Abstract
We had developed Optical Scatter Imaging (OSI) as a method which combines light scattering spectroscopy with microscopic imaging to probe local particle size in situ. Using a variable diameter iris as a Fourier spatial filter, the technique consisted of collecting images that encoded the intensity ratio of wide-to-narrow angle scatter at each pixel in the full field of view. In this paper, we replace the variable diameter Fourier filter with a digital micromirror device (DMD) to extend our assessment of morphology to the characterization of particle shape and orientation. We describe our setup in detail and demonstrate how to eliminate aberrations associated with the placement of the DMD in a conjugate Fourier plane of our microscopic imaging system. Using bacteria and polystyrene spheres, we show how this system can be used to assess particle aspect ratio even when imaged at low resolution. We also show the feasibility of detecting alterations in organelle aspect ratio in situ within living cells. This improved OSI system could be further developed to automate morphological quantification and sorting of non-spherical particles in situ.
Highlights
Alterations in the morphology of subcellular organelles are an important indicator of cellular function
We show pilot data from bacteria and spheres, as well as cells to demonstrate the potential ability of our digital micromirror device (DMD)-based optical scatter imaging approach to differentiate particles based on their aspect ratio
We extended our size-sensitive optical scatter imaging (OSI) method to analyze the shape of the scattering objects
Summary
Alterations in the morphology of subcellular organelles are an important indicator of cellular function. In a transmission microscope utilizing the central dark-ground method, wide and narrow angles of scatter were selected by varying the diameter of a circular iris in a Fourier plane, conjugate to the objective’s back focal plane. Using this method, we showed that changes in subcellular scattering can be correlated with mitochondria-specific treatments such as cyclosporine-A-dependent calcium injury [2] or altered expression of Bcl-2 family proteins which target the mitochondrial outer membrane [3]. To quantify the shape of non-spherical organelles, such as mitochondria, and the anisotropic morphological changes that characterize their activity, we extended the previous OSI method by replacing our variable diameter iris with a digital micromirror device (DMD) as a Fourier filter [5]
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