Open-access database for digital lensless holographic microscopy and its application on the improvement of deep-learning-based autofocusing models.

Among modern optical microscopy techniques, digital lensless holographic microscopy (DLHM) is one of the simplest label-free coherent imaging approaches. However, the hardware simplicity provided by the lensless configuration is often offset by the demanding computational postprocessing required to match the retrieved sample information to the user's expectations. A promising avenue to simplify this stage is the integration of artificial intelligence and machine learning (ML) solutions into the DLHM workflow. The biggest challenge to do so is the preparation of an extensive and high-quality experimental dataset of curated DLHM recordings to train ML models. In this work, a diverse, open-access dataset of DLHM recordings is presented as support for future research, contributing to the data needs of the applied research community. The database comprises 11,760 experimental DLHM holograms of bio and non-bio samples with diversity on the main recording parameters of the DLHM architecture. The database is divided into two datasets of 10 independent imaged samples. The first group, named multi-wavelength dataset, includes 8160 holograms and was recorded using laser diodes emitting at 654 nm, 510 nm, and 405 nm; the second group, named single-wavelength dataset, is composed of 3600 recordings and was acquired using a 633 nm He-Ne laser. All the experimental parameters related to the dataset acquisition, preparation, and calibration are described in this paper. The advantages of this large dataset are validated by re-training an existing autofocusing model for DLHM and as the training set for a simpler architecture that achieves comparable performance, proving its feasibility for improving existing ML-based models and the development of new ones.

Noise reduction in digital holography phase maps by phase-preserving discrete Fourier resampling.

Several methods have been proposed to reduce the detrimental effects of coherent noise in holographic imaging. Among them, the use of spatial-frequency masking or resampling has been widely applied because of its low implementation complexity and well-studied trade-off between denoising effectiveness and spatial resolution. While the digital application of this method has been successfully demonstrated for intensity images, its application to phase maps fails. This work shows that the phase applicability of these methods depends on the use of resampling masks that strictly keep the zero-order spatial frequencies. Alternative masks are proposed that demonstrate effective single-shot noise reduction in experimental phase maps from digital holographic microscopy. The resulting method is potentially extendable to any other complex-valued-field retrieval technique.

Adapting a Blu-ray optical pickup unit as a point source for digital lensless holographic microscopy.

The adaptation of an off-the-shelf Blu-ray optical pickup unit (OPU) into a highly versatile point source for digital lensless holographic microscopy (DLHM) is presented. DLHM performance is mostly determined by the optical properties of the point source of spherical waves used for free-space magnification of the sample's diffraction pattern; in particular, its wavelength and numerical aperture define the achievable resolution, and its distance to the recording medium sets the magnification. Through a set of straightforward modifications, a commercial Blu-ray OPU can be transformed into a DLHM point source with three selectable wavelengths, a numerical aperture of up to 0.85, and integrated micro-displacements in both axial and transversal directions. The functionality of the OPU-based point source is then experimentally validated in the observation of micrometer-sized calibrated samples and biological specimens of common interest, showing the feasibility of obtaining sub-micrometer resolution and offering a versatile option for the development of new cost-effective and portable microscopy devices.

Image enhancement and field of view enlargement in digital lensless holographic microscopy by multi-shot imaging.

A method to improve the quality of reconstructed images while the field of view (FOV) is enlarged in digital lensless holographic microscopy (DLHM) is presented. Multiple DLHM holograms are recorded while a still sample is located at different places of the plane containing it. The different locations of the sample must produce a set of DLHM holograms that share an overlapped area with a fixed DLHM hologram. The relative displacement among multiple DLHM holograms is computed by means of a normalized cross-correlation. The value of the computed displacement is utilized to produce a new DLHM hologram resulting from the coordinated addition of multi-shot DLHM holograms with the corresponding compensated displacement. The composed DLHM hologram carries enhanced information of the sample in a larger format, leading to a reconstructed image with improved quality and larger FOV. The feasibility of the method is illustrated and validated with results obtained from imaging a calibration test target and a biological specimen.

