José Ángel Picazo-Bueno

Improved quantitative phase imaging in lensless microscopy by single-shot multi-wavelength illumination using a fast convergence algorithm

We report on a novel algorithm for high-resolution quantitative phase imaging in a new concept of lensless holographic microscope based on single-shot multi-wavelength illumination. This new microscope layout, reported by Noom et al. along the past year and named by us as MISHELF (initials incoming from Multi-Illumination Single-Holographic-Exposure Lensless Fresnel) microscopy, rises from the simultaneous illumination and recording of multiple diffraction patterns in the Fresnel domain. In combination with a novel and fast iterative phase retrieval algorithm, MISHELF microscopy is capable of high-resolution (micron range) phase-retrieved (twin image elimination) biological imaging of dynamic events. In this contribution, MISHELF microscopy is demonstrated through qualitative concept description, algorithm implementation, and experimental validation using both a synthetic object (resolution test target) and a biological sample (swine sperm sample) for the case of three (RGB) illumination wavelengths. The proposed method becomes in an alternative instrument improving the capabilities of existing lensless microscopes.

Spatially multiplexed interferometric microscopy with partially coherent illumination

Abstract. We have recently reported on a simple, low cost, and highly stable way to convert a standard microscope into a holographic one [Opt. Express 22, 14929 (2014)]. The method, named spatially multiplexed interferometric microscopy (SMIM), proposes an off-axis holographic architecture implemented onto a regular (nonholographic) microscope with minimum modifications: the use of coherent illumination and a properly placed and selected one-dimensional diffraction grating. In this contribution, we report on the implementation of partially (temporally reduced) coherent illumination in SMIM as a way to improve quantitative phase imaging. The use of low coherence sources forces the application of phase shifting algorithm instead of off-axis holographic recording to recover the sample’s phase information but improves phase reconstruction due to coherence noise reduction. In addition, a less restrictive field of view limitation (1/2) is implemented in comparison with our previously reported scheme (1/3). The proposed modification is experimentally validated in a regular Olympus BX-60 upright microscope considering a wide range of samples (resolution test, microbeads, swine sperm cells, red blood cells, and prostate cancer cells).

Compact, cost-effective and field-portable microscope prototype based on MISHELF microscopy

We report on a reduced cost, portable and compact prototype design of lensless holographic microscope with an illumination/detection scheme based on wavelength multiplexing, working with single hologram acquisition and using a fast convergence algorithm for image processing. All together, MISHELF (initials coming from Multi-Illumination Single-Holographic-Exposure Lensless Fresnel) microscopy allows the recording of three Fresnel domain diffraction patterns in a single camera snap-shot incoming from illuminating the sample with three coherent lights at once. Previous implementations have proposed an illumination/detection procedure based on a tuned (illumination wavelengths centered at the maximum sensitivity of the camera detection channels) configuration but here we report on a detuned (non-centered ones) scheme resulting in prototype miniaturization and cost reduction. Thus, MISHELF microscopy in combination with a novel and fast iterative algorithm allows high-resolution (μm range) phase-retrieved (twin image elimination) quantitative phase imaging of dynamic events (video rate recording speed). The performance of this microscope prototype is validated through experiments using both amplitude (USAF resolution test) and complex (live swine sperm cells and flowing microbeads) samples. The proposed method becomes in an alternative instrument improving some capabilities of existing lensless microscopes.

Superresolved spatially multiplexed interferometric microscopy

Superresolution capability by angular and time multiplexing is implemented onto a regular microscope. The technique, named superresolved spatially multiplexed interferometric microscopy (S2MIM), follows our previously reported SMIM technique [Opt. Express22, 14929 (2014)OPEXFF1094-408710.1364/OE.22.014929, J. Biomed. Opt.21, 106007 (2016)JBOPFO1083-366810.1117/1.JBO.21.10.106007] improved with superresolved imaging. All together, S2MIM updates a commercially available non-holographic microscope into a superresolved holographic one. Validation is presented for an Olympus BX-60 upright microscope with resolution test targets.

