Multicolor multiphoton in vivo imaging flow cytometry

Abstract

Abstract:In vivo flow cytometry provides a non-invasive way of probing the biology of circulating cells during disease progression and studying cellular response to therapy. However, current methods provide little morphological information which potentially could be new biological marker for early disease diagnosis, and fail to reveal intercellular interactions. Here we report a multi-color, multiphoton in vivo imaging flow cytometry, to image circulating cells within the vasculature of scattering tissues at high spatiotemporal resolution. We apply it in imaging of cellular dynamics in bone marrow through the intact mouse skull, in situ deformability cytometry, distinguishing cellular clusters, and simultaneously monitoring multiple types of trafficking cells based on their morphologies and fluorescence emission colors. Cote, and C. P. Lin, "In vivo cell tracking with video rate multimodality laser scanning microscopy," IEEE J. Sel. Top. Quantum Electron. 14(1), 10-18 (2008). ©2016 Optical Society of America

Unbiased Analysis Method for Measurement of Red Blood Cell Size and Velocity With Laser Scanning Microscopy

“…This deformability is impaired in many pathological conditions as hereditary disorders (for example spherocytosis, elliptocytosis, ovalocytosis, and stomatocytosis), diabetes, hypercholesterolemia (reviewed by Tomaiuolo, 2014), or during infection by plasmodium (Tiburcio et al, 2012). At cellular resolution, RBCs flow, velocity, and shape are usually investigated with laser scanning microscopy, either with one-photon excitation and confocal detection for superficial vessels or transparent samples (Dirnagl et al, 1992; Villringer et al, 1994), or with multiphoton excitation for scattering tissue (Kleinfeld et al, 1998; Chaigneau et al, 2003; Shih et al, 2012; Kong et al, 2016). RBC velocity measurements are now commonly used to quantify changes of vascular dynamics in brain pathological models (Hutchinson et al, 2006; Schaffer et al, 2006; Shih et al, 2009; Autio et al, 2011; Kisler et al, 2017).…”

In vivo longitudinal visualization of bone marrow engraftment process in mouse calvaria using two-photon microscopy

“…It has been used to monitor the distribution and behaviour of HSCs in mouse calvarial BM at the single-cell level 18 , and has revealed the regulation of HSCs through cell-to-cell interaction or regulator molecule-to-cell interaction 18 20 26 . In addition, studies of local oxygen tension 27 , BM vasculature, and BM endothelial cells 24 28 29 30 31 32 have been conducted and HSCs have been tracked successfully the first few days after transplantation using intravital microscopy of mouse calvarial BM 18 33 . Intravital microscopy has been used to study molecular and cellular regulators of HSC homing 18 , and diabetic conditions was examined 34 .…”

Volumetric Lissajous Confocal Microscopy

“…Alternatively, our current configuration reduces 3D scan time by limiting the travel range of the galvo mirror by having less pixels along the y-direction. This original method of scanning could find applications in imaging flow cytometry [24], particle imaging velocimetry [25] or other instances where fast x-z imaging is preferred. As an extreme example, we imaged an object moving along the z-direction with a static galvo mirror, acquiring 2D sections across the x-z plane.…”