Digital Lensless Holographic Microscopy Research Articles

Quantitative phase imaging verification in large field-of-view lensless holographic microscopy via two-photon 3D printing

Large field-of-view (FOV) microscopic imaging (over 100 mm2) with high lateral resolution (1–2 μm) plays a pivotal role in biomedicine and biophotonics, especially within the label-free regime. Lensless digital holographic microscopy (LDHM) is promising in this context but ensuring accurate quantitative phase imaging (QPI) in large FOV LDHM is challenging. While phantoms, 3D printed by two-photon polymerization (TPP), have facilitated testing small FOV lens-based QPI systems, an equivalent evaluation for lensless techniques remains elusive, compounded by issues such as twin-image and beam distortions, particularly towards the detector’s edges. Here, we propose an application of TPP over large area to examine phase consistency in LDHM. Our research involves fabricating widefield phase test targets with galvo and piezo scanning, scrutinizing them under single-shot twin-image corrupted conditions and multi-frame iterative twin-image minimization scenarios. By measuring the structures near the detector’s edges, we verified LDHM phase imaging errors across the entire FOV, with less than 12% phase value difference between areas. Our findings indicate that TPP, followed by LDHM and Linnik interferometry cross-verification, requires new design considerations for precise large-area photonic manufacturing. This research paves the way for quantitative benchmarking of large FOV lensless phase imaging, enhancing understanding and further development of LDHM technique.

Averaging fractional Fourier domains for background noise removal applied to digital lensless holographic microscopy

A background noise removal method based on averaging fractional Fourier domains is presented. The method is applied to Digital Lensless Holographic Microscopy (DLHM) intensity reconstructions, where the background is perturbed by the weak yet detrimental presence of information of the twin image. A set of fractional Fourier domains of a DLHM intensity reconstruction is computed and thereafter averaged leading to a sensible reduction of the background noise and, therefore, an increase in the overall contrast of the resulting image. The maximum reach of the fractional rotations used in the method is determined by measuring the spatial resolution in a regular star test target such that the spatial resolution is kept within the span of interest for a given application. The set of images to be averaged is composed of fractional rotations of the original intensity reconstruction that are smaller than the previously determined maximum reach. The number of fractional rotations that are finally averaged is determined by the sought increase in the contrast of the resulting image. Experimental samples of micrometer-sized objects and an intricate biological specimen have been used to validate the proposal.

Off-axis digital lensless holographic microscopy based on spatially multiplexed interferometry.

Digital holographic microscopy (DHM) is a label-free microscopy technique that provides time-resolved quantitative phase imaging (QPI) by measuring the optical path delay of light induced by transparent biological samples. DHM has been utilized for various biomedical applications, such as cancer research and sperm cell assessment, as well as for in vitro drug or toxicity testing. Its lensless version, digital lensless holographic microscopy (DLHM), is an emerging technology that offers size-reduced, lightweight, and cost-effective imaging systems. These features make DLHM applicable, for example, in limited resource laboratories, remote areas, and point-of-care applications. In addition to the abovementioned advantages, in-line arrangements for DLHM also include the limitation of the twin-image presence, which can restrict accurate QPI. We therefore propose a compact lensless common-path interferometric off-axis approach that is capable of quantitative imaging of fast-moving biological specimens, such as living cells in flow. We suggest lensless spatially multiplexed interferometric microscopy (LESSMIM) as a lens-free variant of the previously reported spatially multiplexed interferometric microscopy (SMIM) concept. LESSMIM comprises a common-path interferometric architecture that is based on a single diffraction grating to achieve digital off-axis holography. From a series of single-shot off-axis holograms, twin-image free and time-resolved QPI is achieved by commonly used methods for Fourier filtering-based reconstruction, aberration compensation, and numerical propagation. Initially, the LESSMIM concept is experimentally demonstrated by results from a resolution test chart and investigations on temporal stability. Then, the accuracy of QPI and capabilities for imaging of living adherent cell cultures is characterized. Finally, utilizing a microfluidic channel, the cytometry of suspended cells in flow is evaluated. LESSMIM overcomes several limitations of in-line DLHM and provides fast time-resolved QPI in a compact optical arrangement. In summary, LESSMIM represents a promising technique with potential biomedical applications for fast imaging such as in imaging flow cytometry or sperm cell analysis.

Multi-view occlusion removal in digital lensless holographic microscopy

Multi-view occlusion removal in digital lensless holographic microscopy

Phase retrieval via conjugate gradient minimization in double-plane lensless holographic microscopy.

