Two‐photon image correlation spectroscopy and image cross‐correlation spectroscopy

Journal of Microscopy - Tập 200 Số 1 - Trang 14-25 - 2000
Paul W. Wiseman1, Jeffrey A. Squier2, Mark H. Ellisman3, Kent R. Wilson1
1Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0339, U.S.A.
2Department of Electrical & Computer Engineering University of California, San Diego, La Jolla, CA 92093‐0339, U.S.A.
3National Center for Microscopy & Imaging Research (NCMIR), Department of Neurosciences, University of California, San Diego, La Jolla, CA 92093‐0608, U.S.A.

Tóm tắt

We introduce two‐photon image correlation spectroscopy (ICS) using a video rate capable multiphoton microscope. We demonstrate how video rate two‐photon microscopic imaging and image correlation analysis may be combined to measure molecular transport properties over ranges typical of biomolecules in membrane environments. Using two‐photon ICS, we measured diffusion coefficients as large as 10−8 cm2 s−1 that matched theoretical predictions for samples of fluorescent microspheres suspended in aqueous sucrose solutions. We also show the sensitivity of the method for measuring microscopic flow using analogous test samples. We demonstrate explicitly the advantages of the image correlation approach for measurement of correlation functions with high signal‐to‐noise in relatively short time periods and discuss situations when these methods represent improvements over non‐imaging fluorescence correlation spectroscopy. We present the first demonstration of two‐photon image cross‐correlation spectroscopy where we simultaneously excite (via two‐photon absorption) non‐identical fluorophores with a single pulsed laser. We also demonstrate cellular application of two‐photon ICS for measurements of slow diffusion of green fluorescent protein/adhesion receptor constructs within the basal membrane of live CHO fibroblast cells.

Từ khóa


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Journal of Microscopy

Tập 200 Số 1

14-25

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