Optical Fourier processing microscope to quantify subcellular structure and dynamics

Fundamental biological processes, such as cell death, involve time-dependent dynamic alterations in the morphology and organization of organelles. Since organelles are at the limit of optical image resolution, electron microscopy (EM) remains the method of choice to assess subcellular structure. However, sample fixation precludes analysis of time dependent processes within the same specimen with EM, and the use of high magnification with nanoscale resolution often results in the analysis of a few limited fields of view. To address these limitations and assess morphological change over large fields of view with sub-wavelength sensitivity in unstained living cells, we demonstrate a microscope for optical processing with high spatial frequency resolution. We use a digital micromirror device as a Fourier spatial filter to implement two-dimensional Gabor-like filters that can characterize particle size, orientation and aspect ratio. Quantitative object maps that encode these local morphological parameters are generated. We demonstrate the nanoscale sensitivity of this technique to object structure using periodic phase masks and spheres, and show how the size, aspect ratio and orientation of finite objects may be assessed in situ with our approach. In contrast to digital image processing, the sensitivity of our method to local morphological change is determined by frequency resolution in the Fourier plane, and is gained through a trade-off with spatial resolution in the image, therefore making it independent of image blurring or camera pixel size. Applications include rapid throughput analysis of non-spherical scatterers, such as mitochondria, within living cells.