Volumetric chemical imaging by clearing-enhanced stimulated Raman scattering microscopy

Three-dimensional visualization of tissue structures using optical microscopy facilitates the understanding of biological functions. However, optical microscopy is limited in tissue penetration due to severe light scattering. Recently, a series of tissue-clearing techniques have emerged to allow significant depth-extension for fluorescence imaging. Inspired by these advances, we develop a volumetric chemical imaging technique that couples Raman-tailored tissue-clearing with stimulated Raman scattering (SRS) microscopy. Compared with the standard SRS, the clearing-enhanced SRS achieves greater than 10-times depth increase. Based on the extracted spatial distribution of proteins and lipids, our method reveals intricate 3D organizations of tumor spheroids, mouse brain tissues, and tumor xenografts. We further develop volumetric phasor analysis of multispectral SRS images for chemically specific clustering and segmentation in 3D. Moreover, going beyond the conventional label-free paradigm, we demonstrate metabolic volumetric chemical imaging, which allows us to simultaneously map out metabolic activities of protein and lipid synthesis in glioblastoma. Together, these results support volumetric chemical imaging as a valuable tool for elucidating comprehensive 3D structures, compositions, and functions in diverse biological contexts, complementing the prevailing volumetric fluorescence microscopy

Volumetric chemical imaging by clearing-enhanced stimulated Raman scattering microscopy

Three-dimensional visualization of tissue structures using optical microscopy facilitates the understanding of biological functions. However, optical microscopy is limited in tissue penetration due to severe light scattering. Recently, a series of tissue-clearing techniques have emerged to allow significant depth-extension for fluorescence imaging. Inspired by these advances, we develop a volumetric chemical imaging technique that couples Raman-tailored tissue-clearing with stimulated Raman scattering (SRS) microscopy. Compared with the standard SRS, the clearing-enhanced SRS achieves greater than 10-times depth increase. Based on the extracted spatial distribution of proteins and lipids, our method reveals intricate 3D organizations of tumor spheroids, mouse brain tissues, and tumor xenografts. We further develop volumetric phasor analysis of multispectral SRS images for chemically specific clustering and segmentation in 3D. Moreover, going beyond the conventional label-free paradigm, we demonstrate metabolic volumetric chemical imaging, which allows us to simultaneously map out metabolic activities of protein and lipid synthesis in glioblastoma. Together, these results support volumetric chemical imaging as a valuable tool for elucidating comprehensive 3D structures, compositions, and functions in diverse biological contexts, complementing the prevailing volumetric fluorescence microscopy

DISC-3D: dual-hydrogel system enhances optical imaging and enables correlative mass spectrometry imaging of invading multicellular tumor spheroids

Multicellular tumor spheroids embedded in collagen I matrices are common in vitro systems for the study of solid tumors that reflect the physiological environment and complexities of the in vivo environment. While collagen I environments are physiologically relevant and permissive of cell invasion, studying spheroids in such hydrogels presents challenges to key analytical assays and to a wide array of imaging modalities. While this is largely due to the thickness of the 3D hydrogels that in other samples can typically be overcome by sectioning, because of their highly porous nature, collagen I hydrogels are very challenging to section, especially in a manner that preserves the hydrogel network including cell invasion patterns. Here, we describe a novel method for preparing and cryosectioning invasive spheroids in a two-component (collagen I and gelatin) matrix, a technique we term dual-hydrogel in vitro spheroid cryosectioning of three-dimensional samples (DISC-3D). DISC-3D does not require cell fixation, preserves the architecture of invasive spheroids and their surroundings, eliminates imaging challenges, and allows for use of techniques that have infrequently been applied in three-dimensional spheroid analysis, including super-resolution microscopy and mass spectrometry imaging

Breast Cancer Cell Line Aggregate Morphology Does Not Predict Invasive Capacity.

