Quantitative phase microscopy (QPM), which quantifies the complex amplitude of forward-scattered (FS) light induced by specimens, enables the visualization of the macroscopic refractive index distribution in transparent samples and has been used for various applications such as dry mass analysis and cell cycle estimation. Conversely, interferometric scattering microscopy (iSCAT), which captures backward-scattered (BS) light, offers higher sensitivity compared to QPM and can effectively detect nanoparticles such as individual proteins and viruses, with the drawback of being unable to provide quantitative information on macrostructure. These microscopy techniques provide complementary information and have undergone independent advancements. In this study, we present bidirectional quantitative scattering microscopy (bidirectional QSM) for wide dynamic range imaging by simultaneously performing FS and BS measurements with a single image sensor using a digital holographic technique. This approach enables quantitative measurement of macrostructures as well as high sensitivity to nanoparticles. Moreover, digital holography facilitates the acquisition of complex amplitude FS and BS images, which allows simultaneous computational z-axis focusing, enabling subsequent quantitative correlation analysis between them. To provide a practical demonstration, time-lapse imaging of a cell is performed to characterize temporal changes in the state of cellular vital activity.
Label-free optical imaging is valuable for studying fragile biological phenomena where chemical and/or optical damages associated with exogenous labelling of biomolecules are not wanted. Molecular vibrational (MVI) and quantitative phase imaging (QPI) are the two most-established label-free imaging methods that provide biochemical and morphological information of the sample, respectively. While these methods have pioneered numerous important biological analyses along their intensive technological development over the past twenty years, their inherent limitations are still left unresolved. In this contribution, we present a unified imaging scheme that bridges the technological gap between MVI and QPI, achieving simultaneous and in-situ integration of the two complementary label-free contrasts using the midinfrared (MIR) photothermal effect. Our method is a super-resolution MIR imaging where vibrational resonances induced by wide-field MIR excitation and the resulting photothermal RI changes are detected and localized with the spatial resolution determined by a visible-light-based QPI system. We demonstrate applicability of this method, termed MV-sensitive QPI (MV-QPI), to live-cell imaging. Our MV-QPI method could allow for quantitative mapping of subcellular biomolecular distributions within the global cellular morphology in a label-free and damage-less manner, providing more comprehensive pictures of complex and fragile biological activities.
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