Optimization-based phase retrieval method for digital lensless holographic microscopy in the double-plane recording configuration is proposed. In our method the phase retrieval is framed as an optimization problem that can be efficiently and rigorously tackled with gradient decent tools. This is done with the conjugate gradient method that possesses excellent theoretical features such as global and fast convergence (compared to steepest descent) and relatively low computational cost (compared to second order optimizers). The proposed method is extensively tested with simulations and experimental measurements that show superiority of our method over the Gerchberg-Saxton algorithm, especially in terms of reconstruction of problematic low frequency components of viable phase information.

Miniaturized quantitative detection of particles in transformer oil based on lensless holographic microscopy

The insulation effectiveness of transformer oil has a substantial impact on the safety of transformers. In the presence of particles within the transformer oil, they are able to adversely affect its performance, potentially originating from electrical arcs or severe carbonization processes. These particles are commonly capable of reducing the insulating capability of the transformer oil, thereby enhancing the risk of transformer failure. To overcome this dilemma, we propose a miniaturized quantitative particle detection approach in transformer oil based on a lensless digital holographic microscope. In general, methods based on traditional high-resolution optical microscopy are essentially limited by their fairly huge cost and importability. However, the proposed approach utilizes a homemade miniaturized model combined with a CMOS image sensor to achieve microscopic imaging. The deployed device here weighs ∼ 50 g, making it portable and affordable for on-the-go detection. By simplifying the microscope system, we can quickly and accurately detect tiny particles in transformer oils. This enables us to efficiently assess the status of impurities in power transformers and to identify potential issues in advance, thereby enhancing the reliability of transformer oil. This detection method not only features simplicity of operation, convenient on-the-go detection, low cost, and high precision but also provides a convenient approach for accurately assessing the condition of impurity particles in power transformers and preemptively identifying potential issues.

Illumination system contributing zooming function to lensless digital holographic microscope by using lightguide incorporated with volume holographic optical elements

Illumination systems play a crucial role in determining the image quality of lens- less images. In this paper, we propose an illumination system that uses a light guide with a volume holographic optical element (VHOE) and apply it to a lensless digital holographic microscope (DHM). The light guide has an in-coupler that is maturely developed for AR/MR applications. A VHOE serves as an in-coupler and directs the laser light into the guiding mode. Unlike the conventional light guide in the AR/MR application, the out-coupler is changed to a functional VHOE to diffract a divergent spherical wave. Through clever arrangement, the interference fringe caught by the CMOS image sensor can be used to retrieve the tested image. By controlling the focusing point of the diffracted spherical wave, the field of view (FOV) and the resolution can be adjusted. We have demonstrated the use of different VHOEs to perform different FOV and image resolutions. Thus, a zoomable-compact lensless DHM with holographic light guide technology is achievable.

Comparative analysis of digital holographic microscopy and digital lensless holographic microscopy for quantitative phase imaging

This study provides a detailed comparison of two widely used quantitative phase imaging (QPI) techniques: single-shot off-axis digital holographic microscopy (DHM) and digital lensless holographic microscopy (DLHM). The primary aim is to evaluate and contrast critical aspects of their imaging performance, including spatial phase sensitivity, phase measurement accuracy, and spatial lateral resolution. Employing typical configurations for both DHM and DLHM, the study utilizes a customized phase test target featuring linear phase changes introduced by a specially designed linear density attenuation filter. Ground truth data from an atomic force microscope is incorporated to validate the experimental findings. The comparative analysis reveals that DHM and DLHM exhibit nearly identical spatial phase sensitivity, with DHM demonstrating a minimal 3.2% measurement error compared to DLHM's 4% in height measurement accuracy. Notably, DHM achieves a finer spatial lateral resolution down to 3.1 µm, surpassing DLHM's 5.52 µm. While DHM outperforms DLHM in precision and resolution, the latter offers advantages in terms of portability and cost-effectiveness. These findings provide valuable insights for researchers and practitioners, aiding in the informed selection of QPI methods based on specific application requirements.

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 654nm, 510nm, and 405nm; the second group, named single-wavelength dataset, is composed of 3600 recordings and was acquired using a 633nm 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.

