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High‐sensitivity chemical imaging offers a window to decipher the molecular orchestra inside a living system. Based on vibrational fingerprint signatures, coherent Raman scattering microscopy provides a label‐free approach to map biomolecules and drug molecules inside a cell. Yet, by near‐infrared (NIR) pulse excitation, the sensitivity is limited to millimolar concentration for endogenous biomolecules. Here, the imaging sensitivity of stimulated Raman scattering (SRS) is significantly boosted for retinoid molecules to 34 micromolar via electronic preresonance in the visible wavelength regime. Retinoids play critical roles in development, immunity, stem cell differentiation, and lipid metabolism. By visible preresonance SRS (VP‐SRS) imaging, retinoid distribution in single embryonic neurons and mouse brain tissues is mapped, retinoid storage in chemoresistant pancreatic and ovarian cancers is revealed, and retinoids stored in protein network and lipid droplets ofCaenorahbditis elegansare identified. These results demonstrate VP‐SRS microscopy as an ultrasensitive label‐free chemical imaging tool and collectively open new opportunities of understanding the function of retinoids in biological systems.

 

Stimulated Raman projection tomography is a label-free volumetric chemical imaging technology allowing three-dimensional (3D) reconstruction of chemical distribution in a biological sample from the angle-dependent stimulated Raman scattering projection images. However, the projection image acquisition process requires rotating the sample contained in a capillary glass held by a complicated sample rotation stage, limiting the volumetric imaging speed, and inhibiting the study of living samples. Here, we report a tilt-angle stimulated Raman projection tomography (TSPRT) system which acquires angle-dependent projection images by utilizing tilt-angle beams to image the sample from different azimuth angles sequentially. The TSRPT system, which is free of sample rotation, enables rapid scanning of different views by a tailor-designed four-galvo-mirror scanning system. We present the design of the optical system, the theory, and calibration procedure for chemical tomographic reconstruction. 3D vibrational images of polystyrene beads and C. elegans are demonstrated in the C-H vibrational region.

 

Operable under ambient light and providing chemical selectivity, stimulated Raman scattering (SRS) microscopy opens a new window for imaging molecular events on a human subject, such as filtration of topical drugs through the skin. A typical approach for volumetric SRS imaging is through piezo scanning of an objective lens, which often disturbs the sample and offers a low axial scan rate. To address these challenges, we have developed a deformable mirror-based remote-focusing SRS microscope, which not only enables high-quality volumetric chemical imaging without mechanical scanning of the objective but also corrects the system aberrations simultaneously. Using the remote-focusing SRS microscope, we performed volumetric chemical imaging of living cells and captured in real time the dynamic diffusion of topical chemicals into human sweat pores.

 

Abstract: Thin tissue slice based histology has been used as a gold standard for disease diagnosis since over a hundred years ago. However, histopathological evaluation on two-dimensional slides suffers from large variations due to limited sampling. To improve the diagnostic accuracy, three-dimensional (3D) histology is performed through serial sectioning, staining, imaging and reconstruction of individual slices, which is highly time-consuming and labor intensive. We developed a volumetric stimulated Raman scattering (SRS) imaging method, which provides histology-like information in 3D context without the need for staining with dyes. Using a small molecule clearing agent, formamide, we performed tissue clearing within 30 min and achieved an imaging depth up to 500 µm in highly scattered tissues, including brain, kidney, liver and lung. Through a two-color SRS imaging scheme, we obtained histology-like images in cleared brain tissue slices. Our method has the potential for 3D tissue histopathology to improve the accuracy of histopathological examination. https://doi.org/10.1364/BOE.10.004329