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Multifunctional fluorescence correlation microscope for intracellular and microfluidic measurements
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Image of FIG. 1.
FIG. 1.

The schematic diagram of FCM. APD: avalanche photodiode; PMT: photomultiplier tube; F: emission filter; L: lens; BS: beam splitter; M: mirror; DM: dichroic mirror; PH: pinhole; S: scanning mirrors; P: reflective prism; and O: objective.

Image of FIG. 2.
FIG. 2.

The contour maps of (A) diffusion time (s) and (B) structural factor measured in Atto 565 solution are shown in the CLSM image area. (C) ACF curves at the center (solid line) and the periphery (dashed line) of the image are compared. The respective count rates (inset) and fitting residues are also shown. (D) Atto 565 (solid line) and TMR (dashed line) were used at a concentration of to calibrate the performance of FCM.

Image of FIG. 3.
FIG. 3.

SW-FCCS was performed by the FCM. The amplitude of cross correlation curve decreases with the increasing concentration of BA488, which corresponds to the increasing number of binding complexes (SA-QR and BA488). The concentration of SA-QR was . The curves were fitted using Eq. (7) and the residuals are shown.

Image of FIG. 4.
FIG. 4.

Single pinhole spatial FCCS was realized by the displacement of two detectors in the opposite direction. (A) CCF curves for different line scan speeds and the linear dependence of measured “flow” speeds on the scan lengths (inset). (B) Effect of flow angles on the cross correlation amplitudes and the change of with flow angles (inset).

Image of FIG. 5.
FIG. 5.

Translational and rotational diffusions on CHO cell membranes can be measured by the FCM. Cross: FCS measurement point; arrows (from left to right): rotational diffusion in time range, triplet state relaxation in , and translational diffusion in ; and bar: .

Image of FIG. 6.
FIG. 6.

Two-photon excitation line scan FCS for flow direction measurements. ACF curves for laser line scan, microfluidic flow, and net flow of the previous two are plotted.


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Table I.

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752b84549af89a08dbdd7fdb8b9568b5 journal.articlezxybnytfddd
Scitation: Multifunctional fluorescence correlation microscope for intracellular and microfluidic measurements