FCS in Laser Scanning Microscopes

It should be noted that FCS measurements in cells are more difficult than in solution. Especially in transfected cells the fiuorophore concentration cannot be accurately controlled. It is usually much higher than required for FCS. The number of molecules in the focus can easily be of the order of 100, resulting in an extremely small amplitude of the correlation function. Moreover, there is usually motion in living cells that shows up in the FCS curves at a time scale above 100 ms. 

A frequently asked question is whether or not FCS data recording and scanning a sample can be combined. It is generally impossible to scan a sample at a scan rate of the order of the photon times to be correlated. The frame rate must be faster than the shortest correlation time, which is practically impossible. FCS is therefore incompatible with the pixel dwell times and frame rates used in laser scanning microscopes. 

It has been shown that FCS with millisecond resolution can be obtained from a circular line scan. FCS images of moderate pixel numbers can be obtained by scanning a sample at a pixel dwell time much longer than the longest time to be correlated. A possible solution is shown in Fig. 5.115. 

Theoretically, the same procedure can also be used in a commercial confocal or two-photon laser scanning microscope if a sufficiently slow scan speed can be selected. To distinguish the individual pixels of the scan, the short scan control 

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