Zhi Li

Optical imaging technologies are critical in biomedical studies for their ability to obtain both morphological and functional information from biological specimens at high spatial resolution. These optical processes exploit various light-molecule interactions, such as scattering, absorption, emission, and harmonic generation, between photons and the molecules within cells, tissues, or organs. While conventional biomedical imaging has historically focused on applying a single modality, recent research has shown that these diverse techniques provide complementary insights, and their combined outputs offer a more comprehensive understanding of molecular changes in aging processes and disease development and fundamentals in cell biology. In the past decades, label-free optical imaging methods have advanced, enabling detailed exploration of cellular and subcellular environments. For instance, multiphoton fluorescence (MPF) not only facilitates targeted protein imaging but also quantifies metabolic activity through autofluorescent coenzymes, achieving high penetration depth and spatial resolution. Second Harmonic Generation (SHG) is used to image structures like collagen in the extracellular matrix, while Stimulated Raman Scattering (SRS) maps chemical bonds and molecular composition in situ with subcellular resolution. We have developed a multimodal imaging platform that combines MPF, SHG, and SRS modalities. The integration of these modalities into a single platform enables the acquisition of multifaceted information from the same localization within cells, tissues, organs, or even bodies, facilitating a more detailed exploration of the intricate relationships between cellular metabolism, extracellular matrix structure, and molecular composition. This multimodal system offers subcellular resolution, deep tissue penetration, in situ live-cell/tissue imaging, as well as label-free detection and instantaneous coregistration without the need for position adjustments, device switching, or postanalysis alignment. Here, we present a protocol for label-free imaging with this multimodal platform and demonstrate its application in characterizing cellular metabolism, and molecular heterogeneity in cells and tissues for studying aging and diseases.

Optical imaging technologies are critical in biomedical studies for their ability to obtain both morphological and functional information from biological specimens at high spatial resolution. These optical processes exploit various light-molecule interactions, such as scattering, absorption, emission, and harmonic generation, between photons and the molecules within cells, tissues, or organs. While conventional biomedical imaging has historically focused on applying a single modality, recent research has shown that these diverse techniques provide complementary insights, and their combined outputs offer a more comprehensive understanding of molecular changes in aging processes and disease development and fundamentals in cell biology. In the past decades, label-free optical imaging methods have advanced, enabling detailed exploration of cellular and subcellular environments. For instance, multiphoton fluorescence (MPF) not only facilitates targeted protein imaging but also quantifies metabolic activity through autofluorescent coenzymes, achieving high penetration depth and spatial resolution. Second Harmonic Generation (SHG) is used to image structures like collagen in the extracellular matrix, while Stimulated Raman Scattering (SRS) maps chemical bonds and molecular composition in situ with subcellular resolution. We have developed a multimodal imaging platform that combines MPF, SHG, and SRS modalities. The integration of these modalities into a single platform enables the acquisition of multifaceted information from the same localization within cells, tissues, organs, or even bodies, facilitating a more detailed exploration of the intricate relationships between cellular metabolism, extracellular matrix structure, and molecular composition. This multimodal system offers subcellular resolution, deep tissue penetration, in situ live-cell/tissue imaging, as well as label-free detection and instantaneous coregistration without the need for position adjustments, device switching, or postanalysis alignment. Here, we present a protocol for label-free imaging with this multimodal platform and demonstrate its application in characterizing cellular metabolism, and molecular heterogeneity in cells and tissues for studying aging and diseases.

Stimulated Raman Scattering (SRS) nanoscopy imaging offers unprecedented insights into tissue molecular architecture but often requires stitching multiple high-resolution tiles to capture large fields of view. This process is time-consuming and frequently introduces vignetting artifacts—grid-like intensity fluctuations that degrade image quality and hinder downstream quantitative analyses and processing such as super-resolution deconvolution. We present VIgnetted Stitched-Tile Adjustment using Morphological Adaptive Processing (VISTAmap), a simple tool that corrects these shading artifacts directly on the final stitched image. VISTAmap automatically detects the tile grid configuration by analyzing intensity frequency variations and then applies sequential morphological operations to homogenize the image. In contrast to conventional approaches that require increased tile overlap or pre-acquisition background sampling, VISTAmap offers a pragmatic, post-processing solution without the need for separate individual tile images. This work addresses pressing concerns by delivering a robust, efficient strategy for enhancing mosaic image uniformity in modern nanoscopy, where the smallest details make tremendous impacts.