Stain-free histopathology by programmable supercontinuum pulses

Abstract

The preparation, staining, visualization, and interpretation of histological images of tissue is well-accepted as the gold standard process for the diagnosis of disease. These methods were developed historically, and are used ubiquitously in pathology, despite being highly time and labor intensive. Here we introduce a unique optical imaging platform and methodology for label-free multimodal multiphoton microscopy that uses a novel photonic crystal fiber source to generate tailored chemical contrast based on programmable supercontinuum pulses. We demonstrate collection of optical signatures of the tumor microenvironment, including evidence of mesoscopic biological organization, tumor cell migration, and (lymph-)angiogenesis collected directly from fresh ex vivo mammary tissue. Acquisition of these optical signatures and other cellular or extracellular features, which are largely absent from histologically processed and stained tissue, combined with an adaptable platform for optical alignment-free programmable-contrast imaging, offers the potential to translate stain-free molecular histopathology into routine clinical use.

Figure 1. Mesoscopic organization of biological microstructures revealed in co-localized multiphoton images of two rat mammary specimens and absent from corresponding FFPE-H&E histology images

Area-integrated CARS spectra over 34 hyperspectral images confirm the presence of a significant R3050 peak (see text for details) as a potential cancer biomarker. (Left): Different histochemical components of a tumor are selectively revealed by different single-modality images. Cell cluster 1 (red outline) is identifiable in a 2PAF image, and also in 3PAF, R3050, and R2850 images, reflecting tumor-associated metabolism (see text for details). The hexagonal radar multiphoton profile (9-pixel average) of a specific type of isolated vesicle approximates that of vesicles aligned in a tubular formation, suggesting that these vesicles are distributed more diffusely before organizing into a tube (indicative of angiogenesis, see text for details). (Right): Cell cluster 2 (red outline) in a specimen with histologically unidentified cancer from a carcinogen-injected rat is identifiable in a 2PAF image, but not in R3050 and other images, reflecting normal cellular metabolism. Elastin fibers widely observed in connective tissue are shown to be organized into a “basket”, which can be linked to lymphangiogenesis (see text for details). Multiphoton image size: 380 × 380 pixels with 0.5 µm pixel size.

Figure 2. Optical signatures in co-localized multiphoton images of five mammary specimens that are absent from corresponding FFPE-H&E histology images

Area-integrated CARS spectra over 30 hyperspectral images reveal common molecular vibrations of CH₂ (2850 cm⁻¹), CH₃ (2930 cm⁻¹), =CH (3015 cm⁻¹), and OH stretches (3000–3200 cm⁻¹), and exhibit a cancer biomarker at R3050, whereas cross-modality visibilities of 2PAF-enhanced cells (encircled in red and labeled #3–7) represent distinct cellular metabolic states. (Bottom row): histology delineates characteristic adipose and stromal regions of control mammary tissue, and reveals cell nuclei located in interstitial spaces among lipid vacuoles of adipocytes with no correspondence between cell nuclei and lipids. In multiphoton images, however, 2PAF-visible cell bodies in the background of 3PAF-visible lipids point to the unique relationship between cell bodies and lipids (red arrows) that form complete adipocytes. (Bottom row, second left): histology of a stroma-only region from a normal appearing mammary specimen (no palpable tumor) from a carcinogen-injected rat displays only some cell nuclei scattered in a distorted collagen fiber network, whereas multiphoton images show how several cells orient themselves in the voids of the collagen fiber network. (Bottom row, middle): histology identifies fibroblasts (blue arrows) from mast cells (magenta arrows) at a tumor margin approximately 1 mm away from a palpable week 6 tumor. In multiphoton images, however, spindle-shaped collagen-producing fibroblasts are aligned with SHG-visible collagen fibers and can be easily differentiated from mast cells near a lymphatic vessel (LV) with flowing lymph (Supplementary Video 4) and a leaky 2PAF-visible basement membrane containing elastin fibers. (Second right): multiphoton images of the inside of a non-palpable tumor reveals cross-linked collagen network that assists tumor invasion. (Right): multiphoton images at a tumor-stroma boundary exhibit vital signatures of local tumor invasion across TACS-2 collagen. Abbreviations: EF – elastin fibers; N– nerve; LV – lymphatic vessel; TACS – tumor-associated collagen structure. Multiphoton image size: 380 × 380 pixels with 0.5 µm pixel size.

Figure 3. Optical signatures of local tumor invasion in large-area tri-modal multiphoton images of two rat mammary specimens that are absent from corresponding FFPE-H&E histology images

The margin specimen was excised approximately 1 mm away from the tumor. Directed local tumor invasion (downward in image) at the tumor-stroma boundary (left) and degradation of adipose tissue by vascularized stroma at the tumor margin (right) are recognized in the multiphoton images, but only the former can be revealed in standard H&E histology. In the latter case, the 3PAF-visible angiogenesis (An) is absent and naturally branched 2PAF-visible blood vessels (BV) become distorted and fragmented in the histology image so that the tumor-associated angiogenesis is hardly detectable. In both cases, several vital optical signatures (arrows) of local tumor invasion are largely obscured by a strong interfering background in the tri-modal multiphoton images, just like in histology, but emerge when the contrasts of individual multiphoton modalities are unmixed (see Supplementary Figs. S6–S9 and text for details). Abbreviations: AACR – angiogenesis-accommodating collagen reorganization; An – angiogenesis; BV – blood vessel; CTCI – collective tumor cell invasion; LA – lymphangiogenesis; TACS – tumor-associated collagen structure. Multiphoton image size: 1100 × 1100 pixels with 0.5 µm pixel size.