Digital Lensless Holographic Microscopy Research Articles

Dynamic Imaging and Characterization of Volatile Aerosols in E-Cigarette Emissions Using Deep Learning-Based Holographic Microscopy.

Various volatile aerosols have been associated with adverse health effects; however, characterization of these aerosols is challenging due to their dynamic nature. Here, we present a method that directly measures the volatility of particulate matter (PM) using computational microscopy and deep learning. This method was applied to aerosols generated by electronic cigarettes (e-cigs), which vaporize a liquid mixture (e-liquid) that mainly consists of propylene glycol (PG), vegetable glycerin (VG), nicotine, and flavoring compounds. E-cig-generated aerosols were recorded by a field-portable computational microscope, using an impaction-based air sampler. A lensless digital holographic microscope inside this mobile device continuously records the inline holograms of the collected particles. A deep learning-based algorithm is used to automatically reconstruct the microscopic images of e-cig-generated particles from their holograms and rapidly quantify their volatility. To evaluate the effects of e-liquid composition on aerosol dynamics, we measured the volatility of the particles generated by flavorless, nicotine-free e-liquids with various PG/VG volumetric ratios, revealing a negative correlation between the particles' volatility and the volumetric ratio of VG in the e-liquid. For a given PG/VG composition, the addition of nicotine dominated the evaporation dynamics of the e-cig aerosol and the aforementioned negative correlation was no longer observed. We also revealed that flavoring additives in e-liquids significantly decrease the volatility of e-cig aerosol. The presented holographic volatility measurement technique and the associated mobile device might provide new insights on the volatility of e-cig-generated particles and can be applied to characterize various volatile PM.

Lensless digital holographic microscopy as an efficient method to monitor enzymatic plastic degradation

A big challenge of the 21st century is to cope with the huge amounts of plastic waste on Earth. Especially the oceans are heavily polluted with plastics. To counteract this issue, biological (enzymatic) plastic decomposition is increasingly gaining attention. Recently it was shown that polyethylene terephthalate (PET) can be degraded in a saltwater-based environment using bacterial PETase produced by a marine diatom. At moderate temperatures, plastic biodegradation is slow and requires sensitive methods for detection, at least at initial stages. However, conventional methods for verifying the plastic degradation are either complex, expensive, time-consuming or they interfere with the degradation process. Here, we adapt lensless digital holographic microscopy (LDHM) as a new application for efficiently monitoring enzymatic degradation of a PET glycol copolymer (PETG). LDHM is a cost-effective, compact and sensitive optical method. We demonstrate enzymatic PETG degradation over a time course of 43 days employing numerical analysis of LDHM images.

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.

An edge-lit volume holographic optical element for an objective turret in a lensless digital holographic microscope

In this paper, we propose and demonstrate the use of an edge-lit volume holographic optical element (EL-VHOE) as a reference waveguide to reduce the volume of a lensless digital holographic microscope. Additionally, a hybrid lensless Fourier transform digital holography is applied to make the EL-VHOE function as an objective turret. It used a spherical wave in the object beam of the EL-VHOE, which served as the reference beam of the microscope. Another sheared spherical wave was used to illuminate the sample. The longitudinal position of the spherical reference beam is changeable. It was shown that the tradeoff between resolution and field of view can be adjusted by changing the longitudinal position of the spherical reference beam. The corresponding experimental results matched the simulational and theoretical predictions. A resolution of approximately 3.11 μm was achieved when the object distance was 6 mm and the longitudinal distance of the spherical reference was 10 mm.

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.

Sizing calibration in digital lensless holographic microscopy via iterative Talbot self-imaging

In this work, an iterative method to calibrate the length measurements in digital lensless holographic microscopy (DLHM) is presented. In DLHM, the correct sizing is controlled by the accurate knowledge of the illumination wavelength, the digital camera specifications, and the source-to-screen distance. While the two formers are provided by the manufacturers, the latter has to be somehow measured, usually by mechanical means. As an alternative for performing that key measurement in DLHM, the Talbot self-imaging effect, and hence the Talbot distance, is used as a measurement tool in this work. Amplitude reconstructions of a monolayer of self-organized spheres of polystyrene produce the self-imaging phenomenon, with the Talbot distance controlled by the illuminating wavelength, the reconstruction distance, and the period of the monolayer. As the latter can be varied in DLHM by changing the source-to-screen distance while keeping fixed the wavelength and the camera specifications, the source-to-screen distance is iteratively varied until the correct Talbot distance is found. The proposed method has the advantages of i) increased sensitivity because of the axial quadratic dependency on transversal measurements, ii) not requiring in-focus images that demand the use of focusing criteria, and iii) can use non-approximated propagation methods to produce axial intensity profiles to determine the correct source-to-screen distance. The feasibility of the calibration method is tested by reconstructing a USAF 1951 test of resolution.

