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.
[Show abstract][Hide abstract] ABSTRACT: Intravital microscopy is an extremely powerful tool that enables imaging several biological processes in live animals. Recently, the ability to image subcellular structures in several organs combined with the development of sophisticated genetic tools has made possible extending this approach to investigate several aspects of cell biology. Here we provide a general overview of intravital microscopy with the goal of highlighting its potential and challenges. Specifically, this review is geared toward researchers that are new to intravital microscopy and focuses on practical aspects of carrying out imaging in live animals. Here we share the know-how that comes from first-hand experience, including topics such as choosing the right imaging platform and modality, surgery and stabilization techniques, anesthesia and temperature control. Moreover, we highlight some of the approaches that facilitate subcellular imaging in live animals by providing numerous examples of imaging selected organelles and the actin cytoskeleton in multiple organs.
[Show abstract][Hide abstract] ABSTRACT: The aqueous outflow system (AOS), including the trabecular meshwork
(TM), the collector channels (CC) and the Schlemm's canal (SC),
regulates intraocular pressure (IOP) through the drainage of the aqueous
humor (AH). Abnormal IOP elevation leads to increased pressure stress to
retinal ganglion cells, resulting in cell loss that can ultimately lead
to complete loss of eyesight. Therefore, development of imaging tools to
detect abnormal structural and functional changes of the AOS is
important in early diagnosis and prevention of glaucoma. Multiphoton
microscopy (MPM), including twophoton autofluorescence (TPAF) and second
harmonic generation (SHG), is a label-free microscopic technique that
allows molecular specific imaging of biological tissues like the TM.
Since the TM and other AOS structures are located behind the highly
scattering scleral tissue, transscleral imaging of the TM does not
provide enough optical resolution. In this work, a gonioscopic lens is
used to allow direct optical access of the TM through the cornea for MPM
imaging. Compared to transscleral imaging, the acquired MPM images show
improved resolution as individual collagen fiber bundles of the TM can
be observed. MPM gonioscopy may have the potential to be developed as a
future clinical imaging tool for glaucoma diagnostics.
Proceedings of SPIE - The International Society for Optical Engineering 03/2013; 8567. DOI:10.1117/12.2003031 · 0.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We present a fast and flexible fluorescence lifetime imaging microscopy which uses a two-dimensional acousto-optic deflector to achieve fast beam scanning across the sample and provides random access to the regions of interests (ROI). Experimental results using standard fluorescent dye and biological samples show that this system can make addressable fluorescence lifetime measurements and perform fast and flexible fluorescence lifetime imaging particularly to the discontinuous ROI in the sample.
Zsuzsanna Nemeth, Mailin Li, Eva Csizmadia, Balazs Döme, Martin Johansson, Jenny Liao Persson, Pankaj Seth, Leo Otterbein, Barbara Wegiel
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