Abstract

The technique of fluorescence photobleaching recovery is used to determine the diffusion coefficients of fluorescently labeled molecules. A three-dimensionally defined spot of dye is bleached out and the fluorescence recovery is monitored as unbleached molecules diffuse back into the spot. The shape of the recovery curve yields the diffusion coefficient and information about immobile fraction and particle flow. Conventional one-photon fpr is restricted to the study of cell membranes or very thin regions of cells (lamellapodia) because the laser beam is able to excite fluorescence all along the beam length. In order to excite a three-dimensionally defined volume the third dimension must be limited by the sample itself. Two photon fluorescent excitation is confined to a three-dimensionally defined ellipsoid around the focal spot. This enables TPFPR to be performed within the cytoplasm of living cells. We have developed a fast user-friendly instrument that measures the diffusion coefficient of fluorescently labeled molecules in solution, and have verified its performance by measuring the diffusion coefficient of fluorescein-labeled BSA, finding a value of 6.8 e-7 cm^2 /s, in excellent agreement with published values.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR004224-09
Application #
5224724
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
9
Fiscal Year
1996
Total Cost
Indirect Cost

  Publications

Migone, Fernando F; Cowan, Robert G; Williams, Rebecca M et al. (2016) In vivo imaging reveals an essential role of vasoconstriction in rupture of the ovarian follicle at ovulation. Proc Natl Acad Sci U S A 113:2294-9
O'Dell, Ryan S; Cameron, David A; Zipfel, Warren R et al. (2015) Reelin Prevents Apical Neurite Retraction during Terminal Translocation and Dendrite Initiation. J Neurosci 35:10659-74
Byrnes, Laura J; Singh, Avtar; Szeto, Kylan et al. (2013) Structural basis for conformational switching and GTP loading of the large G protein atlastin. EMBO J 32:369-84
Jain, Manu; Robinson, Brian D; Scherr, Douglas S et al. (2012) Multiphoton microscopy in the evaluation of human bladder biopsies. Arch Pathol Lab Med 136:517-26
Degala, Satish; Williams, Rebecca; Zipfel, Warren et al. (2012) Calcium signaling in response to fluid flow by chondrocytes in 3D alginate culture. J Orthop Res 30:793-9
O'Dell, Ryan S; Ustine, Candida J M; Cameron, David A et al. (2012) Layer 6 cortical neurons require Reelin-Dab1 signaling for cellular orientation, Golgi deployment, and directed neurite growth into the marginal zone. Neural Dev 7:25
McMullen, J D; Kwan, A C; Williams, R M et al. (2011) Enhancing collection efficiency in large field of view multiphoton microscopy. J Microsc 241:119-24
Kim, Sally A; Sanabria, Hugo; Digman, Michelle A et al. (2010) Quantifying translational mobility in neurons: comparison between current optical techniques. J Neurosci 30:16409-16
Bowles, Robby D; Williams, Rebecca M; Zipfel, Warren R et al. (2010) Self-assembly of aligned tissue-engineered annulus fibrosus and intervertebral disc composite via collagen gel contraction. Tissue Eng Part A 16:1339-48
McMullen, Jesse D; Zipfel, Warren R (2010) A multiphoton objective design with incorporated beam splitter for enhanced fluorescence collection. Opt Express 18:5390-8

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