Molecular histopathology of matrix proteins through autofluorescence super-resolution microscopy

Extracellular matrix diseases like fibrosis are elusive to diagnose early on, to avoid complete loss of organ function or even cancer progression, making early diagnosis crucial. Imaging the matrix densities of proteins like collagen in fixed tissue sections with suitable stains and labels is a standard for diagnosis and staging. However, fine changes in matrix density are difficult to realize by conventional histological staining and microscopy as the matrix fibrils are finer than the resolving capacity of these microscopes. The dyes further blur the outline of the matrix and add a background that bottlenecks high-precision early diagnosis of matrix diseases. Here we demonstrate the multiple signal classification method-MUSICAL-otherwise a computational super-resolution microscopy technique to precisely estimate matrix density in fixed tissue sections using fibril autofluorescence with image stacks acquired on a conventional epifluorescence microscope. We validated the diagnostic and staging performance of the method in extracted collagen fibrils, mouse skin during repair, and pre-cancers in human oral mucosa. The method enables early high-precision label-free diagnosis of matrix-associated fibrotic diseases without needing additional infrastructure or rigorous clinical training.

. Overview of the numbers of samples, images and ROI's used in the study.*The pathologists select these 25 samples according to their exclusion criteria, where they choose samples that do not have other complications.

Metrics Explanation and Clinical relevance Human Oral
The overall collagen density change in sub-epithelium with respect to keratin density change in Submucous SE/E Epithelium.
Provides an overview of how the density of these two molecules is effected when the pre-cancer Fibrosis progresses to cancer.The density change of keratin in upper epithelium relative to lower epithelium.

UE/LE
The disease originates in lower epithelium with high keratin expression relative to upper epithelium.Lower value denotes normal and the value increases with disease progression.The density of collagen in upper sub-epithelium relative to keratin in lower epithelium.USE/LE These two sub-layers are closely interacting with each-other and is at the junction of the site of invasion.This is an important metric to evaluate the relative collage keratin interaction in early onset of pre-cancer and therefore, a valuable early-marker.
The density change of collagen in upper sub-epithelium relative to lower sub-epithelium.LSE/USE This represents papillary and reticular regions of sub-epithelium which increasingly gets indistinguishable with advancement of oral sub-mucous fibrosis.Human Oral PKL/UE The density of keratin in para-keratinized layer relative to upper epithelium.

Leukoplakia
The density of para-keratinized layer at this junction indicates progression of the disease.
The keratin in the PKL layer are highly compressed and difficult to effectively stain due to antigen blocking, but are easily discernible with auto-fluorescence.

UE/LE
The density of keratin in upper epithelium relative to lower epithelium.
Table S2.Overview of the clinical metrics used in the study.This table refers to the metrics used in Fig 4 and Fig 5 of the article.For mouse dermal fibrosis, all intensity measurements are taken relative to the stratum corneum of the skin (outer most exposed layer of skin) in the Fig 7 of the article .

Figure S2 .
Figure S2.Effect of epitope retrieval in reducing the formalin-induced fluorescence (a,b) are the autofluorescence images of the same mouse skin tissue section taken before and after heat-induced epitope retrieval (same tissue section, image captured at similar location before and after processing).The yellow arrows show the regions with non-uniform fluorescence before epitope retrieval.(c) FTIR spectra of the adjacent mouse skin tissue sections with and without epitope retrieval.The spectra shows that the peaks of methylene bridges (formalin-derived crosslinks that cause unspecific autofluorescence) are substantially reduced upon epitope retrieval.

Figure S3 .
Figure S3.Benchmarking MUSI-tAF super-resolution in collagen nanofibers.A region of thinly deposited collagen-I under (a) diffraction-limited (mag 20×), (b) MUSI-tAF, and (c) SEM showing that MUSI-tAF images collagen nanofibers and can distinguish collagen spaced 70-80 nm apart (edge to edge).A denser region of rat tail collagen-I fibers under (d) diffraction-limited, (e) MUSI-tAF, and (f) SEM showing matching regions.The (g) diffraction-limited and (h) MUSI-tAF images of the same region demonstrate enhancement of resolution in closely spaced collagen fibers (yellow arrows).The profiles along the white line is shown in (i) demonstrating that MUSI-tAF super-resolves a nearly flat profile in DL imaging of four closely spaced collagen bundles.The colored boxes in the images (d,e,f) highlight the regions of the fiber structure visualizing the matching between the DL, MUSI-tAF and the SEM images respectively.The boxes of the same color show the matching region.

