Quantitative Multiscale Cell Imaging in Controlled 3D Microenvironments

Abstract

The microenvironment determines cell behavior, but the underlying molecular mechanisms are poorly understood because quantitative studies of cell signaling and behavior have been challenging due to insufficient spatial and/or temporal resolution and limitations on microenvironmental control. Here we introduce microenvironmental selective plane illumination microscopy (meSPIM) for imaging and quantification of intracellular signaling and submicrometer cellular structures as well as large-scale cell morphological and environmental features. We demonstrate the utility of this approach by showing that the mechanical properties of the microenvironment regulate the transition of melanoma cells from actin-driven protrusion to blebbing, and we present tools to quantify how cells manipulate individual collagen fibers. We leverage the nearly isotropic resolution of meSPIM to quantify the local concentration of actin and phosphatidylinositol 3-kinase signaling on the surfaces of cells deep within 3D collagen matrices and track the many small membrane protrusions that appear in these more physiologically relevant environments.

Light-sheet microscopy: a tutorial

“…16 and 19. A similar finding was reported by Welf et al [121]. A LLS has been successfully demonstrated for fast high-resolution imaging of mitosis and locomotion of cells and the development of early stage embryos of small invertebrates [75].…”

“…DSLM with BBs has been successfully used for imaging a variety of biological samples, from cells [63] and small invertebrates [64] to tissue-mimic constructs [70]. This modality can be further combined with the confocal line detection that may provide an additional increase of contrast and optical sectioning [121].…”

“…To keep the sample hydrated and to allow the flux of nutrients toward the living sample inside the chamber, samples are embedded in a transparent porous substrate. Depending on the sample and on the application, this substrate can be made up of agarose gels, molten phytagel [43], and collagen fibrous microenvironments [121], among others. More recently, FEP materials have proven to be useful since their refractive index is close to that of water, helping to minimize aberrations.…”

Seeing is believing: multi-scale spatio-temporal imaging towards in vivo cell biology

“…In addition, by imaging living zebrafish embryos with LSFM, the interaction between Schwann cells and axons during neuron damage repair was observed in vivo (Xiao et al, 2015). More recently, two-photon Bessel beam light-sheet microscopy was optimized to study how cells maintained in a 3D culture mechanically react to changes in their microenvironment, at a subcellular level and without spatial constraint (Welf et al, 2016). Taken together, these recent studies demonstrate that LFSM is a particularly promising tool for studying mechano-and cell biology.…”

Meeting report – Cellular dynamics: membrane–cytoskeleton interface

“…These initial talks provided a glimpse into what the future of cellular imaging might hold, but also clearly indicated a significant need for data management and analysis. Toward this end, Erik Welf from Gaudenz Danuser's lab (University of Texas Southwestern, Dallas, USA) presented his analysis of cellular migration using microenvironmental selective plane illumination microscopy (meSPIM), which allows for isotropic resolution, combined with sophisticated image analysis tools that can segment surfaces of cells (Welf et al, 2016). This analysis is capable of defining different types of protrusions, thereby allowing precise quantification of cell dynamics and potentially providing insights into microdomains where signaling networks function to regulate changes in cell shape.…”