We describe the setup and apply two algorithms for fast imaging in a sample raster scanning two photon microscope. Imaging can be performed at a rate of 1-100 lines per second with a closed loop piezo actuator, and the detection is performed via avalanche photodiodes. This allows to investigate single molecule emission with 50 ms time resolution. In a slow scanning algorithm we have implemented fluorescence fluctuation analysis by computing the photon counting histogram (PCH) on each pixel of the image. In a fast-scan acquistion method the image acquistion rate is 5 lines per second on a large field of view and high resolution(50 nm scanning step, 100x100 mum(2) field of view) and congruent to100 lines per second on smaller field of views with optically limited resolution (200 nm scanning step, 20x20 mum(2) field of view). This figure, which is lower than the typical value for normal confocal scanning imaging (congruent to500 lines per second), allows nevertheless to perform imaging studies of extended samples in reasonable times for intracellular kinetics and interactions. With this setup and by means of the PCH analysis we are able to discriminate between local concentration and molecular brightness on extended samples also at the level of the single molecule. (C) 2004 American Institute of Physics.
Malengo, G., Milani, R., Cannone, F., Krol, S., Diaspro, A., Chirico, G. (2004). High sensitivity optical microscope for single molecule spectroscopy studies. REVIEW OF SCIENTIFIC INSTRUMENTS, 75(8), 2746-2751 [10.1063/1.1777385].
High sensitivity optical microscope for single molecule spectroscopy studies
MILANI, ROBERTO;CHIRICO, GIUSEPPE
2004
Abstract
We describe the setup and apply two algorithms for fast imaging in a sample raster scanning two photon microscope. Imaging can be performed at a rate of 1-100 lines per second with a closed loop piezo actuator, and the detection is performed via avalanche photodiodes. This allows to investigate single molecule emission with 50 ms time resolution. In a slow scanning algorithm we have implemented fluorescence fluctuation analysis by computing the photon counting histogram (PCH) on each pixel of the image. In a fast-scan acquistion method the image acquistion rate is 5 lines per second on a large field of view and high resolution(50 nm scanning step, 100x100 mum(2) field of view) and congruent to100 lines per second on smaller field of views with optically limited resolution (200 nm scanning step, 20x20 mum(2) field of view). This figure, which is lower than the typical value for normal confocal scanning imaging (congruent to500 lines per second), allows nevertheless to perform imaging studies of extended samples in reasonable times for intracellular kinetics and interactions. With this setup and by means of the PCH analysis we are able to discriminate between local concentration and molecular brightness on extended samples also at the level of the single molecule. (C) 2004 American Institute of Physics.
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