Patterned illumination single molecule localization microscopy (piSMLM): user defined blinking regions of interest cellSTORM – Cost-effective Super-Resolution on a Cellphone using dSTORM

Single molecule localization microscopy (SMLM) has been established as an important super-resolution technique for studying subcellular structures with a resolution down to a lateral scale of 10 nm. Usually samples are illuminated with a Gaussian shaped laser beam, which results in spatially different blink kinetics of fluorophores in the desired area due to inhomogeneous excitation in the sample. This leads to artifacts in the reconstructed image, which should be minimized in order to provide appropriate quantification of fluorescence signals in the super-resolution images. We present a newly developed patterned illumination single molecule localization microscope (piSMLM) to overcome the problem of uneven illumination by computer-generated holography (CGH). By utilizing a phase-only spatial light modulator (SLM) in combination with a modified Gerchberg-Saxton algorithm, a user-defined pattern with homogeneous and nearly speckle-free illumination is obtained. Our experimental results show that an intensity of 1 to 5 kW/cm2 was achieved by using a laser with an output power of 200 mW. Higher intensities up to 20 kW/cm2 can be reached by simply reducing the size of the region of interest (ROI). To demonstrate piSMLM, nuclear structures were imaged based on fluctuation binding-activated localization microscopy (fBALM). The super-resolution fBALM images reveal nuclear structures at a nanometer scale.