Single-shot capture bright-field and digital holographic microscopy for the analysis of living cells

Picazo-Bueno, J.Á.; Barroso, Á.; Ketelhut, S.; Schnekenburger, J.; Micó, V.; Kemper, B.

Abstract

Digital holographic microscopy (DHM) is an emerging label-free modality for quantitative imaging of semi-transparent biological specimens. DHM provides quantitative phase imaging (QPI) of the light transmitted or scattered by an object, which allows to extract information about morphology and dynamics of investigated specimens. DHM can be implemented with other optical microscopy techniques like bright field (BF) or fluorescence, for example, to provide complementary information about cellular processes. Multimodal microscopy is often achieved by sequential acquisition of information from different imaging modalities or simultaneously by capturing images with different acquisition devices. However, such arrangements can be cost-intensive and/or limited in speed for imaging of dynamic processes. Here, we investigate a single-snapshot multimodal approach that enables simultaneous acquisition of DHM-based QPI and BF images and is compatible with common optical microscopes for the analysis of living cells. The technique is based on spatially multiplexed interferometric microscopy (SMIM), where off-axis holograms are generated by spatial multiplexing of the illuminated sample plane utilizing a diffraction grating, which is extended by additional superposition of complementary white-light image information. Image separation is achieved by Fourier-transformation-based numerical demultiplexing procedures. The technique is firstly characterized by investigations on microspheres. Then, the application on living adherent and suspended cells is illustrated.