Search Results (20)

Imaging of microscopic objects is of fundamental importance, especially in life sciences. Recent fast progress in electronic detection and control, numerical computation, and digital image processing, has been crucial in advancing modern microscopy. Digital holography is a new field in three-dimensional imaging. Digital reconstruction of a hologram offers the remarkable capability to refocus at different depths inside a transparent or semi-transparent object. Thus, this technique is very suitable for biological cell studies in vivo and could have many biomedical and biological applications. A comprehensive review of the research carried out in the area of digital holographic microscopy (DHM) for live-cell imaging is presented. The novel microscopic technique is non-destructive and label-free and offers unmatched imaging capabilities for biological and bio-medical applications. It is also suitable for imaging and modelling of key metabolic processes in living cells, microbial communities or multicellular plant tissues. Live-cell imaging by DHM allows investigation of the dynamic processes underlying the function and morphology of cells. Future applications of DHM can include real-time cell monitoring in response to clinically relevant compounds. The effect of drugs on migration, proliferation, and apoptosis of abnormal cells is an emerging field of this novel microscopic technique. Full article

In-line digital holography is a powerful tool widely used for microscopic object imaging. Usually, in-line and out-line configurations are used to implement holographic systems, but in-line-based set-ups are preferable as they are less sensitive to mechanical vibrations and refraction index variations. However, non-desired blurred conjugate images are superposed to the reconstructed object image by using in-line systems. One strategy to remove the conjugate image contribution is to include a double-sideband filter at the Fourier plane of the system. After using the filter, data obtained at the CCD are processed to retrieve the magnitude and phase (hologram) of the diffracted wavefront while removing the conjugated image. Afterwards, a diffraction integral equation is used to digitally propagate the hologram. Despite the above-mentioned factors, there is not a thorough analysis in the literature of magnification parameters associated with the final reconstructed image, this aspect being crucial for the experimental application of the above-stated approach. Under this scenario, a theoretical analysis of the longitudinal and transverse magnifications of the reconstructed images is provided in this work. The method is validated through the simulation and experimental results of different microscopic objects: glass microspheres, a micrometric reticle, and a resolution test chart USAF 1951. The obtained results provide that the combination of magnification relations with methods for hologram propagation and optimal focused image identification is effective for object position determination. This approach could be useful for 3D microparticle localization and monitoring with optimized magnification within real-time applications. Full article

Permanent engravings on contact lenses provide information about the manufacturing process and lens positioning when they are placed on the eye. The inspection of their morphological characteristics is important, since they can affect the user’s comfort and deposit adhesion. Therefore, an inverted wavefront holoscope (a lensless microscope based on Gabor’s principle of in-line digital holography) is explored for the characterization of the permanent marks of soft contact lenses. The device, based on an in-line transmission configuration, uses a partially coherent laser source to illuminate the soft contact lens placed in a cuvette filled with a saline solution for lens preservation. Holograms were recorded on a digital sensor and reconstructed by back propagation to the image plane based on the angular spectrum method. In addition, a phase-retrieval algorithm was used to enhance the quality of the recovered images. The instrument was experimentally validated through a calibration process in terms of spatial resolution and thickness estimation, showing values that perfectly agree with those that were theoretically expected. Finally, phase maps of different engravings for three commercial soft contact lenses were successfully reconstructed, validating the inverted wavefront holoscope as a potential instrument for the characterization of the permanent marks of soft contact lenses. To improve the final image quality of reconstructions, the geometry of lenses should be considered to avoid induced aberration effects. Full article

This work studies enhancing the capabilities of compact laser spectroscopes integrated into space-exploration rovers by adding 3D topography measurement techniques. Laser spectroscopy enables the in situ analysis of sample composition, aiding in the understanding of the geological history of extraterrestrial bodies. To complement spectroscopic data, the inclusion of 3D imaging is proposed to provide unprecedented contextual information. The morphological information aids material characterization and hence the constraining of rock and mineral histories. Assigning height information to lateral pixels creates topographies, which offer a more complete spatial dataset than contextual 2D imaging. To aid the integration of 3D measurement into future proposals for rover-based laser spectrometers, the relevant scientific, rover, and sample constraints are outlined. The candidate 3D technologies are discussed, and estimates of performance, weight, and power consumptions guide the down-selection process in three application examples. Technology choice is discussed from different perspectives. Inline microscopic fringe-projection profilometry, incoherent digital holography, and multiwavelength digital holography are found to be promising candidates for further development. Full article

