The mobility and reaction parameters of molecules inside living cells can be conveniently measured using fluorescent probes. Typically fluorescence correlation spectroscopy (FCS) based on confocal microscopy is used for such measurements.
This implies high time-resolution but only for a single spot at a time. So in order to use this technique to image mobility parameters, we have to scan the focus of the confocal microscope and measure the image spot by spot sequentially. In order to overcome this tedious scheme, cameras can be used to detect fluorescence and then an FCS evaluation for every pixel can be performed. This requires an illumination scheme, that excites fluorescence at all pixels of the field of view at the same time. Currently two schemes have shown to be useful:
Single Plane Illumination Microscopy (SPIM) is a fluorescence microscopy technique, where the illumination is done perpendicularly to the detection. The technique shapes the illumination laser beam into a rectangle and then focuses it down only in one direction, using a cylindrical lens. This forms a thin "sheet of light" right in the focal plane of the detection objective. As the lightsheet can be tailored to be thin (< 2μm FWHM), we achieve good sectioning of the sample and out-of-focus light suppression. The lateral resolution is given by the detection objective only. Our setup currently uses a 60x/NA1.0 objective which leads to a lateral resolution of around 0.65μm.
The sample is mounted from above (although other implementations exist) inside a water- or buffer-filled sample chamber (stainless steal) which can also be heated to 37°C in order to create near-physiological conditions. It is embedded in a low concentration agarose gel (0.5-0.7%), or on a cover slip. As the detection objective can not be moved in our setup, the sample is mounted on a computer controlled XYZ translation stage (100nm resolution).
As an imaging detector, we either use an electron-multiplying CCD camera (EMCCD, Andor iXon X3 850), or a custom-made single-photon avalanche diode (SPAD) array (see below).
Image series taken with a SPIM at high frame rates (>500fps) can be used for an FCS evaluation, which then yields a set of autocorrelation functions. After a model fit to each of these functions, we get a map of the mobility parameters inside the sample. As fluorescence fluctuations were not only measured at a single spot, we can also calculate pixel-pixel-crosscorrelation functions, which e.g. yield information about flow patterns in the sample.
In addition we recently introduced two-color fluorescence crosscorrelation spectroscopy on our lightsheet microscope, which allows us to measure molecular interactions in different compartments of the cell (cytosol, nucleoplasm, membrane) in a spatially resolved manner.
Our SPIM-FCS data evaluation software QuickFit 3.0 is available for free.
Our EMCCD camera (500fps @ 128×128 pixels, 14000fps @ 128×1 pixels) is reasonably fast, but not fast enough for FCS measurements on small proteins in cells (e.g. free eGFP). So we also use a 32×32 pixel array of single-photon avalnche diodes (SPAD array) as a secondary image detector. Our SPAD array was developed in the group of Prof. Edoardo Charbon at the TU Delft. It's features are:
In order to improve the usability of the SPAD array for SPIM-FCS, we use the two programmable logic devices to do an online-calculation of the autocorrelation functions. Our system achieves real-time processing of all 1024 pixels at a readout rate of 100000fps. The correlators span a dynamic range of 10μs up to 1.2s, which matches the range of interest for FCS in live cells (see our OptEx publication on this topic).