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

Abstract

Lensless holographic microscopy (LHM) comes out as a promising label-free technique since it supplies high-quality imaging and adaptive magnification in a lens-free, compact and cost-effective way. Compact sizes and reduced prices of LHMs make them a perfect instrument for point-of-care diagnosis and increase their usability in limited-resource laboratories, remote areas, and poor countries. LHM can provide excellent intensity and phase imaging when the twin image is removed. In that sense, multi-illumination single-holographic-exposure lensless Fresnel (MISHELF) microscopy appears as a single-shot and phase-retrieved imaging technique employing multiple illumination/detection channels and a fast-iterative phase-retrieval algorithm. In this contribution, we review MISHELF microscopy through the description of the principles, the analysis of the performance, the presentation of the microscope prototypes and the inclusion of the main biomedical applications reported so far.

Keywords: Gabor holography; biomedical imaging; digital holography; digital image processing; in-line holographic microscopy; label-free imaging; lensless microscopy; multiplexed holography.

Figure 1

Optical scheme implemented in MISHELF microscopy.

Figure 2

Flow chart of the algorithm implemented by MISHELF microscopy. In the chart, the variables $I$, $A$ and $B$ mean intensity and complex amplitudes at the hologram plane (HP). $O$ represents the complex amplitude at the object plane (OP) and $F\left\{\right\}$ is the Fourier transform operator. All variables are (x, y) spatially dependent.

Figure 3

Example of the digital processing performed in MISHELF microscopy involving an USAF test target [22].

Figure 4

Performance analysis of MISHELF microscopy involving a positive USAF resolution test target [47]. Rows (a,b): results provided by B-LHM and MISHELF microscopy, respectively. (a1,b1) in-line color holograms, (a2,b2) amplitude reconstructions of the region of interest (yellow square in (a1)), (a3,b3) twin images, and (a4,b4) magnified central regions of images in (a2,b2) (green square in (a2)). (c) plots along blue and red arrows in (a4,b4) for spatial resolution comparison. (d) plots along blue and red arrows in (a2,b2) for contrast comparison. White rectangle area in (a1) is considered for coherence noise calculation.

Figure 5

MISHELF microscopy implemented using different illumination/detection channels [49]. Rows (a,b): color holograms and amplitude reconstructions involving an USAF test target, respectively; row (c): phase images involving several 90 μm microspheres. (a1–c1): conventional LHM (V illumination); (a2–c2), (a3–c3) and (a4–c4): MISHELF microscopy implemented with two (V-G), three (V-G-R) and four (V-B-G-R) illumination wavelengths, respectively. Yellow scale bars in column (1) represent 100 μm.

Figure 6

Microscope prototypes developed for MISHELF microscopy implementation. (a–c) High performance and (d–f) compact and cost-effective microscope prototypes, respectively. (a,d) Optical schemes of the microscopes including sensor pixel ordination, spectral sensitivity and wavelength line emissions; (b,e) perspective views of the prototypes design including units in mm; (c,f) pictures of fabricated microscopes.

Figure 7

Sperm motility analysis performed with MISHELF microscopy involving different types of sperm cells. Rows (a,b) human and starry skate fish spermatozoa, respectively. (a1,b1) reconstructed amplitude images in a sample plane; (a2,b2) perspective views of three different planes showing sperm cells in focus; (a3,b3) 3D trajectories of some spermatozoa. Scale bars in (a1,b1) represent 100 and 200 μm, respectively.

Figure 8

Example of a 3D trajectory of a boar sperm cell provided by MISHELF microscopy [33]: (a) perspective and (b) lateral views, respectively. Black arrow in (b) highlights the axial movement of the spermatozoon.