Fig. 1: Design and capabilities of the label-free optical platform span targeted regions of interest to whole slide imaging. | Nature Communications

Fig. 1: Design and capabilities of the label-free optical platform span targeted regions of interest to whole slide imaging.

From: Label-free multimodal optical biopsy reveals biomolecular and morphological features of diabetic kidney tissue in 2D and 3D

Fig. 1

a Diagram of the label-free microscopy system showing the transmission detection of stimulated Raman loss and epi detection of fluorescence emission and second harmonic generation. Created in BioRender. Fung, T. (2025) https://BioRender.com/oh1d3qp. b Example images corresponding to each modality in 2D and 3D, along with their respective Jablonski diagram. c A whole slide image of a renal core biopsy from a patient diagnosed with Diabetic Nephropathy (DN) imaged using the platform described in (a). The image is the result of overlaying seven discrete channels ascribed to saturated fatty acids (SRS at 2880 cm−1), unsaturated fatty acids (SRS 3011 cm−1), proteins (SRS 2940 cm−1), lipids (SRS 2850 cm−1), collagen (SHG at 1031 nm/515 nm), flavins (TPF 860 nm/515 nm), and NAD(P)H (TPF 780 nm/460 nm). d An unsupervised k-means clustering of the multimodal WSI. e A PCA-initialized, standardized t-SNE plot of the pixels in the multimodal image color coded to the respective k-means cluster. Colors correspond to cluster identity 1–6. f The stacked bar graph shows the simplex-normalized average pixel intensity’s channel contribution in each cluster. Color corresponds to discrete channel of the multimodal image stack. g Regions of interest are selected along the cortex-medulla axis and a full SRS hyperspectral image sweep (512 × 512 pixels) of the C-H stretching region are obtained at the same time. The mean SRS spectra and 1 standard deviation error bands are plotted for each region. h Phasor plots of all pixels in the ROIs separate spectra based on phase and amplitude. The plot is duplicated, and color coded according to region. Scale bar is 700 µm, corresponding to the large WSI in (a). DM Dichroic Mirror, EOM Electro-Optic Modulator, OB Objective Lens, CO Oil Condenser, SF Stokes Filter, PD Photodiode, SRL Stimulated Raman Loss, SRS Stimulated Raman Scattering, TPF Two Photon Fluorescence, SHG Second Harmonic Generation.

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