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.

Optical Fiber Point-Source for Digital Lensless Holographic Microscopy

In this paper, cone-shaped optical fibers as point light sources of spherical waves for digital lensless holographic microscopy (DLHM) are developed and evaluated. The resolution characterization of DLHM with cone-shaped fibers was carried out and a comparative analysis with respect to the pinhole illumination system was developed. The results are very encouraging and support the viability of these cone-shape fibers as a technological alternative to the pinhole illumination system for DLHM.

Numerical dark field illumination applied to experimental digital lensless holographic microscopy for reconstructions with enhanced contrast.

The implementation of a numerical dark field illumination to obtain reconstructions with enhanced contrast in digital lensless holographic (DLHM) microscopy is presented. In this proposal, an opaque circular stop is numerically inserted in the light path between the sample and hologram plane. The contrast of the reconstructed holograms is increased at least 46% in comparison with that obtained by the regular reconstruction methods. This proposal has been validated using experimental holograms of a paramecium in water and a section of the head of a Drosophila melanogaster fly recorded in DLHM.

High-precision rotation angle measurement method based on a lensless digital holographic microscope

To accurately measure ultrasmall rotation angles, a robust and effective method based on lensless digital holographic microscopy is proposed in this paper. The method combines holographic microscopy, solid geometry, and 3D measurement, including holographic measurement and angle measurement processes. We can calculate the 3D shape by the angular spectrum algorithm and the least-squares phase-unwrapping algorithm in the holographic process. According to the relationship between the surface shape and rotation angles, the real-time rotation angles can be calculated. To validate the feasibility and practicability of the proposed approach, numerical noise simulations and experiments were performed. The measurement precision of rotation angle can reach 0.5″ in the range of 1000″ in this paper's experiments. The holographic method has high measurement precision and good stability. In addition, the compact small volume has great potential in small-angle sensor applications.

Resolution optimization of an off-axis lensless digital holographic microscope.

Microscopes aimed at detecting cellular life in extreme environments such as ocean-bearing solar system moons must provide high resolution in a compact, robust instrument. Here, we consider the resolution optimization of a compact off-axis lensless digital holographic microscope (DHM) that consists of a sample placed between an input point-source pair and a detector array. Two optimal high-resolution regimes are identified at opposite extremes-a low-magnification regime with the sample located near a small-pixel detector array, and a high-magnification regime with the sample near the input plane. In the former, resolution improves with smaller pixels, while in the latter, the effect of the finite pixel size is obviated, and the spatial resolution improves with detector array size. Using an off-axis lensless DHM with a 2 k×2 k array of 5.5μm-pixels in the high-magnification regime, and standard aberration correction software, a resolution of ∼0.95 μm has been demonstrated, a factor of 5.8 smaller than the pixel size. Our analysis further suggests that with yet larger detector arrays, a lensless DHM should be capable of near wavelength-scale resolution.

Lensless digital holographic microscopy and its applications in biomedicine and environmental monitoring.

Optical compound microscope has been a major tool in biomedical imaging for centuries. Its performance relies on relatively complicated, bulky and expensive lenses and alignment mechanics. In contrast, the lensless microscope digitally reconstructs microscopic images of specimens without using any lenses, as a result of which it can be made much smaller, lighter and lower-cost. Furthermore, the limited space-bandwidth product of objective lenses in a conventional microscope can be significantly surpassed by a lensless microscope. Such lensless imaging designs have enabled high-resolution and high-throughput imaging of specimens using compact, portable and cost-effective devices to potentially address various point-of-care, global-health and telemedicine related challenges. In this review, we discuss the operation principles and the methods behind lensless digital holographic on-chip microscopy. We also go over various applications that are enabled by cost-effective and compact implementations of lensless microscopy, including some recent work on air quality monitoring, which utilized machine learning for high-throughput and accurate quantification of particulate matter in air. Finally, we conclude with a brief future outlook of this computational imaging technology.

Cooperative execution of auto-focusing metrics in digital lensless holographic microscopy for internal-structured samples.

