Label-Free Complete Absorption Microscopy Using Second Generation Photoacoustic Remote Sensing

Abstract

In the past decades, absorption modalities have emerged as powerful tools for label-free functional and structural imaging of cells and tissues. Many biomolecules present unique absorption spectra providing chromophore-specific information on properties such as chemical bonding, and sample composition. As chromophores absorb photons the absorbed energy is emitted as photons (radiative relaxation) or converted to heat and under specific conditions pressure (non-radiative relaxation). Modalities like fluorescence microscopy may capture radiative relaxation to provide contrast, while modalities like photoacoustic microscopy may leverage non-radiative heat and pressures. Here we show an all-optical non-contact total-absorption photoacoustic remote sensing (TA-PARS) microscope, which can capture both radiative and non-radiative absorption effects in a single acquisition. The TA-PARS yields an absorption metric proposed as the quantum efficiency ratio (QER), which visualizes a biomolecules proportional radiative and non-radiative absorption response. The TA-PARS provides label-free visualization of a range of biomolecules enabling convincing analogues to traditional histochemical staining of tissues, effectively providing label-free Hematoxylin and Eosin (H&E)-like visualizations. These findings represent the establishment of an effective all-optical non-contact total-absorption microscope for label-free inspection of biological media.

Virtual Histological Staining of Label-Free Total Absorption Photoacoustic Remote Sensing (TA-PARS)

“…A newly developed modality, photoacoustic remote sensing (PARS) [32][33][34] , overcomes the drawbacks of PAM. In PARS, the acoustically coupled ultrasound transducer is replaced with a secondary co-focused detection laser, offering robust all-optical, non-contact, label-free photoacoustic imaging 35 .…”

“…In PARS, the acoustically coupled ultrasound transducer is replaced with a secondary co-focused detection laser, offering robust all-optical, non-contact, label-free photoacoustic imaging 35 . Over the past few years, PARS has emerged as a powerful contender in the label-free histological imaging space [32][33][34]36 . PARS may emulate current histochemical staining, by targeting the UV absorption of DNA (analogous to hematoxylin staining) 32,33 , and by targeting the 420 nm absorption of cytochrome (analogous to hematoxylin staining) 32 .…”

“…PARS may emulate current histochemical staining, by targeting the UV absorption of DNA (analogous to hematoxylin staining) 32,33 , and by targeting the 420 nm absorption of cytochrome (analogous to hematoxylin staining) 32 . Recent works have shown that PARS may offer high contrast and high spatial resolution in freshly resected tissue, formalin xed tissue, and FFPE tissue sections 33,34 . In resected tissues, PARS has even been shown to provide 3D imaging of subsurface nuclei 33 .…”

“…In this paper, we employ a second-generation PARS architecture, total-absorption photoacoustic remote sensing (TA-PARS) 34 , to provide label-free histology-like images. As outlined by Ecclestone et al the TA-PARS architecture incorporates a number of advances providing improved sensitivity and contrast when imaging in tissues specimen 34 . Applied to resected tissues, TA-PARS directly measures analogous contrast to H&E staining, capturing nuclei and cytoplasm independently.…”

“…To the extent of our knowledge, the TA-PARS is the only modality that can provide all three contrast mechanisms in a single capture. This rich array of input data provides simultaneous recovery of structures including cell nuclei, brin, connective tissues, adipocytes, and neurons 34 in a single excitation event. The diagnostic con dence of deep learning-based virtual histological staining can be improved using this more informative dataset for network training and colorization.…”