With the development of digital holography, the accuracy requirements for the reconstruction phase are becoming increasingly high. The transfer function of the double fast transform (D-FFT) algorithm is distorted when the diffraction distance is larger than the criterion distance ${\mathrm{d}}_{\mathrm{t}}$, which reduces the accuracy of solving the phase. In this paper, the Fresnel diffraction integration algorithm is improved by using the low-pass Tukey window to obtain more accurate reconstructed phases. The improved algorithm is called the D-FFT (Tukey) algorithm. The D-FFT (Tukey) algorithm adjusts the degree of edge smoothing of the Tukey window, using the peak signal-to-noise ratio (PSNR) and the structural similarity (SSIM) to remove the ringing effect and obtain a more accurate reconstructed phase. In a simulation of USAF1951, the longitudinal resolution of the reconstructed phase obtained by D-FFT (Tukey) reached 1.5 $\mathsf{\mu }\mathrm{m}$, which was lower than the 3 $\mathsf{\mu }\mathrm{m}$ obtained by the T-FFT algorithm. The results of Fresnel holography experiments on lung cancer cell slices also demonstrated that the phase quality obtained by the D-FFT (Tukey) algorithm was superior to that of the T-FFT algorithm. D-FFT (Tukey) algorithm has potential applications in phase correction, structured illumination digital holographic microscopy, and microscopic digital holography. Full article

In 3D microsphere tracking, unlike in-plane motion that can be measured directly by a microscope, axial displacements are resolved by optical interference or a diffraction model. As a result, the axial results are affected by the environmental noise. The immunity to environmental noise increases with measurement accuracy and the signal-to-noise ratio (SNR). In compound digital holography microscopy (CDHM)-based measurements, precise identification of the tracking marker is critical to ensuring measurement precision. The reconstruction centering method (RCM) was proposed to suppress the drawbacks caused by installation errors and, at the same time, improve the correct identification of the tracking marker. The reconstructed center is considered to be the center of the microsphere, rather than the center of imaging in conventional digital holographic microscopy. This method was verified by simulation of rays tracing through microspheres and axial moving experiments. The axial displacements of silica microspheres with diameters of 5 μm and 10 μm were tested by CDHM in combination with the RCM. As a result, the SNR of the proposed method was improved by around 30%. In addition, the method was successfully applied to axial displacement measurements of overlapped microspheres with a resolution of 2 nm. Full article

Recently, research on disease diagnosis using red blood cells (RBCs) has been active due to the advantage that it is possible to diagnose many diseases with a drop of blood in a short time. Representatively, there are disease diagnosis technologies that utilize deep learning techniques and digital holographic microscope (DHM) techniques. However, three-dimensional (3D) profile obtained by DHM has a problem of random noise caused by the overlapping DC spectrum and sideband in the Fourier domain, which has the probability of misjudging diseases in deep learning technology. To reduce random noise and obtain a more accurate 3D profile, in this paper, we propose a novel image processing method which randomly selects the center of the high-frequency sideband (RaCoHS) in the Fourier domain. This proposed algorithm has the advantage of filtering while using only recorded hologram information to maintain high-frequency information. We compared and analyzed the conventional filtering method and the general image processing method to verify the effectiveness of the proposed method. In addition, the proposed image processing algorithm can be applied to all digital holography technologies including DHM, and in particular, it is expected to have a great effect on the accuracy of disease diagnosis technologies using DHM. Full article

Though microscopy is most often intended as a technique for providing qualitative assessment of cellular and subcellular properties, when coupled with other instruments such as wavelength selectors, lasers, photoelectric devices and computers, it can perform a wide variety of quantitative measurements, which are demanding in establishing relationships between the properties and structures of biological material in all their spatial and temporal complexities. These combinations of instruments are a powerful approach to improve non-destructive investigations of cellular and subcellular properties (both physical and chemical) at a macromolecular scale resolution. Since many subcellular compartments in living cells are characterized by structurally organized molecules, this review deals with three advanced microscopy techniques well-suited for these kind of investigations, i.e., microspectrophotometry (MSP), super-resolution localization microscopy (SRLM) and holotomographic microscopy (HTM). These techniques can achieve an insight view into the role intracellular molecular organizations such as photoreceptive and photosynthetic structures and lipid bodies play in many cellular processes as well as their biophysical properties. Microspectrophotometry uses a set-up based on the combination of a wide-field microscope and a polychromator, which allows the measurement of spectroscopic features such as absorption spectra. Super resolution localization microscopy combines dedicated optics and sophisticated software algorithms to overcome the diffraction limit of light and allow the visualization of subcellular structures and dynamics in greater detail with respect to conventional optical microscopy. Holotomographic microscopy combines holography and tomography techniques into a single microscopy set-up, and allows 3D reconstruction by means of the phase separation of biomolecule condensates. This review is organized in sections, which for each technique describe some general aspects, a peculiar theoretical aspect, a specific experimental configuration and examples of applications (fish and algae photoreceptors, single labeled proteins and endocellular aggregates of lipids). Full article