The cooperative execution of two metrics to automatically determine the best focal plane in digital lensless holographic microscopy (DLHM) is presented. This proposal is comprised of two stages: first, a quick coarse search over the whole reconstruction range by using Dubois's metric allows the finding of a range in which the best focal plane can be found. In a second stage, the modified enclosed energy (MEE) metric is used within the found range in the former stage to finely determine the best focal plane. While this cooperative implementation keeps the proven effectiveness of the MEE in DLHM, it reduces by at least 11 times the total computational complexity of the auto-focusing method with respect to the MEE method only. This proposal has been validated experimentally with DLHM holograms of a paramecium specimen, polystyrene beads, and the section of the head of a Drosophila melanogaster fly.

Microscopia holográfica digital sin lentes y aplicaciones

The principles and some applications of the digital lensless holographic microscopy (DLHM) to study the microworld are shown in this paper. The recording and reconstruction processes of the DLHM are analyzed to study its lateral resolution power. The effect of the spatial coherence in the study of a section of the head of a Drosophila melanogaster fly are presented. DLHM is applied to study dwynamic and static colloidal systems as a proof of the capability of achieving micrometer spatial lateral resolution without the use of lenses.

Compact, lensless digital holographic microscope for remote microbiology.

In situ investigation of microbial life in extreme environments can be carried out with microscopes capable of imaging 3-dimensional volumes and tracking particle motion. Here we present a lensless digital holographic microscope approach that provides roughly 1.5 micron resolution in a compact, robust package suitable for remote deployment. High resolution is achieved by generating high numerical-aperture input beams with radial gradient-index rod lenses. The ability to detect and track prokaryotes was explored using bacterial strains of two different sizes. In the larger strain, a variety of motions were seen, while the smaller strain was used to demonstrate a detection capability down to micron scales.

Comparison of two broadband illumination sources in digital lensless holographic microscopy to study biological samples

The use of two broadband illumination sources in digital lensless holographic microscopy (DLHM) is presented. A femtosecond laser and a white light emitting diode, both with spectral widths of hundreds of nanometers, are the optical sources utilized for illuminating biological samples. Despite both sources have similar spectral widths, while the former is fully spatially coherent, the latter is spatially incoherent; this difference determines a completely dissimilar performance of the DLHM. The resulting images of samples obtained with DLHM by using both light sources are analyzed on the framework of basic coherence theory. Sections of the head of a Drosophila Melanogaster fly are imaged with both broadband illumination sources in the digital lensless holographic microscope.

Color digital lensless holographic microscopy: laser versus LED illumination.

A comparison of the performance of color digital lensless holographic microscopy (CDLHM) as utilized for illumination of RGB lasers or a super-bright white-light LED with a set of spectral filters is presented. As the use of lasers in CDLHM conceals the possibility of having a compact, lightweight, portable, and low cost microscope, and additionally the limited available laser radiation wavelengths limit a real multispectral imaging microscope, here we present the use of super-bright white-light LED and spectral filters for illuminating the sample. The performance of RGB laser-CDLHM and LED-CDLHM is evaluated on imaging a section of the head of a Drosophila melanogaster fly. This comparison shows that there is trade-off between the spatial resolution of the microscope and the light sources utilized, which can be understood with regard to the coherence properties of the illuminating light. Despite the smaller spatial coherence features of LED-CDLHM in comparison with laser-CDLHM, the former shows promise as a portable RGB digital lensless holographic microscope that could be extended to other wavelengths by the use of different spectral filters.

Compact Lens-less Digital Holographic Microscope for MEMS Inspection and Characterization.

A micro-electro-mechanical-system (MEMS) is a widely used component in many industries, including energy, biotechnology, medical, communications, and automotive. However, effective inspection and characterization metrology systems are needed to ensure the functional reliability of MEMS. This study presents a system based on digital holography as a tool for MEMS metrology. Digital holography has gained increasing attention in the past 20 years. With the fast development and decreasing cost of sensor arrays, resolution of such systems has increased broadening potential applications. Thus, it has attracted attention from both research and industry sides as a potential reliable tool for industrial metrology. Indeed, by recording the interference pattern between an object beam (which contains sample height information) and a reference beam on a CCD camera, one can retrieve the quantitative phase information of an object. However, most of digital holographic systems are bulky and thus not easy to implement on industry production lines. The novelty of the system presented is that it is lens-less and thus very compact. In this study, it is shown that the Compact Digital Holographic Microscope (CDHM) can be used to evaluate several characteristics typically consider as criteria in MEMS inspections. The surface profiles of MEMS in both static and dynamic conditions are presented. Comparison with AFM is investigated to validate the accuracy of the CDHM.