Thickness and surface profiling of optically transparent and reflecting samples using lens-less self-referencing digital holographic microscopy

Thickness and surface profiling of transparent/semi-transparent specimens are vital in various applications, including electronics, optics, healthcare, and biotechnology. Surface profiling techniques characterize and analyze surface thickness, morphology, and roughness. Developing easy-to-use, single-shot, wide field-of-view techniques that provide nanometer level surface thickness and profiling is vital for these applications. Digital holography is a state-of-the-art technique that provides the quantitative phase images of transparent objects, from which their thickness profiles could be extracted and used for surface profiling. It has the added advantage of numerical focusing. The present manuscript details the development of a compact wide field of view, self-referencing, lens-less digital holographic microscope for surface profiling of transparent/semi-transparent samples in transmission and reflection mode. The developed microscope requires only a glass plate to generate holograms and can be used to study the dynamics of the surfaces also. It provides a field of view of 3.2mm x 2.5 mm along with a thickness measurement resolution of 2.8 nm and temporal stability of 1.1 nm over a period of 120 s. The developed microscope was tested by measuring the thickness of GeSe semiconductor thin films grown on glass substrates and comparing it with AFM measurements. The microscope was then used to quantify spatially varying thickness profiles of overlapped thin films, junction formed by heterogenous compounds and metal thin films. The microscope was also tested for dynamic studies of surface profiles by thermally loading ink markings on glass slides.

Single-shot experimental-numerical twin-image removal in lensless digital holographic microscopy

Lensless digital holographic microscopy (LDHM) offers very large field of view label-free imaging crucial, e.g., in high-throughput particle tracking and biomedical examination of cells and tissues. Compact layouts promote point-of-case and out-of-laboratory applications. The LDHM, based on the Gabor in-line holographic principle, is inherently spoiled by the twin-image effect, which complicates the quantitative analysis of reconstructed phase and amplitude maps. Popular family of solutions consists of numerical methods, which tend to minimize twin-image upon iterative process based on data redundancy. Additional hologram recordings are needed, and final results heavily depend on the algorithmic parameters, however. In this contribution we present a novel single-shot experimental-numerical twin-image removal technique for LDHM. It leverages two-source off-axis hologram recording deploying simple fiber splitter. Additionally, we introduce a novel phase retrieval numerical algorithm specifically tailored to the acquired holograms, that provides twin-image-free reconstruction without compromising the resolution. We quantitatively and qualitatively verify proposed method employing phase test target and cheek cells biosample. The results demonstrate that the proposed technique enables low-cost compact LDHM imaging with enhanced precision, achieved through the elimination of twin-image errors. This advancement opens new avenues for more accurate technical and biomedical imaging applications using LDHM, particularly in scenarios where cost-effective and portable imaging solutions are desired.

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.

FocusNET: An autofocusing learning‐based model for digital lensless holographic microscopy

This paper reports on a convolutional neural network (CNN) – based regression model, called FocusNET, to predict the accurate reconstruction distance of raw holograms in Digital Lensless Holographic Microscopy (DLHM). This proposal provides a physical-mathematical formulation to extend its use to different DLHM setups than the optical and geometrical conditions utilized for recording the training dataset; this unique feature is tested by applying the proposal to holograms of diverse samples recorded with different DLHM setups. Additionally, a comparison between FocusNET and conventional autofocusing methods in terms of processing times and accuracy is provided. Although the proposed method predicts reconstruction distances with approximately 54 µm standard deviation, accurate information about the samples in the validation dataset is still retrieved. When compared to a method that utilizes a stack of reconstructions to find the best focal plane, FocusNET performs 600 times faster, as no hologram reconstruction is needed. When implemented in batches, the network can achieve up to a 1200-fold reduction in processing time, depending on the number of holograms to be processed. The training and validation datasets, and the code implementations, are hosted on a public GitHub repository that can be freely accessed.

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.

Experimental optimization of lensless digital holographic microscopy with rotating diffuser-based coherent noise reduction.

Laser-based lensless digital holographic microscopy (LDHM) is often spoiled by considerable coherent noise factor. We propose a novel LDHM method with significantly limited coherent artifacts, e.g., speckle noise and parasitic interference fringes. It is achieved by incorporating a rotating diffuser, which introduces partial spatial coherence and preserves high temporal coherence of laser light, crucial for credible in-line hologram reconstruction. We present the first implementation of the classical rotating diffuser concept in LDHM, significantly increasing the signal-to-noise ratio while preserving the straightforwardness and compactness of the LDHM imaging device. Prior to the introduction of the rotating diffusor, we performed LDHM experimental hardware optimization employing 4 light sources, 4 cameras, and 3 different optical magnifications (camera-sample distances). It was guided by the quantitative assessment of numerical amplitude/phase reconstruction of test targets, conducted upon standard deviation calculation (noise factor quantification), and resolution evaluation (information throughput quantification). Optimized rotating diffuser LDHM (RD-LDHM) method was successfully corroborated in technical test target imaging and examination of challenging biomedical sample (60 µm thick mouse brain tissue slice). Physical minimization of coherent noise (up to 50%) was positively verified, while preserving optimal spatial resolution of phase and amplitude imaging. Coherent noise removal, ensured by proposed RD-LDHM method, is especially important in biomedical inference, as speckles can falsely imitate valid biological features. Combining this favorable outcome with large field-of-view imaging can promote the use of reported RD-LDHM technique in high-throughput stain-free biomedical screening.