Optics in South America: introduction.

South American optics research has seen remarkable growth over the past 50 years, with significant contributions in areas such as quantum optics, holography, spectroscopy, nonlinear optics, statistical optics, nanophotonics and integrated photonics. The research has driven economic development in sectors like telecom, biophotonics, biometrics, and agri-sensing. This joint feature issue between JOSA A and JOSA B exhibits cutting-edge optics research from the region, fostering a sense of community and promoting collaboration among researchers.

Robust and compact digital Lensless Holographic microscope for Label-Free blood smear imaging.

The lack of equipped healthcare infrastructure in isolated hard-to-reach zones exposes their population to a higher risk of complications in common diseases. With a timely diagnosis setting a life-altering difference, worldwide efforts have been conducted for the development of point-of-care testing (PoCT) with cost-effective devices. Among the most common interests in PoCT is the analysis of blood smear samples, as they can help to detect, diagnose, and monitor a wide range of diseases and disorders. With microscopy being the traditional tool for these analyses, a significative advance has been the development of cost-effective digital holographic microscopy systems, driven in part by its label-free imaging capabilities that waive the need for any sample preprocessing. Here, a robust and portable digital lensless holographic microscope, functionalized for the analysis of non-preprocessed blood smear samples in PoCT environments, is presented, and its viability is tested in the observation of red blood cells. The device uses an optical fiber with a cone-shaped tip instead of a pinhole, which ensures the sturdiness of the system and eliminates the need for challenging alignment. While the distances of the microscope can be tuned before fabrication, the herein-reported operational parameters are functionalized for the specific analysis of blood samples.

Resolution limit in opto-digital systems revisited.

The resolution limit achievable with an optical system is a fundamental piece of information when characterizing its performance, mainly in case of microscopy imaging. Usually this information is given in the form of a distance, often expressed in microns, or in the form of a cutoff spatial frequency, often expressed in line pairs per mm. In modern imaging systems, where the final image is collected by pixelated digital cameras, the resolution limit is determined by the performance of both, the optical systems and the digital sensor. Usually, one of these factors is considered to be prevalent over the other for estimating the spatial resolution, leading to the global performance of the imaging system ruled by either the classical Abbe resolution limit, based on physical diffraction, or by the Nyquist resolution limit, based on the digital sensor features. This estimation fails significantly to predict the global performance of opto-digital imaging systems, like 3D microscopes, where none of the factors is negligible. In that case, which indeed is the most common, neither the Abbe formula nor the Nyquist formula provide by themselves a reliable prediction for the resolution limit. This is a serious drawback since systems designers often use those formulae as design input parameters. Aiming to overcome this lack, a simple mathematical expression obtained by finely articulating the Abbe and Nyquist formulas, to easily predict the spatial resolution limit of opto-digital imaging systems, is proposed here. The derived expression is tested experimentally, and shows to be valid in a broad range of opto-digital combinations.

Competitive fiber optic sensors for the highly selective detection of mercury in water.

Two competitive fiber optic sensors for the rapid, sensitive, and highly selective detection of mercury in water are designed, fabricated, and evaluated. A wavelength-modulated sensor based on an etched single-mode-multimode-single-mode (E-SMS) optical fiber structure and an intensity-modulated sensor based on fiber optics with a slanted end were fabricated by readily reproducible methods. The sensors were activated with a nanostructured chitosan/maghemite (CS/Fe2O3) composite thin film for the selective detection of mercury ions (Hg2+) in water. The functionalized sensors were implemented to experimentally validate the potential of CS/Fe2O3 thin film for optical sensing of Hg2+ in drinking water. The sensor based on the E-SMS structure exhibited a wavelength-modulated response with a sensitivity of up to 290 pm/(µg/mL), and the sensor based on the slanted end structure showed an intensity-modulated response with a sensitivity of -0.07dBm/(µg/mL). Validation of the experimental assay method proves the ability to selectively detect chemical interactions as low as 1 ng/mL (one part per billion) of Hg2+ in water for both sensors. The high specificity of the two sensors was demonstrated by evaluating their responses to a number of potentially interfering metal ions in water. These sensors are cost-effective, simple to construct, and easy to implement, which makes them very promising for the on-site detection and monitoring of mercury in bodies of water.

