of biological samples in a noninvasive and nondestructive way. We have found that depolarization information is of special interest in turbid media such as plant tissue. In this manuscript we use polarimetric observables for plant inspection. In particular, we provide enhanced visualization of certain plant pathologies by constructing depolarization based pseudocolored images of pathological leaves where the pathological areas are revealed.