IMART software for correction of motion artifacts in images collected in intravital microscopy
Intravital microscopy is a uniquely powerful tool, providing the ability to characterize cell and organ physiology in the natural context of the intact, living animal. With the recent development of high-resolution microscopy techniques such as confocal and multiphoton microscopy, intravital microscopy can now characterize structures at subcellular resolution and capture events at sub-second temporal resolution. However, realizing the potential for high resolution requires remarkable stability in the tissue. Whereas the rigid structure of the skull facilitates high-resolution imaging of the brain, organs of the viscera are free to move with respiration and heartbeat, requiring additional apparatus for immobilization. In our experience, these methods are variably effective, so that many studies are compromised by residual motion artifacts. Here we demonstrate the use of IMART, a software tool for removing motion artifacts from intravital microscopy images collected in time series or in three dimensions.
Optimization of the dorsal skinfold window chamber model and multi-parametric characterization of tumor-associated vasculature
The dorsal skinfold window chamber (DSWC) model is a unique tool that enables analysis of various aspects of tumor biology and therapeutic response. Although the protocol for the murine DSWC model is standardized, certain tumors fail to grow or require a particular environment to promote growth. Given such limitations, we optimized the DSWC model for a slow-growing tumor that regresses spontaneously in the standard protocol. We further characterized the vascular network in the tumor model compared with that of non-tumor-bearing mice and observed significant differences in multiple parameters related to vascular structure and function.
Semi-automated vitality analysis of human trabecular meshwork
Glaucoma is associated with cell loss in the trabecular meshwork of the eye. Multiphoton microscopy aided by intravital dye labeling permits visualization of live and dead cells within the intact trabecular meshwork. We have developed a semi-automated method to quantify cellular viability within the human trabecular meshwork based on three-dimensional software-assisted tissue reconstruction and isosurface modeling. Live cellularity counts by the semi-automated method were obtained quickly and agreed with that of manual counting in the same group of tissues (1.6% group difference; n = 13), with counts in individual tissues showing a mean coefficient of variation of 10%.
Automated analysis of clonal cancer cells by intravital imaging
Longitudinal analyses of single cell lineages over prolonged periods have been challenging particularly in processes characterized by high cell turn-over such as inflammation, proliferation, or cancer. RGB marking has emerged as an elegant approach for enabling such investigations. However, methods for automated image analysis continue to be lacking. Here, to address this, we created a number of different multicolored poly- and monoclonal cancer cell lines for in vitro and in vivo use. To classify these cells in large scale data sets, we subsequently developed and tested an automated algorithm based on hue selection. Our results showed that this method allows accurate analyses at a fraction of the computational time required by more complex color classification methods. Moreover, the methodology should be broadly applicable to both in vitro and in vivo analyses.
Intravital multiphoton imaging reveals multicellular streaming as a crucial component of in vivo cell migration in human breast tumors
Metastasis is the main cause of death in breast cancer patients. Cell migration is an essential component of almost every step of the metastatic cascade, especially the early step of invasion inside the primary tumor. In this report, we have used intravital multiphoton microscopy to visualize the different migration patterns of human breast tumor cells in live primary tumors. We used xenograft tumors of MDA-MB-231 cells as well as a low passage xenograft tumor from orthotopically injected patient-derived breast tumor cells. Direct visualization of human tumor cells in vivo shows two patterns of high-speed migration inside primary tumors: (1) single cells and (2) multicellular streams (i.e., cells following each other in a single file but without cohesive cell junctions). Critically, we found that only streaming and not random migration of single cells was significantly correlated with proximity to vessels, with intravasation and with numbers of elevated circulating tumor cells in the bloodstream. Finally, although the two human tumors were derived from diverse genetic backgrounds, we found that their migratory tumor cells exhibited coordinated gene expression changes that led to the same end-phenotype of enhanced migration involving activating actin polymerization and myosin contraction. Our data are the first direct visualization and assessment of in vivo migration within a live patient-derived breast xenograft tumor.
Four-dimensional microglia response to anti-Aβ treatment in APP/PS1xCX3CR1/GFP mice
Senile plaques, mainly composed of amyloid-β (Aβ), are a major hallmark of Alzheimer disease (AD), and immunotherapy is a leading therapeutic approach for Aβ clearance. Although the ultimate mechanisms for Aβ clearance are not well known, characteristic microglia clusters are observed in the surround of senile plaques, and are implicated both in the elimination of Aβ as well as the deleterious inflammatory effects observed in AD patients after active immunization. Therefore, analyzing the direct effect of immunotherapy on microglia, using longitudinal in vivo multiphoton microscopy can provide important information regarding the role of microglia in immunotherapy. While microglia were observed to surround senile plaques, topical anti-Aβ antibody administration, which led to a reduction in plaque size, directed microglia toward senile plaques, and the overall size of microglia and number of processes were increased. In some cases, we observed clusters of microglia in areas of the brain that did not have detectable amyloid aggregates, but this did not predict the deposition of new plaques in the area within a week of imaging, indicating that microglia react to but do not precipitate amyloid aggregation. The long-term presence of large microglial clusters in the surrounding area of senile plaques suggests that microglia cannot effectively remove Aβ unless anti-Aβ antibody is administered. All together, these data suggest that although there is a role for microglia in Aβ clearance, it requires an intervention like immunotherapy to be effective.