Video-rate resonant scanning multiphoton microscopy: An emerging technique for intravital imaging of the tumor microenvironment

Abstract

The abnormal tumor microenvironment fuels tumor progression, metastasis, immune suppression, and treatment resistance. Over last several decades, developments in and applications of intravital microscopy have provided unprecedented insights into the dynamics of the tumor microenvironment. In particular, intravital multiphoton microscopy has revealed the abnormal structure and function of tumor-associated blood and lymphatic vessels, the role of aberrant tumor matrix in drug delivery, invasion and metastasis of tumor cells, the dynamics of immune cell trafficking to and within tumors, and gene expression in tumors. However, traditional multiphoton microscopy suffers from inherently slow imaging rates—only a few frames per second, thus unable to capture more rapid events such as blood flow, lymphatic flow, and cell movement within vessels. Here, we report the development and implementation of a video-rate multiphoton microscope (VR-MPLSM) based on resonant galvanometer mirror scanning that is capable of recording at 30 frames per second and acquiring intravital multispectral images. We show that the design of the system can be readily implemented and is adaptable to various experimental models. As examples, we demonstrate the utility of the system to directly measure flow within tumors, capture metastatic cancer cells moving within the brain vasculature and cells in lymphatic vessels, and image acute responses to changes in a vascular network. VR-MPLSM thus has the potential to further advance intravital imaging and provide new insight into the biology of the tumor microenvironment.

A Perspective on Studying G-Protein–Coupled Receptor Signaling with Resonance Energy Transfer Biosensors in Living Organisms

“…Several recent advances in MP microscopy have provided improved acquisition speeds for FLIM-based approaches (Kirkpatrick et al, 2012;Rinnenthal et al, 2013;Poland et al, 2014). Additionally, commercially available enhancements to microscopy are now available, including gallium arsenide phosphide multidetector units, which have higher quantum efficiency and wavelength sensitivity while still enabling lownoise high-speed imaging.…”

Imaging the Dynamics of Endocytosis in Live Mammalian Tissues

“…Superresolution microscopy (e.g., PALM) is not suited for animal work, as well, because it requires the complete stability of the specimen and long acquisition times. However, this issue will probably be overcome by the development of confocal and two-photon microscopes equipped with fast resonant scanners (Kirkpatrick et al 2012) or by using spinning disk microscopy, which has been recently introduced to in vivo imaging (Jenne et al 2011).…”

Molecular mobility and activity in an intravital imaging setting – implications for cancer progression and targeting

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