Measurement of the Point- and Line-Spread Functions Enables Deconvolution in Bright Field Light Microscopy

Abstract

Bright field is the simplest and most widespread light microscopy modality. However, its use in cellular biology has been limited due to lack of contrast in the imaging of thin, transparent samples such as cells. Instead, more involved microscopy techniques (e. g. differential interference contrast, dark field, phase contrast, among others) have been used. An alternative to increase image contrast is deconvolution processing, a powerful method often used in fluorescence microscopy. However, application of deconvolution processing to bright field images has been scarce, mainly because acquisition of the point-spread function (PSF) has been difficult. In this work, we present direct measurements of the point- and line-spread functions of a high-aperture microscope operating in bright field. Polystyrene nanoparticles of 100 nm in diameter and cytoskeletal microtubules serve as the point and line objects, respectively, that are imaged with high contrast and low noise using conventional microscopy plus digital image processing. To our knowledge, this is the first report that describes the experimental assessment of these functions. Our experimental results are in good agreement with a proposed model for both point- and line-spread functions. The measured PSF allows us to demonstrate conventional deconvolution on the bright field images of living, unstained Escherichia coli cells, showing improved definition of cell boundaries and sub-cellular features. Our studies provide a simple method to acquire the PSF, making possible the extension of deconvolution beyond fluorescence to include bright field as well.