Effect of counting chamber depth on the accuracy of lensless microscopy for the assessment of boar sperm motility

Sperm motility is one of the most significant parameters in the prediction of male fertility. Until now, both motility analysis using an optical microscope and computer-aided sperm analysis (CASA-Mot) entailed the use of counting chambers with a depth to 20µm. Chamber depth significantly affects the intrinsic sperm movement, leading to an artificial motility pattern. For the first time, laser microscopy offers the possibility of avoiding this interference with sperm movement. The aims of the present study were to determine the different motility patterns observed in chambers with depths of 10, 20 and 100µm using a new holographic approach and to compare the results obtained in the 20-µm chamber with those of the laser and optical CASA-Mot systems. The ISAS®3D-Track results showed that values for curvilinear velocity (VCL), straight line velocity, wobble and beat cross frequency were higher for the 100-µm chambers than for the 10- and 20-µm chambers. Only VCL showed a positive correlation between chambers. In addition, Bayesian analysis confirmed that the kinematic parameters observed with the 100-µm chamber were significantly different to those obtained using chambers with depths of 10 and 20µm. When an optical analyser CASA-Mot system was used, all kinematic parameters, except VCL, were higher with ISAS®3D-Track, but were not relevant after Bayesian analysis. Finally, almost three different three-dimensional motility patterns were recognised. In conclusion, the use of the ISAS®3D-Track allows for the analysis of the natural three-dimensional pattern of sperm movement.

Single-shot dual-mode water-immersion microscopy platform for biological applications

A single-shot water-immersion digital holographic microscope combined with broadband (white light) illumination mode is presented. This double imaging platform allows conventional incoherent visualization with phase holographic imaging of inspected samples. The holographic architecture is implemented at the image space (that is, after passing the microscope lens), thus reducing the sensitivity of the system to vibrations and/or thermal changes in comparison to regular interferometers. Because of the off-axis holographic recording principle, quantitative phase images of live biosamples can be recorded in a single camera snapshot at full-field geometry without any moving parts. And, the use of water-immersion imaging lenses maximizes the achievable resolution limit. This dual-mode microscope platform is first calibrated using microbeads, then applied to the characterization of fixed cells (neuroblastoma, breast cancer, and hippocampal neuronal cells) and, finally, validated for visualization of dynamic living cells (hippocampal neurons).

Modified variational image decomposition algorithm aided by the Hilbert transform as an alternative to 2D Hilbert-Huang transform for fringe pattern phase retrieval

The process of information recovering from fringe pattern can be divided into two main parts: filtration (fringe pattern background and noise removal) and phase (or amplitude) demodulation. In recent years the 2D Hilbert spiral transform (HST) has become one of the most popular phase demodulation techniques. Together with empirical mode decomposition used for fringe pattern preprocessing forms a strong fringe pattern analysis algorithm called 2D HilbertHuang transform (HHT). Variational image decomposition was recently adapted for fringe pattern filtration. In combination with the 2D Hilbert spiral transform and after some modifications it might become an excellent tool for fringe pattern analysis purpose and can compete with well-developed HHT. Proposed modification is the first attempt to automate the variational image decomposition in terms of fringe pattern filtration. Received results show that VID-HST can compete with HHT and may become an excellent alternative for fringe pattern evaluation. Another fact encouraging the development of VID is a wide range of applications that have been proposed up to now, i.e., image denoising, fringe pattern filtration and phase filtration.