Fourier lightfield microscopy: a practical design guide.

In this work, a practical guide for the design of a Fourier lightfield microscope is reported. The fundamentals of the Fourier lightfield are presented and condensed on a set of contour plots from which the user can select the design values of the spatial resolution, the field of view, and the depth of field, as function of the specifications of the hardware of the host microscope. This work guides the reader to select the parameters of the infinity-corrected microscope objective, the optical relay lenses, the aperture stop, the microlens array, and the digital camera. A user-friendly graphic calculator is included to ease the design, even to those who are not familiar with the lightfield technology. The guide is aimed to simplify the design process of a Fourier lightfield microscope, which sometimes could be a daunting task, and in this way, to invite the widespread use of this technology. An example of a design and experimental results on imaging different types of samples is also presented.

Realistic simulation and real-time reconstruction of digital holographic microscopy experiments in ImageJ.

The description, implementation, and validation of an ImageJ plugin that allows the realistic simulation and real-time reconstruction of digital holographic microscopy (DHM) experiments are presented. The simulation module implements a telecentric image-plane DHM recording scheme with fully configurable imaging system, interference, and scaling parameters, including the possibility of defining an estimate of the roughness distribution of the sample to produce realistic coherent-noise affectations. The reconstruction module allows the computation of amplitude, intensity, or phase, from digital holograms' input as either single images or video streams for real-time processing; this module also implements user-defined fine-tuning parameters, allowing subpixel linear phase compensations and digital refocusing of the complex-valued reconstructed fields. In this note, the functionality of the plugin is illustrated by simulating the noisy DHM recording of a phase-only resolution test target and the reconstruction of both the resulting synthetic hologram and an equivalent experimental recording; the results show good agreement between the simulation and the experimental recording, and accurate measurements on the reconstructed information, thus granting the use of either module with full confidence according to needs and possibilities.

High-speed measurement of mechanical micro-deformations with an extended phase range using dual-wavelength digital holographic interferometry.

The implementation of a digital holographic interferometry setup for high-speed micro-deformation measurement is presented. This proposal uses a dual-wavelength recording strategy to reconstruct micro-deformations up to 4.85 µm with no phase wrapping. The numerical processing required to recover the phase maps containing the information of micro-deformations is carried out in a general-purpose computing on graphics processing unit environment to boost its performance. The method completely processes recorded holograms of 1024×1024pixels in 48 ms, i.e., 21 frames per second (FPS) for a single-wavelength acquisition and 96 ms or 11 FPS for dual-wavelength recordings. The method is experimentally evaluated measuring deformations ranging from 0.033 µm to 4.85 µm with no need for phase unwrapping algorithms for an 8 cm diameter aluminum plate in a 110cm2 field of view.

Handheld and Cost-Effective Fourier Lightfield Microscope.

In this work, the design, building, and testing of the most portable, easy-to-build, robust, handheld, and cost-effective Fourier Lightfield Microscope (FLMic) to date is reported. The FLMic is built by means of a surveillance camera lens and additional off-the-shelf optical elements, resulting in a cost-effective FLMic exhibiting all the regular sought features in lightfield microscopy, such as refocusing and gathering 3D information of samples by means of a single-shot approach. The proposed FLMic features reduced dimensions and light weight, which, combined with its low cost, turn the presented FLMic into a strong candidate for in-field application where 3D imaging capabilities are pursued. The use of cost-effective optical elements has a relatively low impact on the optical performance, regarding the figures dictated by the theory, while its price can be at least 100 times lower than that of a regular FLMic. The system operability is tested in both bright-field and fluorescent modes by imaging a resolution target, a honeybee wing, and a knot of dyed cotton fibers.

