Search

Exposure to laser light alters cell culture examination via optical microscopic imaging techniques, also based on label-free coherent digital holography. To mitigate this detrimental feature, researchers tend to use a broader spectrum and lower intensity of illumination, which can decrease the quality of holographic imaging due to lower resolution and higher noise. We study the lensless digital holographic microscopy (LDHM) ability to operate in the low photon budget (LPB) regime to enable imaging of unimpaired live cells with minimized sample interaction. Low-cost off-the-shelf components are used, promoting the usability of such a straightforward approach. We show that recording data in the LPB regime (down to 7 uW of illumination power) does not limit the contrast nor resolution of the hologram phase and amplitude reconstruction compared to the regular illumination. The LPB generates hardware camera shot noise, however, to be effectively minimized via numerical denoising. The ability to obtain high-quality, high-resolution optical complex field reconstruction was confirmed using the USAF 1951 amplitude sample, phase resolution test target, and finally, live glial restricted progenitor cells (as a challenging strongly absorbing and scattering biomedical sample). The proposed approach based on severely limiting the photon budget in lensless holographic microscopy method can open new avenues in high-throughout (optimal resolution, large field-of-view and high signal-to-noise-ratio single-hologram reconstruction) cell culture imaging with minimized sample interaction.

Laser-based lensless digital holographic microscopy (LDHM) is often spoiled by considerable coherent noise factor. We propose a novel LDHM method with significantly limited coherent artifacts, e.g., speckle noise and parasitic interference fringes. It is achieved by incorporating a rotating diffuser, which introduces partial spatial coherence and preserves high temporal coherence of laser light, crucial for credible in-line hologram reconstruction. We present the first implementation of the classical rotating diffuser concept in LDHM, significantly increasing the signal-to-noise ratio while preserving the straightforwardness and compactness of the LDHM imaging device. Prior to the introduction of the rotating diffusor, we performed LDHM experimental hardware optimization employing 4 light sources, 4 cameras, and 3 different optical magnifications (camera-sample distances). It was guided by the quantitative assessment of numerical amplitude/phase reconstruction of test targets, conducted upon standard deviation calculation (noise factor quantification), and resolution evaluation (information throughput quantification). Optimized rotating diffuser LDHM (RD-LDHM) method was successfully corroborated in technical test target imaging and examination of challenging biomedical sample (60um thick mouse brain tissue slice). Physical minimization of coherent noise (up to 50%) was positively verified, while preserving optimal spatial resolution of phase and amplitude imaging. Coherent noise removal, ensured by proposed RD-LDHM method, is especially important in biomedical inference, as speckles can falsely imitate valid biological features. Combining this favorable outcome with large field-of-view imaging can promote the use of reported RD-LDHM technique in high-throughput stain-free biomedical screening.