Design, Calibration, and Application of a Robust, Cost-Effective, and High-Resolution Lensless Holographic Microscope

Abstract

Lensless holographic microscope (LHM) is an emerging very promising technology that provides high-quality imaging and analysis of biological samples without utilizing any lens for imaging. Due to its small size and reduced price, LHM can be a very useful tool for the point-of-care diagnosis of diseases, sperm assessment, or microfluidics, among others, not only employed in advanced laboratories but also in poor and/or remote areas. Recently, several LHMs have been reported in the literature. However, complete characterization of their optical parameters remains not much presented yet. Hence, we present a complete analysis of the performance of a compact, reduced cost, and high-resolution LHM. In particular, optical parameters such as lateral and axial resolutions, lateral magnification, and field of view are discussed into detail, comparing the experimental results with the expected theoretical values for different layout configurations. We use high-resolution amplitude and phase test targets and several microbeads to characterize the proposed microscope. This characterization is used to define a balanced and matched setup showing a good compromise between the involved parameters. Finally, such a microscope is utilized for visualization of static, as well as dynamic biosamples.

Multi-Illumination Single-Holographic-Exposure Lensless Fresnel (MISHELF) Microscopy: Principles and Biomedical Applications

“…In MISHELF microscopy, several ways to produce a multi-wavelength coherent point source have been reported [ 22 , 47 , 48 , 49 ]. First, the light coming from different lasers with different wavelengths has been combined before arriving at a pinhole with dichroic beam combiners [ 47 ].…”

“…In that regard, lensless holographic microscopes are ideal candidates since they digitally reconstruct images of microscopic specimens without using any lenses, so that their designs are compact, lightweight and inexpensive [ 3 , 4 , 5 ]. New findings in economical optoelectronic components have recently pushed towards a strong development of lensless holographic microscopes [ 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 ] and a huge variety of biological applications, including cancer, SARS-CoV-2 and disease diagnosis [ 23 , 24 , 25 ], water and air quality monitoring [ 7 , 15 , 26 , 27 ], microbial viability testing [ 21 , 28 ], or 3D motion tracking of biological samples [ 20 , 29 , 30 , 31 , 32 , 33 ], among others [ 9 , 10 , 11 , 17 , 34 ].…”

“…Inside the extreme simplicity of LHM configurations, there exists great flexibility in the choice of both point light sources [ 6 , 7 , 22 , 34 , 36 , 40 , 41 , 42 , 43 , 44 , 45 ] and layout geometries [ 6 , 7 , 15 , 16 , 19 , 20 , 34 , 40 , 42 , 43 , 44 , 46 ]. Concerning illumination, point sources have been generated in LHM mainly using pinholes in combination with laser beams [ 7 , 40 , 41 , 47 ] or LEDs [ 15 , 34 ], high-numerical-aperture (NA) focusing lenses together with either LEDs [ 6 ] or laser diodes [ 14 , 22 , 48 ], and also employing different types of lenses and light sources such as GRIN lenses [ 21 , 42 ], tunable lenses [ 36 ], optical fibers [ 43 , 49 ], pulsed lasers [ 44 ], SLED sources [ 45 ] and terahertz lasers [ 50 ]. Regarding layout geometry, LHM has been typically implemented under two opposite configurations [ 3 ].…”

“…In line with those single-shot phase-retrieval methods applied to LHM, the technique called multi-illumination single-holographic-exposure lensless Fresnel (MISHELF) microscopy proposes a new concept of LHM based on wavelength multiplexing and the use of a rapid-convergence iterative phase-retrieval algorithm for coherent noise reduction and twin-image mitigation [ 22 , 47 , 48 , 49 ]. MISHELF microscopy illuminates the sample with various wavelengths and records the different in-line Fresnel holograms in a single camera acquisition.…”

“…MISHELF microscopy illuminates the sample with various wavelengths and records the different in-line Fresnel holograms in a single camera acquisition. So far, MISHELF microscopy has been reported using two [ 48 ], three [ 22 , 47 ] and four [ 49 ] illumination/detection channels, validated either at the laboratory level on an optical table within a well-controlled environment [ 47 , 48 ], or at the field level in different 3D-printed prototype microscopes [ 22 , 49 ], as well as applied in biomedical research for sperm motility assessment [ 33 ].…”