Figure S4 .
Figure S4.Collagen autofluorescence provides a faithful distribution and localization than its labeled microscopy.The left column shows collagen autofluorescence (blue channel) and the right column is the collagen labeling (red channel) taken from the same region.The zoomed images show that although labeling can provide a visual correspondence of collagen density it has poor localization of fibers.The images were acquired at 20× magnification, 0.8 NA.

Figure S5 .
Figure S5.Comparing collagen nanostructures in MUSI-tAF autofluorescence and labelled fluorescence.MUSI-tAF images of (a-d) purified collagen-I and (e-h) mouse skin tissue sections.Images derived from (a,b,e,f) tissue autofluorescence (tAF) in blue emission, (c,d,g,h) same tissue immunolabeled with collagen-I and fluorescent probe having red emission.All insets show the corresponding source (diffraction-limited) images (20×, 0.8 NA objective).(a,c,e,g) are full-field super-resolved images and (b,d,f,h) are a small region illustrating super-resolved structures.Co-localization of structures of (i,j) pure collagen-I and (m,n)tissue from autofluorescence (blue) and labeled (red) fluorescence.Similarity maps of MUSICAL structures in the red and blue colors images in (i,j) are shown in (k,l) for Collagen-I, and similarly similarity maps of the structures in red and blue colored images in (m,n) are shown in (o,p) for the mouse skin tissue.

Figure S6 .
Figure S6.Simulated sample showing emitters placed along a straight line at a distance of 10 µm, showing the rejection of out-of-focus light property in MUSI-tAF.The imaging process was simulated using a 20× and 100× objective with Poisson noise.(a) The line passes through several planes at different z-positions and is the ground-truth.The color bar indicates the distance of the emitter and the coverslip.(b) 20× objective diffraction limited image over 100 frames created for the sample.The edges of the line (arrows) show the effect of the point-spread-function being wider at off-focus regions.(c) MUSI-tAF results for the 20× objective where the focal section is filtered from the off-focus emission.The dotted line is the profile of the corresponding diffraction limited line showing the region of rejection on the edges.(d) Profile plot of a 100× diffraction limited image.

Figure S7 .
Figure S7.Histology of representative full oral tissues sections with varying levels of pathology associated with oral carcinoma.(a) Hematoxylin and eosin (HE) stain of the normal oral mucosa (NOM), the darker stained outer epithelium (black arrows), and sub-epithelium (red arrow).The region below the black line is majorly muscle tissues; the bold black arrow shows the epithelium's rete pegs, i.e., undulations at the base of the epithelial layer.(b) HE of oral submucous fibrosis (OSF) shows denser epithelium and flattened rete pegs (bold arrow).(c)HE of oral submucous fibrosis with dysplasia (OSFD), ) with flattened rete pegs (bold arrow), dense sub-epithelium, and perivascular fibrosis (red arrows).(m) HE of oral squamous cell carcinoma (OSCC), with the epithelial basement membrane, lost continuity and invaded into subepithelium (bold arrows).

Figure S8 .
Figure S8.Multi-scale MUSI-tAF images of healthy and fibrotic skin tissues.(a-d) MUSI-tAF images of a healthy skin tissue visualized at image sizes of (a) 10000×13400 (full field of view), (b) 4000×5000, (c) 2000×3000, and (d) 1000×1000.The four image dimensions have been illustrated for progressive pathological fibrosis at (e-h) 18 days, (i-l) 30 days, (m-p) 60 days, and (q-t) 180 days; here, while 18 days and 30 days treatment are early fibrosis, 60 days and 180 days are advanced fibrosis.(u-t) images of scar tissue collected from 60 days of wound healing at the same dimensions as healthy skin.scale bar= 2µm.

Figure S9 .
Figure S9.Comparison of epifluorescence and MUSI-tAF intensity ratios in different regions of the oral tissue.The figure highlights how MUSI-tAF can make better delination of disease stages compared to epifluorescence imaging of autofluorescent tissue matrix.This figure corresponds to Fig 4 (a-e) in the main manuscript. Table