Open-source, cost-effective, portable, 3D-printed digital lensless holographic microscope.

In this work, the design, construction, and testing of the most cost-effective digital lensless holographic microscope to date are presented. The architecture of digital lensless holographic microscopy (DLHM) is built by means of a 3D-printed setup and utilizing off-the-shelf materials to produce a DLHM microscope costing US$52.82. For the processing of the recorded in-line holograms, an open-source software specifically developed to process this type of recordings is utilized. The presented DLHM setup has all the degrees of freedom needed to achieve different fields of view, levels of spatial resolution, and 2D scanning of the sample. The feasibility of the presented platform is tested by imaging non-bio and bio samples; the resolution test targets, a section of the head of a Drosophila melanogaster fly, red blood cells, and cheek cells are imaged on the built microscope.

Preprocessing in digital lensless holographic microscopy for intensity reconstructions with enhanced contrast.

In this work, a numerical method to enhance the contrast of intensity hologram reconstructions of digital lensless holographic microscopy (DLHM) is presented. The method manipulates the in-line hologram and reference images through mathematical operations between them; additionally, a sharpening operation, functionalized in terms of the parameters of the recording setup, is applied to the said images. The preprocessing of the recorded images produces a modified in-line hologram and a reference wave image from which an intensity reconstruction with a 25% improvement of its contrast, with respect to the conventional reconstruction procedure, is achieved. The method is illustrated with intensity reconstructions of a hologram of a monolayer of polystyrene spheres 1.09 µm in diameter. Finally, the preprocessing method is validated with a modeled hologram, successfully applied to holograms of the section of the head a Drosophila melanogaster fly and its results are contrasted with those obtained via bright-field microscopy.

Physical pupil manipulation for speckle reduction in digital holographic microscopy.

The reduction of speckle noise by physically changing the pupil of the imaging system, as first envisioned in optical holography, is experimentally applied to a digital holographic microscope (DHM). The imaging pupil of a DHM, operating in image plane telecentric-afocal architecture, is changed in a controlled way between successive recordings, allowing the shooting of multiple partially-decorrelated holograms. Averaging the numerically reconstructed holograms yields amplitude and/or phase images with reduced speckle noise. Experimental results of biological specimens and a phase-only resolution test show the feasibility to recover micron-sized features in images with reduced speckle noise.

Fast-iterative blind phase-shifting digital holographic microscopy using two images.

Digital holographic microscopy (DHM) has consolidated as a tool for diagnosis and measuring in life sciences, thanks to its capability to perform quantitative phase imaging. The reduction of the acquisition and computation time has driven the development of diverse reconstruction methodologies using a single-shot and two-frame approach. Methods based on the Fourier transform, the Hilbert transform, and the phase derivative are counted among the most utilized. The sensitivity of those methods is highly dependent on the compensation of the phase step, which requires the accurate knowledge of the phase shift between the two recorded holograms. Here, an alternative fast-iterative method based on the demodulation of the different components of the recorded interferograms is presented. The novelties of the proposed two-frame approach are: minimum number of images, since it requires 2 recorded holograms; a minimum phase error of the order of 0.005% independently of the phase step ranging from 0 to 180 deg.; a maximum correlation coefficient equal to 1 between the phase and the retrieved phase image; and, finally, a reduced processing time compared with the previous three-frame approach. Experimental results demonstrate the goodness and feasibility of the proposed technique.

Advantages of Fresnel biprism-based digital holographic microscopy in quantitative phase imaging.

