期刊
NATURE PROTOCOLS
卷 6, 期 10, 页码 1500-1520出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2011.376
关键词
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资金
- US National Institutes of Health [NCI100324, GM073913]
- National Cancer Institute's Tumor Microenvironment Network
- Gruss Lipper Biophotonics Center
- Mouse Models of Human Cancers Consortium
- Charles H. Revson fellowship
- Medical Research Council [G1002033] Funding Source: researchfish
- MRC [G1002033] Funding Source: UKRI
Characterizing biological mechanisms dependent upon the interaction of many cell types in vivo requires both multiphoton microscope systems capable of expanding the number and types of fluorophores that can be imaged simultaneously while removing the wavelength and tunability restrictions of existing systems, and enhanced software for extracting critical cellular parameters from voluminous 4D data sets. We present a procedure for constructing a two-laser multiphoton microscope that extends the wavelength range of excitation light, expands the number of simultaneously usable fluorophores and markedly increases signal to noise via 'over-clocking' of detection. We also utilize a custom-written software plug-in that simplifies the quantitative tracking and analysis of 4D intravital image data. We begin by describing the optics, hardware, electronics and software required, and finally the use of the plug-in for analysis. We demonstrate the use of the setup and plug-in by presenting data collected via intravital imaging of a mouse model of breast cancer. The procedure may be completed in similar to 24 h.
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