Chapter 16 Two‐Photon Microscopy and Multidimensional Analysis of Cell Dynamics

Zinselmeyer, Bernd H.; Dempster, John; Wokosin, David L.; Cannon, Jonathan; Pless, Robert; Parker, Ian; Miller, Mark J.

doi:10.1016/s0076-6879(09)05416-0

Cited by 57 publications

(77 citation statements)

References 54 publications

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“…These cells are more than 95% neutrophils based on flow cytometric analysis (37,38) and intravital microscopy analysis of LysM-GFP mice injected with a neutrophil-specific anti-Ly6G antibody (1A8) (Supplemental Figure 4, A-C). The right kidney was surgically exteriorized and imaged (39), and tissue perfusion was verified by bright-field observation of robust capillary flow and by injection of Q-dots into the retro-orbital sinus. In both control and anti-GBM-treated mice, neutrophils were visible passing through and arresting transiently ("fluttering") in glomerular capillaries, which we show approximated the diameter of a neutrophil ( Figure 5, A and B).…”

Section: Abl1 Regulates F-actin Polymerization Needed For Neutrophil mentioning

confidence: 99%

Neutrophil FcγRIIA promotes IgG-mediated glomerular neutrophil capture via Abl/Src kinases

Nishi

Furuhashi

Culleré

et al. 2017

Journal of Clinical Investigation

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Section: Abl1 Regulates F-actin Polymerization Needed For Neutrophil mentioning

confidence: 99%

Neutrophil FcγRIIA promotes IgG-mediated glomerular neutrophil capture via Abl/Src kinases

Nishi

Furuhashi

Culleré

et al. 2017

Journal of Clinical Investigation

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“…2-Photon excitation produced a second harmonic signal from collagen within the analyzed tissue (40). Multidimensional rendering and manual cell tracking was done with Imaris (Bitplane).…”

Section: Animals Cx3cr1mentioning

confidence: 99%

“…The meandering factor was calculated by dividing the displacement of a cell by its total path length. All track calculations were performed using T cell Analyser version 1.7.0 software (http://spider.science.strath.ac.uk/sipbs/showPage.php?page=software_imag-ing), written by J. Dempster (University of Strathclyde, Glasgow, Scotland) (40). Data were transferred and plotted in GraphPad Prism 6 (GraphPad Software Inc.) for creation of the graphs.…”

Section: Animals Cx3cr1mentioning

confidence: 99%

CCR7 and IRF4-dependent dendritic cells regulate lymphatic collecting vessel permeability

Ivanov¹,

Scallan²,

Kim³

et al. 2016

Journal of Clinical Investigation

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International audienceLymphatic collecting vessels direct lymph into and from lymph nodes (LNs) and can become hyperpermeable as the result of a previous infection. Enhanced permeability has been implicated in compromised immunity due to reduced flow of lymph and immune cells to LNs, which are the primary site of antigen presentation to T cells. Presently, very little is known about the molecular signals that affect lymphatic collecting vessel permeability. Here, we have shown that lymphatic collecting vessel permeability is controlled by CCR7 and that the chronic hyperpermeability of collecting vessels observed in Ccr7–/– mice is followed by vessel fibrosis. Reexpression of CCR7 in DCs, however, was sufficient to reverse the development of such fibrosis. IFN regulatory factor 4–positive (IRF4+) DCs constitutively interacted with collecting lymphatics, and selective ablation of this DC subset in Cd11c-Cre Irf4fl/fl mice also rendered lymphatic collecting vessels hyperpermeable and fibrotic. Together, our data reveal that CCR7 plays multifaceted roles in regulating collecting vessel permeability and fibrosis, with one of the key players being IRF4-dependent DCs

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“…[25][26][27] In some experiments involving red-shifted FPs (tdTomato and mCherry), an optical parametric oscillator (OPO) laser (Chameleon Compact OPO) from Coherent was used to extend the output wavelength range as far as 1030-1300 nm, sequentially or simultaneously with the Ti-Sa laser.…”

Section: Confocal and 2-photon Microscopymentioning

confidence: 99%

Dynamic clonal analysis of murine hematopoietic stem and progenitor cells marked by 5 fluorescent proteins using confocal and multiphoton microscopy

Malide¹,

Métais

Dunbar

2012

Blood

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IntroductionModern microscopy is an invaluable tool, with advances in resolution, contrast, molecular specificity, speed, and biocompatibility to enable visualization of cellular processes in intact tissues and organisms. The use of endogenously produced multicolor fluorescent proteins (FPs) to label cells has emerged as a versatile approach for cell tracking and lineage tracing during morphogenesis or regenerative processes. 1-3 However, many FP variants have similar excitation and emission properties, making unambiguous separation of signals from multiple reporters challenging. The ability to label with multiple FPs in the same experiment and apply high resolution multidimensional imaging can provide insights into complex biologic processes. 4 Recently, the concept of genetically labeling clonal cell populations via fluorescent proteins of distinct colors has been developed. The initial technology known as the "brainbow" 5 was based on controlled transgene recombination, and successive improvements increased the range of applications. [6][7][8][9] Up to 5 different FP were expressed from a single "MultiLabel" expression plasmid using tandem recombineering induced by a tissue-and stage-specific promoter, resulting in homogeneous cell populations all expressing multiple FPs. 10,11 To date, this approach has not been applied to hematopoiesis because of the lack of a highly hematopoietic stem cell-specific promoter. An alternative strategy, known as "RGB marking" uses lentiviral gene ontology (LeGO) vectors encoding red, green, and blue FPs to transduce multiple cell types and track clones after transfer, 12-14 resulting in fluorescence intensities much brighter than most transgenic FPs, and broad combinatorial color diversity.We took advantage of LeGO vectors constitutively expressing 5FPs to mark hematopoietic stem and progenitor cells (HSPCs) and study the process of hematopoiesis at a clonal level over time and in multiple tissues. HSPCs reside within the BM in a complex niche consisting of osteoblasts, stromal cells, adipose tissue, and vascular structures, crucial for maintenance of self-renewal and modulation of differentiation and death pathways. [15][16][17][18] As BM has been inaccessible to direct observation, the interactions between HSPCs and their microenvironment remains largely uncharacterized in vivo. Recently, we established a methodology to visualize the 3D architecture of intact BM using confocal fluorescence and reflection microscopy. 19 We combine generation of a diverse palette of clone colors via cotransduction of HSPCs with 5FPs LeGO vectors with new imaging and analysis technologies to computationally reconstruct the 3D architecture of tissues at high resolution to depths of 150-300 m, elucidating biologically interesting clonal reconstitution patterns. We demonstrate that confocal imaging can be combined with multiphoton microscopy, revealing complementary information from autofluorescent and second-harmonic-generating (SHG) structures. Furthermore, dynamic 4D high-resolution imaging ...

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Chapter 16 Two‐Photon Microscopy and Multidimensional Analysis of Cell Dynamics

Cited by 57 publications

References 54 publications

Neutrophil FcγRIIA promotes IgG-mediated glomerular neutrophil capture via Abl/Src kinases

Neutrophil FcγRIIA promotes IgG-mediated glomerular neutrophil capture via Abl/Src kinases

CCR7 and IRF4-dependent dendritic cells regulate lymphatic collecting vessel permeability

Dynamic clonal analysis of murine hematopoietic stem and progenitor cells marked by 5 fluorescent proteins using confocal and multiphoton microscopy

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