Low-intensity illumination for lensless digital holographic microscopy with minimized sample interaction.

Exposure to laser light alters cell culture examination via optical microscopic imaging techniques based on label-free coherent digital holography. To mitigate this detrimental feature, researchers tend to use a broader spectrum and lower intensity of illumination, which can decrease the quality of holographic imaging due to lower resolution and higher noise. We study the lensless digital holographic microscopy (LDHM) ability to operate in the low photon budget (LPB) regime to enable imaging of unimpaired live cells with minimized sample interaction. Low-cost off-the-shelf components are used, promoting the usability of such a straightforward approach. We show that recording data in the LPB regime (down to 7 µW of illumination power) does not limit the contrast or resolution of the hologram phase and amplitude reconstruction compared to regular illumination. The LPB generates hardware camera shot noise, however, to be effectively minimized via numerical denoising. The ability to obtain high-quality, high-resolution optical complex field reconstruction was confirmed using the USAF 1951 amplitude sample, phase resolution test target, and finally, live glial restricted progenitor cells (as a challenging strongly absorbing and scattering biomedical sample). The proposed approach based on severely limiting the photon budget in lensless holographic microscopy method can open new avenues in high-throughout (optimal resolution, large field-of-view, and high signal-to-noise-ratio single-hologram reconstruction) cell culture imaging with minimized sample interaction.

Holographic point source for digital lensless holographic microscopy.

A holographic point source (HPS) developed for digital lensless holographic microscopy (HPS-DLHM) is presented. The HPS is an off-axis phase transmission hologram of an experimental micrometer pinhole recorded on a photopolymer holographic film. An amplitude division interferometer, adjusted to operate at maximum diffraction efficiency, has been employed to record the hologram. The results of HPS-DLHM have been contrasted with the results obtained via conventional DLHM, and the two techniques were found to give similar measurements. Compared with conventional pinhole-based DLHM illumination, our cost-effective proposal provides increased mechanical stability, the possibility of wider spherical illumination cones, and shorter reconstruction distances. These superior features pave the way to applying this quantitative phase imaging (QPI) technique in biomedical and telemedicine applications. The imaging capabilities of our HPS-DLHM proposal have been tested by using an intricate sample of a honeybee leg, a low-absorption sample of epithelial cheek cells, a 1951 USAF test target, and smeared human erythrocytes.

Resolution and Contrast Enhancement for Lensless Digital Holographic Microscopy and Its Application in Biomedicine

An important imaging technique in biomedicine, the conventional optical microscopy relies on relatively complicated and bulky lens and alignment mechanics. Based on the Gabor holography, the lensless digital holographic microscopy has the advantages of light weight and low cost. It has developed rapidly and received attention in many fields. However, the finite pixel size at the sensor plane limits the spatial resolution. In this study, we first review the principle of lensless digital holography, then go over some methods to improve image contrast and discuss the methods to enhance the image resolution of the lensless holographic image. Moreover, the applications of lensless digital holographic microscopy in biomedicine are reviewed. Finally, we look forward to the future development and prospect of lensless digital holographic technology.

Hologram Correction in Digital Lensless Holographic Microscopy by Sample Plane Scanning

Hologram Correction in Digital Lensless Holographic Microscopy by Sample Plane Scanning

Linear diattenuation imaging of biological samples with digital lensless holographic microscopy.

A digital lensless holographic microscope (DLHM) sensitive to the linear diattenuation produced by biological samples is reported. The insertion of a linear polarization-states generator and a linear polarization-states analyzer in a typical DLHM setup allows the proper linear diattenuation imaging of microscopic samples. The proposal has been validated for simulated and experimental biological samples containing calcium oxalate crystals extracted from agave leaves and potato starch grains. The performance of the proposed method is similar to that of a traditional polarimetric microscope to obtain linear diattenuation images of microscopic samples but with the advantages of DLHM, such as numerical refocusing, cost effectiveness, and the possibility of field-portable implementation.