SIGNIFICANCE: The hallmarks of digital holographic microscopy (DHM) compared with other quantitative phase imaging (QPI) methods are high speed, accuracy, spatial resolution, temporal stability, and polarization-sensitivity (PS) capability. The above features make DHM suitable for real-time quantitative PS phase imaging in a broad number of biological applications aimed at understanding cell growth and dynamic changes occurring during physiological processes and/or in response to pharmaceutical agents. AIM: The insertion of a Fresnel biprism (FB) in the image space of a light microscope potentially turns any commercial system into a DHM system enabling QPI with the five desired features in QPI simultaneously: high temporal sensitivity, high speed, high accuracy, high spatial resolution, and PS. To the best of our knowledge, this is the first FB-based DHM system providing these five features all together. APPROACH: The performance of the proposed system was calibrated with a benchmark phase object. The PS capability has been verified by imaging human U87 glioblastoma cells. RESULTS: The proposed FB-based DHM system provides accurate phase images with high spatial resolution. The temporal stability of our system is in the order of a few nanometers, enabling live-cell studies. Finally, the distinctive behavior of the cells at different polarization angles (e.g., PS capability) can be observed with our system. CONCLUSIONS: We have presented a method to turn any commercial light microscope with monochromatic illumination into a PS QPI system. The proposed system provides accurate quantitative PS phase images in a new, simple, compact, and cost-effective format, thanks to the low cost (a few hundred dollars) involved in implementing this simple architecture, enabling the use of this QPI technique accessible to most laboratories with standard light microscopes.

Digital lensless holographic microscopy: numerical simulation and reconstruction with ImageJ.

The description and validation of an ImageJ open-source plugin to numerically simulate and reconstruct digital lensless holographic microscopy (DLHM) holograms are presented. Two modules compose the presented plugin: the simulation module implements a discrete version of the Rayleigh-Somerfield diffraction formula, which allows the user to directly build a simulated hologram from a known phase and/or amplitude object by just introducing the geometry parameters of the simulated setup; the plugin's reconstruction module implements a discrete version of the Kirchhoff-Helmholtz diffraction integral, thus allowing the user to reconstruct DLHM holograms by introducing the parameters of the acquisition setup and the desired reconstruction distance. The plugin offers the two said modules within the robust environment provided by a complete set of built-in tools for image processing available in ImageJ. While the simulation module has been validated through the evaluation of the forecasted lateral resolution of a DLHM setup in terms of the numerical aperture, the reconstruction module is tested by means of reconstructing experimental DLHM holograms of biological samples.

Cone-shaped optical fiber tip for cost-effective digital lensless holographic microscopy.

In this work, the development and application of a cost-effective and robust digital lensless holographic microscopy (DLHM) system is presented. In the simple architecture of DLHM based on a point source and a digital camera, the production of the former is introduced by means of an engineered step-index optical fiber with a cone-shaped end tip. The conventional and regularly expensive point source in DLHM is produced by means of a high-numerical-aperture microscope objective and a metallic wavelength-sized pinhole. The proposed replacement renders to DLHM additional simplicity of building, in addition to mechanical stability and robustness, and further reduces the cost of the microscope. The simplified cost-effective DLHM architecture is utilized for imaging resolution test targets and samples of human blood and pond water, revealing competitive mechanical stability and trustable phase images of the imaged specimens.

Non-approximated Rayleigh-Sommerfeld diffraction integral: advantages and disadvantages in the propagation of complex wave fields.

Advantages and disadvantages of the non-approximated numerical implementation of the Rayleigh-Sommerfeld diffraction integral (RSD) are revisited. In this work, it is shown that as trade-off for its large computation load, the non-approximated RSD removes any limitation on the propagation range and does not introduce any artifact in the computed wave field. A non-approximated GPU implementation of the RSD is contrasted with the angular spectrum, the Fresnel transform, and a fast Fourier transform implementation of the RSD. The forecasted phase shift introduced in the propagated wave fields as light is diffracted on complementary apertures and utilized as a metric to quantify the performance of the tested methods. An application to numerical reconstructions with arbitrary shape and size of digital recorded holograms from digital lensless holographic microscopy is presented.