Label-free Microscopy of E. coli Sub-cellular Structure in 3D
By combining a novel algorithm with a recently-developed add-on technique for commercial microscopes, researchers at the University of Illinois have created a fast, non-invasive 3D method for visualizing, quantifying, and studying cells without the use of fluorescence or contrast agents. In a paper published online in the journal PLoS ONE, the researchers who developed the technique reported that they were able to use it to visualize the E. coli bacteria with a combination of speed, scale, and resolution unparalleled for a label-free method.
The method is based on a broadband interferometric technique known as Spatial Light Interference Microscopy (SLIM) that was designed by Beckman Institute researcher Gabriel Popescu as an add-on module to a commercial phase contrast microscope. SLIM is extremely fast and sensitive at multiple scales (from 200 nm and up) but, as a linear optical system, its resolution is limited by diffraction.
By applying a novel deconvolution algorithm to retrieve sub-diffraction limited resolution information from the fields measured by SLIM, Popescu and his fellow researchers were able to render tomographic images with a resolution beyond SLIM's diffraction limits. They used the sparse reconstruction method to render 3D reconstructed images of E. coli cells, enabling label-free visualization of the specimens at sub-cellular scales.
Last year the researchers successfully demonstrated a new optical technique that provides 3D measures of complex fields called Spatial Light Interference Tomography (SLIT) on live neurons and photonic crystal structures. In this project they developed a novel algorithm to further extend the three-dimensional capabilities by performing deconvolution on the measured 3D field, based on modeling the image using sparsity principles. This microscopy capability, called dSLIT, was used to visualize coiled sub-cellular structures in E. coli cells.
The researchers said that these structures have only been observed using specialized strains and plasmids and fluorescence techniques, and usually on non-living cells.
These new methods provide a practical way for non-invasive study of such structures.
Measuring the phase shift that the specimen adds to the optical field at each point in the field of view is known as quantitative phase imaging, an imaging method for which Popescu developed the SLIM modality. It provides extremely sensitive phase measurements of thin, transparent structures such as the E. coli cells studied here.
The researchers wrote that the method addresses two major issues in cell microscopy: lack of contrast, due to the thin and optically transparent nature of cells, and diffraction limited resolution. They write that dSLIT's ability to retrieve limited resolution information delivered by SLIM will give researchers a tool to study structures like E. coli cells in a completely new way, thereby providing novel insights into cellular function.
Mustafa Mir, S. Derin Babacan, Michael Bednarz, Minh N. Do, Ido Golding, Gabriel Popescu: Visualizing Escherichia coli Sub-Cellular Structure Using Sparse Deconvolution Spatial Light Interference Tomography, PLoS ONE
Popescu et al.: Optically Measuring Single Cell Mass - Femtogram Sensitivity and Cycle Dependent Growth