FCS Setup

FCS Setup

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Construction of an FCS setup at the Institute of physics is the main technological result of this project. In brief, fluorescence correlation spectroscopy (FCS) allows one to measure the diffusion of fluorescently labeled macromolecules in solution. Importantly, the quantities of molecules are in nanomoles, and the laser is focused into a femtoliter volume. Effectively, this allows to perform measurements of diffusion inside a living cell. We foresee that the biology and biotechnology communities in Zagreb will be interested into using this instrument. FCS will be complementary to the confocal microscope available (for fluorescent cell imaging) in the group of Igor Weber, at the Rudjer Boskovic Institute. Indeed, dr. Weber has kindly provided an inverted microscope (Zeiss Axiovert) body/chassis, around which we have constructed our FCS setup.

Single photon FCS setup scheme

The confocal FCS setup is illustrated schematically in this figure. The excitation laser beam is directed into a microscope objective via a dichroic mirror and focused on the sample. As the sample molecules are usually dissolved in aqueous solution, water immersion objectives with a high numerical aperture (ideally > 0.9) are used. The fluorescence light from the sample is collected by the same objective and passed through the dichroic and the emission filter. The pinhole in the image plane (field aperture) blocks any fluorescence light not originating from the focal region, thus providing axial resolution. Afterwards, the light is focused onto the detector, preferably an avalanche photodiode or a photomultiplier with single photon sensitivity.

FCS, Institute of physics, Zagreb

Red line denotes the laser beam path from the home made diode-laser, through the beam-cleaning device (the intensity profile of the beam has to be Gaussian). The beam is expanded to overfill the back aperture of the objective (Olympus Plan Apochromat 60x NA 1.2) which focuses it on the sample.

Sample fluorescence (in pink) is collected by the same objective, filtered and coupled into an optical fiber, leading to the detector (single photon avalanche diode-SPAD). The optical fiber's end acts as the pinhole and has to be adjustable in the z-direction. Alignment in xy-direction is also important to get all the available fluorescence into the fiber and the detector. This is achieved with a precise mechanical device, "fiber launch" system. It facilitates everyday alignment of the fiber necessary for optimal fluorescence signal recovery.

Femtosecond laser, for the two-photon FCS, is easily coupled into the system, as it is found at the same optical table, serving also other experiments.

SPAD is kept in a black box to remove the ambient light and allow it to count only the fluorescence photons arriving from the fiber. The detector was produced by M. Stipčević from Rudjer Boskovic Institute within his project funded by Croatian Institute for Technology, HIT.

FCS data analysis

The screenshot above shows the photon-counter trace. Binning time is 2 ms, and about 15-20 photons are recorded in this period, leading to 8-10 kHz count rate. This is a rather low for optimal autocorrelation, as may be seen on the left, autocorrelation curve is very noisy. A better data set is shown in the other image, compared to an autocorrelation curve for pure water. The latter demonstrates the appearance of a non-fluorescent sample.