Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Abstract

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

Local remodeling of synthetic extracellular matrix microenvironments by co-cultured endometrial epithelial and stromal cells enables long-term dynamic physiological function

“…In order to further probe ECM composition and structure, we live-imaged the stromal layer with two-photon second harmonic imaging to visualize fibrillar collagens (i.e., collagens I-III and others, excluding the non-fibrillar collagen IV). 78,79 A cell associated second harmonic signal was observed in the presence of matrix binding peptides, especially the FN-binder , but was absent without matrix stabilizing peptides (Supplementary Fig. S5).…”

Macrophage-Dependent Trafficking and Remodeling of the Basement Membrane-Interstitial Matrix Interface

“…Given the compositional and structural integrity of our ex vivo tissue construct, we next sought to characterize the nature of its interactions with primary human macrophages. As such, carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled monocyte-derived macrophages were cultured atop basement membranes that were pre-labeled with fluorescently-tagged anti-laminin antibodies in the presence of F c receptor blocking reagents to prevent direct interactions between the macrophages and the antibody-coated surface (Güç, Fankhauser, Lund, Swartz, & Kilarski, 2014; Kilarski et al, 2013). After a 48 h time period, the macrophage-tissue constructs were imaged for 160 mins using real-time spinning disc confocal microscopy.…”

Evaluation of Sidestream Darkfield Microscopy for Real-Time Imaging Acellular Dermal Matrix Revascularization

“…22 Techniques for intravital microscopy, including 2-photon microscopy and laser tomography, demonstrate promise for visualization of cell-cell interactions with blood, lymphatic, and extracellular matrix compartments of dermis; however, these techniques require the use of exogenous reporters and fluorescent-labeled antibodies and are at present restricted to investigational use. [22][23][24] Photoacoustic microscopy is an advanced imaging technique, which uses a focused laser pulse, which is absorbed by hemoglobin in tissue which is converted to heat and ultimately a detectable acoustic emission, which can be converted to a 3-dimensional map of the microvasculature. This technique is currently being investigated clinically for its unparalleled spatial resolution without processing artifact and allows for a direct assessment of the concentration of hemoglobin in tissue as well as oxygen saturation and blood flow; however, no commercially available imaging system is currently clinically available.…”