ABSTRACT
The article introduces an optical microscopy technique capable of simultaneously acquiring quantitative fluorescence and phase (or equivalently wavefront) images with a single camera sensor, avoiding any delay between both images, or registration of images acquired separately. The method is based on the use of a 2-dimensional diffraction grating (aka cross-grating) positioned at a millimeter distance from a 2-color camera. Fluorescence and wavefront images are extracted from the two color channels of the camera, and retrieved by image demodulation. The applicability of the method is illustrated on various samples, namely fluorescent micro-beads, bacteria and mammalian cells.
ABSTRACT
Quantitative phase microscopy (QPM) represents a noninvasive alternative to fluorescence microscopy for cell observation with high contrast and for the quantitative measurement of dry mass (DM) and growth rate at the single-cell level. While DM measurements using QPM have been widely conducted on mammalian cells, bacteria have been less investigated, presumably due to the high resolution and high sensitivity required by their smaller size. This article demonstrates the use of cross-grating wavefront microscopy, a high-resolution and high-sensitivity QPM, for accurate DM measurement and monitoring of single microorganisms (bacteria and archaea). The article covers strategies for overcoming light diffraction and sample focusing, and introduces the concepts of normalized optical volume and optical polarizability (OP) to gain additional information beyond DM. The algorithms for DM, optical volume, and OP measurements are illustrated through two case studies: monitoring DM evolution in a microscale colony-forming unit as a function of temperature, and using OP as a potential species-specific signature.
