Deconvolution Spatial Light Interference Microscopy for Subdiffraction Imaging of Live Cells

Posted on February 28, 2012 by mmir2

S. D.  Babacan, Z. Wang, M. N. Do and G.Popescu , Cell imaging beyond the diffraction limit using sparse deconvolution spatial light interference microscopy , Biomed. Opt. Exp., 2 (7), (2011).

We developed an imaging method that combines a novel deconvolution algorithm with Spatial Light Interference Microscopy (SLIM), to achieve 2.3x resolution enhancement with respect to the diffraction limit. This method, referred to as deconvolved SLIM (dSLIM) allows, for the first time to our knowledge, imaging very fine structures and motions in live cells below diffraction limit.

The main features associated with SLIM are as follows: it provides speckle-free images, which allows for spatially sensitive optical path-length measurement (0.3 nm); it uses common path interferometry, which enables temporally sensitive optical path-length measurement (0.03nm). Therefore, SLIM is characterized by a very high signal to noise, of the order of 1,000 or more. This feature enables experimental access to accurate PSF, which, in turn can be used for complex field deconvolution operations.

In this work, we significantly improve SLIM’s resolution capability via spatially adaptive complex field deconvolution. By exploiting both the characteristics of biological specimen based on the sparsity principle, and the physics of the image formation in SLIM, we recover the very fine biological structure blurred by the optics. Due to this accurate modeling, dSLIM does not lead to noise amplification or deconvolution artifacts, providing precise localization and quantitative information. Further, it is easy to use with only a single parameter, the noise floor, which is measureable from the acquired images.

We demonstrate that resolution of 238 nm can be obtained for an objective with NA=0.65, which represents an increase by a factor of 2.3 over the diffraction limit. This remarkable result is essentially due to the fact that the image formation can be treated as a linear process in the complex fields. Experiments with primary brain cells, i.e. neurons and glial cells, reveal new subdiffraction structures and motions. This new information is used for studying vesicle transport in neurons, which may shed light on dynamic cell functioning.

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