Search

Super-resolution image acquisition has turned photo-activated far-infrared thermal imaging into a promising tool for the characterization of biological tissues. By the sub-diffraction localization of sparse temperature increments primed by the sample absorption of modulated focused laser light, the distribution of (endogenous or exogenous) photo-thermal biomarkers can be reconstructed at tunable ∼10−50 μm resolution. We focus here on the theoretical modeling of laser-primed temperature variations and provide the guidelines to convert super-resolved temperature-based images into quantitative maps of the absolute molar concentration of photo-thermal probes. We start from camera-based temperature detection via Stefan–Boltzmann’s law, and elucidate the interplay of the camera point-spread-function and pixelated sensor size with the excitation beam waist in defining the amplitude of the measured temperature variations. This can be accomplished by the numerical solution of the three-dimensional heat equation in the presence of modulated laser illumination on the sample, which is characterized in terms of thermal diffusivity, conductivity, thickness, and concentration of photo-thermal species. We apply our data-analysis protocol to murine B16 melanoma biopsies, where melanin is mapped and quantified in label-free configuration at sub-diffraction 40 µm resolution. Our results, validated by an unsupervised machine-learning analysis of hematoxylin-and-eosin images of the same sections, suggest potential impact of super-resolved thermography in complementing standard histopathological analyses of melanocytic lesions.

A recent challenge in bioimaging is the observation and imaging of vital, thick, and complex tissues in real time and in non-invasive mode. In the last decade, non-linear excitation microscopy showed several advantages for in-vivo imaging compared to conventional confocal techniques. Nevertheless, deep tissue imaging remains challenging, especially for thick media, due to spherical aberrations induced on focused beams by the tissue. A low numerical aperture objective lens coupled to high dioptric power microlenses, implanted in the tissue, can be beneficial for the reduction of optical aberrations. In this context, we fabricated a system of plano-convex microlenses and microscaffolds on a single chip by means of two-photon polymerization), to be used for non-linear imaging of biological specimens.

Super‐resolution strategies expand the applicability of imaging modalities toward previously inaccessible spatial scales. Herein, photoactivated far‐infrared thermography is pushed from the millimeter to the micrometer scale by a 2D super‐resolution imaging approach capable of tackling the resolution barriers imposed by both diffraction‐limited signal collection and lateral spatiotemporal heat diffusion. The proposed imaging strategy relies on a full‐wave forward model of far‐infrared thermography and on an image‐inversion approach, which reconstructs the surface distribution of the sample photothermal absorbers via gradient‐descent optimization as the one yielding the best match between predicted and experimental images. With minute‐long acquisitions on a commercial low‐cost far‐infrared camera, less‐than‐5 μm spatial resolution is demonstrated on both synthetic samples and human liver biopsies ex vivo, thereby exemplifying the applicability of super‐resolution photothermal imaging to the fast nondestructive characterization of biological specimens well below the tissue spatial scale. [ABSTRACT FROM AUTHOR]

Proteins belonging to the superfamily of pyridoxal 5'-phosphate-dependent enzymes are currently classified into three functional groups and five distinct structural fold types. The variation within this enzyme group creates an ideal system to investigate the relationships among amino acid sequences, folding pathways, and enzymatic functions. The number of known three-dimensional structures of pyridoxal 5'-phosphate-dependent enzymes is rapidly increasing, but only for relatively few have the folding mechanisms been characterized in detail. The dimeric O-acetylserine sulfhydrylase from Salmonella typhimurium belongs to the beta-family and fold type II group. Here we report the guanidine hydrochloride-induced unfolding of the apo- and holoprotein, investigated using a variety of spectroscopic techniques. Data from absorption, fluorescence, circular dichroism, (31)P nuclear magnetic resonance, time-resolved fluorescence anisotropy, and photon correlation spectroscopy indicate that the O-acetylserine sulfhydrylase undergoes extensive disruption of native secondary and tertiary structure before monomerization. Also, we have observed that the holo-O-acetylserine sulfhydrylase exhibits a greater conformational stability than the apoenzyme form. The data are discussed in light of the fact that the role of the coenzyme in structural stabilization varies among the pyridoxal 5'-phosphate-dependent enzymes and does not seem to be linked to the particular enzyme fold type.