Superresolution optical fluctuation imaging (SOFI)
Statistical analysis techniques based on auto- and cross-correlations/cumulants, of image stacks of fluctuating objects are used to improve resolution beyond the classical diffraction limit and to reduce the background. The time trajectory of every pixel in the image frame is correlated with itself and/or with the time trajectory of an adjacent pixel. The amplitude of these auto- or cross-correlations/cumulants of each pixel, at a given time lag or averaged or integrated over an interval of time lags, is used as the intensity value of that pixel in the generated superresolved optical fluctuation image.
1. A method for analyzing a field-of-view of independently blinking objects, comprising the following steps:
i) selecting a sample or an observation image comprised of objects from the class of optically signaling objects that stochastically and independently fluctuate;
ii) acquiring a sequence of repeated optical images of the object as a function of time, producing an x, y, t image stack of pixels;
iii) calculating at least one of: an autocorrelation function, an autocumulant function, a crosscorrelation function, or a crosscumulant function from the temporal fluctuations of selected pixels of the image stack to at least the second order for at least a chosen time lag or a plurality of chosen time lags in order to generate a superresolved and background-reduced image including pixels having an intensity value being the amplitude of the function for the at least one chosen time lag or a linear combination of amplitudes of the functions for different chosen time lags.
2. A method as set forth in claim 1 , wherein the independently blinking objects could be separated by distances shorter than the diffraction limit-resolved distance of the imaging system used.
3. A method as set forth in claim 1 , wherein the sample objects comprising the sample or the observation image are selected from the class comprising emitting, absorbing or scattering materials and objects labeled with such materials.
4. A method as set forth in claim 1 , wherein the sequence of optical images is produced from a scanning beam or scan sample, in parallel or in series-wide field, or in pixel-by-pixel sequence.
5. A method as set forth in claim 1 , wherein the correlation or cummulant functions are of higher order than the second order.
6. A method as set forth in claim 5 , wherein the acquired sequence of optical images comprises multiple sets of image stacks.
7. A method of superresolution of images of objects being separated by a distance shorter than the optical diffraction limit, comprising the following steps:
introducing observable temporal emission fluctuations in the objects of interest, the fluctuations being stochastic and independent of each other;
generating a magnified image of a field of view encompassing the emitting objects of interest, the magnified image including pixels being substantially smaller than the point spread function introduced in magnification;
sensing temporal fluctuations in the pixels in a succession of image frames corresponding to the temporal fluctuations in the emission of different distributed emitting objects; and
statistically correlating the temporal fluctuations between selected pixels in the image frames to generate a superresolved image in at least two dimensions of the emitting objects in the field of view.