Characterization of a Single-Capture Bright-Field and Off-Axis Digital Holographic Microscope for Biological Applications

We present a single-capture multimodal bright-field (BF) and quantitative phase imaging (QPI) approach that enables the analysis of large, connected, or extended samples, such as confluent cell layers or tissue sections. The proposed imaging concept integrates a fiber-optic Mach-Zehnder interferometer-based off-axis digital holographic microscopy (DHM) with an inverted commercial optical BF microscope. Utilizing 8-bit grayscale dynamic range multiplexing, we simultaneously capture both BF images and digital holograms, which are then demultiplexed numerically via Fourier filtering, phase aberration compensation, and weighted image subtraction procedures. Compared to previous BF-DHM systems, our system avoids synchronization challenges caused by multiple image recording devices, improves acquisition speed, and enhances versatility for fast imaging of large, connected, and rapidly moving samples. Initially, we perform a systematic characterization of the system's multimodal imaging performance by optimizing numerical as well as coherent and incoherent illumination parameters. Subsequently, the application capabilities are evaluated by multimodal imaging of living cells. The results highlight the potential of single-capture BF-DHM for fast biomedical imaging.