To invade and metastasize to distant loci, breast cancer cells must breach the layer of basement membrane surrounding the tumor and then invade through the dense collagen I-rich extracellular environment of breast tissue. Previous studies have shown that breast cancer cell aggregate morphology in basement membrane extract correlated with cell invasive capacity in some contexts. Moreover, cell lines from the same aggregate morphological class exhibited similarities in gene expression patterns. To further assess the capacity of cell and aggregate morphology to predict invasive capacity in physiologically relevant environments, six cell lines with varied cell aggregate morphologies were assessed in a variety of assays including a 3D multicellular invasion assay that recapitulates cell-cell and cell-environment contacts as they exist in vivo in the context of the primary breast tumor. Migratory and invasive capacities as measured through a 2D gap assay and a 3D spheroid invasion assay reveal that breast cancer cell aggregate morphology alone is insufficient to predict migratory speed in 2D or invasive capacity in 3D. Correlations between the 3D spheroid invasion assay and gene expression profiles suggest this assay as an inexpensive functional method to predict breast cancer invasive capacity

Cell line morphological, genetic, and functional characteristics.

<p>Cell line aggregate morphological classes, cancer stem cell (CSC) percentages, subtypes, positivity for wound, hypoxia, and 70 gene signature, and collagen related activities.</p><p>^§CSC percentages obtained from Ref. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139523#pone.0139523.ref050" target="_blank">50</a>]. MDA-MB-468 cells were not included in this study and thus labeled N/A (not available).</p><p>*Subtypes of breast cancer cell lines identified by unsupervised hierarchical clustering: BB = basal B, BA = basal A, L = luminal [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139523#pone.0139523.ref021" target="_blank">21</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139523#pone.0139523.ref046" target="_blank">46</a>].</p><p>**Subtypes of breast cancer cell lines using tumor cell classifications described in Ref. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139523#pone.0139523.ref043" target="_blank">43</a>] as reported in Ref. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139523#pone.0139523.ref046" target="_blank">46</a>]: ERBB2 = ERBB2 signature; B = basal, LA = luminal A, LB = luminal B. ***Subtypes of breast cancer cell lines using tumor cell classifications as reported in Ref. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139523#pone.0139523.ref047" target="_blank">47</a>]: CLL = claudin-low; B = basal; L = luminal.</p><p>⁺+ = positive and— = negative for wound or hypoxia gene signatures or poor prognosis signature in the 70 gene assay as reported in Ref. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139523#pone.0139523.ref046" target="_blank">46</a>].</p><p>^#This study: + indicates moderate to high collagen contractile capacity (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139523#pone.0139523.g005" target="_blank">Fig 5</a>), moderate to high levels of collagen-relevant integrins (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139523#pone.0139523.g004" target="_blank">Fig 4</a>), and capacity to efficiently invade collagen I gels from spheroid culture (Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139523#pone.0139523.g003" target="_blank">3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139523#pone.0139523.g005" target="_blank">5</a>).</p><p>Cell line morphological, genetic, and functional characteristics.</p

Transmittance images of cells from each cell line investigated in a gap assay at 24 hours.

<p>Initial gap distance is 500 μm, the height of the images. Scale bar is 200 μm.</p

Spheroids of each investigated cell line at 2 hours and 24 hours after implantation in 1.0 mg/mL collagen I.

<p>(a) Transmittance images at 2 hours, (b) confocal reflectance images of collagen surrounding the spheroids (and a portion of the spheroid in the lower left corner) at 2 hours, (c) transmittance images at 24 hours, (d) magnified view of cells invading from the spheroid at 24 hours. Additional magnification shown in the inset highlights invading cell morphology. Black scale bars are 200 μm and white scale bars are 50 μm. Confocal reflectance images (row b) have had an optical artifact that causes a bright spot in the center of the field of view removed by replacement of this spot with a representative area from elsewhere in the image.</p

Morphology of representative isolated cells and aggregates of each investigated cell line in 3D 10 mg/mL lrECM, on 2D glass, and in 3D 1.0 mg/mL collagen I matrices.

<p>Cells were fixed after 24 hours of culture and stained for F-actin. Scale bar is 50 μm.</p