Two-photon fluorescence correlation spectroscopy: method and application to the intracellular environment.

Abstract:

:We report on the application of two photon molecular excitation to fluorescence correlation spectroscopy. We demonstrate the first fluorescence correlation spectroscopy measurements of translational mobility in the cytoplasm of living cells. Two-photon excitation inherently excites small sample volumes in three dimensions, providing depth discrimination similar to confocal microscopy, without emission pinholes. We demonstrated accurate measurements of the diffusion constant, D, for particles of several different known sizes, in bulk solutions of different viscosity. We then showed measurements of translational diffusion for 7- and 15-nm radius latex beads in the cytoplasm of mouse fibroblast cells. We measured time-dependent diffusion coefficients. When first injected in the cells, the spheres moved from two to five times slower than in water, with average rates of 18 x 10(-8) cm2/s for the 7 nm and 5 x 10(-8) cm2/s for the 15 nm radius spheres. After a few hours, spheres stick to the cells, and the motion slows down 10 to 100 times.

journal_name

Biophys J

journal_title

Biophysical journal

authors

Berland KM,So PT,Gratton E

doi

10.1016/S0006-3495(95)80230-4

subject

Has Abstract

pub_date

1995-02-01 00:00:00

pages

694-701

issue

2

eissn

0006-3495

issn

1542-0086

pii

S0006-3495(95)80230-4

journal_volume

68

pub_type

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