Multi-beam spatially multiplexed interference microscopy for phase objects examination

Multi-beam Spatially Multiplexed Interferometric Microscopy (MB-SMIM) is presented as the implementation of a robust, fast and accurate single-shot phase-estimation algorithm with a simple, low-cost and stable way to convert a bright-field microscope into a holographic one using partially-coherent illumination. Ronchi diffraction grating is used to generate two-beam and multi-beam interference. To ensure effectiveness of the process object field should be divided into analyzed specimen area (1/3) and clear reference area (other 2/3). Using partially coherent light source one is able to produce pure three-beam interference of -1 diffraction order of specimen area, and 0/+1 diffraction orders of both clear reference areas. Opposite sign orders create doubled spatial frequency term overlapping with the regular SMIM interferogram. Phase demodulation based on the single-frame two-dimensional Hilbert-Huang transform or regular multi-frame phase-shifting follows. Single-frame processing is more beneficial for increased spatial frequency term when doubled number of fringes “samples” the same phase variation as in regular SMIM interferogram. Adding (or subtracting) two three-beam interferograms phase shifted by π allows one to accurately isolate doubled (or single) spatial frequency fringe pattern. Altogether, MB-SMIM adds quantitative phase imaging capability to a commercially available non-holographic microscope with improved phase reconstruction: noise reduction, spatial frequency doubling and phase change disambiguation capabilities. Proposed method enables common-path optical generation of doubled spatial frequency fringes only by slight object field manipulation – no mechanical doubling of incident angle is needed. The technique has been validated using a 20X/0.46NA objective in Olympus BX-60 upright microscope for prostate cancer cells phase imaging.

Moiré effect-based interference microscopy for biospecimen characterization

Interference microscopy for biospecimen characterization based on the moiré phenomena is described. Two scenarios of incoherent multiplicative superimposition are employed: (1) object information carrying interferogram is superimposed with reference one or (2) two object interferograms are superimposed (with equal or opposite phase signs). Second strategy provides additional doubling of underlying phase function of interest. Superimposition can be performed using two experimental interferograms or single real interferogram and numerically designed reference structure in so-called digital moiré regime. Multiplicative superimposition of two periodic intensity distribution yields moiré pattern containing low spatial frequency difference beat term (moiré fringes – macrostructure) and high spatial frequency sum beat term (microstructure). The macrostructure was studied in great majority of previously reported moiré techniques. In this contribution we point attention onto the sum beat spatial frequency component and report efficient means for its recovery using numerically advanced digital filtering (variational/empirical decomposition approaches). Its single-frame (single-pattern) phase demodulation by 2D Hilbert spiral transform with enhanced accuracy follows - this feature comes from the fact that sum beat moiré term has high spatial frequency which is generally beneficial in single-frame fringe analysis and its phase function is be doubled. In particular spatial phase change is better sampled by fringes for denser interferogram and more importantly numerical filtering of fringe term of interest from background intensity is easier. We propose and preliminarily evaluate experimental proof-of-concept strategy where two interferograms with slight difference in spatial frequency are simultaneously recorded in two halves of CCD camera and superimposed multiplicatively. Proposed moiré technique opens up new possibilities in interference microscopy based bio-phase imaging mainly due to its data-driven enhanced phase sensitivity (fringe doubling effect) and real-time operation. Evaluation employing numerical simulations and validation using experimental recordings of phase bio-samples, i.e., prostate cancer cells are enclosed.

Superresolution imaging in spatially multiplexed interferometric microscopy by using time multiplexing

We report on the merging between our recently introduced SMIM (initials incoming from Spatially-Multiplexed Interferometric Microscopy) technique and superresolution imaging. SMIM has been previously reported [Opt. Express 22, 14929 (2014); J. Bio. Opt. 21, 106007 (2016)] as a low cost, extremely simple, and highly stable scheme to update a standard microscope into a holographic microscope but, as consequence, the usable FOV is reduced. Superresolution capability enables to enhance the resolution limit in the usable FOV thus compensating the FOV reduction. In this contribution, superresolution is implemented joint together with SMIM defining a new method named as S2MIM (initials incoming from Superresolved Spatially Multiplexed Interferometric Microscopy) which updates a commercially available non-holographic microscope into a superresolved holographic one. Experimental validation is presented for an Olympus BX-60 upright microscope with a USAF resolution test target as calibration object.