The priority of modern dentistry is to keep patients’ teeth for as long as possible. Tooth extraction is a procedure performed as a last resort when conservative methods and endodontic surgery procedures have not brought the expected results. As a consequence, the number of patients in dental offices, who require first and repeated endodontic treatment, is increasing. Thus, the development of new technologies in the medical industry, including microscopy, computer tomography (CT), as well as diode and neodymium-YAG-erbium lasers, enables dentists to increase the percentage of successful treatments. Moreover, mixed reality (MR) is a very new technology, in which the 3D view can help plan or simulate various types of tasks before they will be carried out in real life. In dentistry, 3D holography can be applied to display CT data to plan endodontic treatment. The most important element in effective root canal treatment is the precise imaging of the root canal. The CT scans allow dentists to view the anatomy of the patient’s tooth with much higher precision and understanding than using 2D radiography (RTG-radiographic photo) pictures. Recently, the development of new 3D technologies allows dentists to obtain even more data from existing CT scans. In this paper, the CT scan data were applied to generate patient teeth in 3D and simulate the view of the root canal’s anatomy in MR devices, i.e., Microsoft HoloLens 2. Using DICOM RAW data from the CT exam, we generated a 3D model of the jaw with a tooth. In the next step, the crown of the tooth was removed in a similar way to how a dentist would do this using a dental handpiece. Furthermore, all root canals were cleaned of everything inside. This way we achieved empty tunnels, namely root canals. Finally, we added appropriate lighting, similar to the type of lighting that dentists use. The proposed approach enables to display of the root canals in the same way as during the endodontic procedure using a microscope. It allows for the visualization of the root canal and changing its direction, in which dimensional accuracy is crucial. It turns out that mixed reality can be considered a complementary method to the traditional approach, which reduces the amount of time for the root canal treatment procedure by up to 72.25%, depending on the complexity of the case, and increases its effectiveness. Thus, the mixed reality-based system can be considered an effective tool for planning dental treatment. Full article

We previously developed an incoherent holography technique for use in lattice light sheet (LLS) microscopes that represents a specialized adaptation of light sheet microscopy. Light sheet instruments resolve 3D information by illuminating the sample at 90° to the imaging plane with a sheet of laser light that excites fluorophores in the sample only in a narrow plane. Imaging this plane and then moving it in the imaging z-axis allows construction of the sample volume. Among these types of instruments, LLS microscopy gives higher z-axis resolution and tissue depth penetration. It has a similar working principle to light sheet fluorescence microscopy but uses a lattice configuration of Bessel beams instead of Gaussian beams. Our incoherent light detection technique replaces the glass tube lens of the original LLS with a dual diffractive lens system to retrieve the axial depth of the sample. Here, we show that the system is applicable to all light sheet instruments. To make a direct comparison in the same emission light path, we can imitate the nature of non-Bessel light sheet systems by altering the mask annuli used to create Bessel beams in the LLS system. We change the diffractive mask annuli from a higher NA anulus to a smaller NA anulus. This generates a Gaussian excitation beam similar to conventional light sheet systems. Using this approach, we propose an incoherent light detection system for light sheet 3D imaging by choosing a variable NA and moving only the light sheet while keeping the sample stage and detection microscope objective stationary. Full article

Optical imaging has been utilized in nature and technology for decades. Recently, new methods of optical imaging assisted by computational imaging techniques have been proposed and demonstrated. We describe several new methods of three-dimensional optical imaging, from Fresnel incoherent correlation holography (FINCH) to interferenceless coded aperture correlation holography (COACH). FINCH and COACH are methods for recording digital holograms of a three-dimensional scene. However, COACH can be used for other incoherent and coherent optical applications. The possible applications for these imaging methods, ranging from a new generation of fluorescence microscopes to noninvasive imaging methods through a scattering medium, are mentioned. Full article

Lensless holographic microscope (LHM) is an emerging very promising technology that provides high-quality imaging and analysis of biological samples without utilizing any lens for imaging. Due to its small size and reduced price, LHM can be a very useful tool for the point-of-care diagnosis of diseases, sperm assessment, or microfluidics, among others, not only employed in advanced laboratories but also in poor and/or remote areas. Recently, several LHMs have been reported in the literature. However, complete characterization of their optical parameters remains not much presented yet. Hence, we present a complete analysis of the performance of a compact, reduced cost, and high-resolution LHM. In particular, optical parameters such as lateral and axial resolutions, lateral magnification, and field of view are discussed into detail, comparing the experimental results with the expected theoretical values for different layout configurations. We use high-resolution amplitude and phase test targets and several microbeads to characterize the proposed microscope. This characterization is used to define a balanced and matched setup showing a good compromise between the involved parameters. Finally, such a microscope is utilized for visualization of static, as well as dynamic biosamples. Full article

Stimuli to excitable cells and various cellular processes can cause cell surface deformations; for example, when excitable cell membrane potentials are altered during action potentials. However, these cellular changes may be at or below the diffraction limit (in dendrites the structures measured are as small as 1 µm), and imaging by traditional methods is challenging. Using dual lenses incoherent holography lattice light-sheet (IHLLS-2L) detection with holographic phase imaging of selective fluorescent markers, we can extract the full-field cellular morphology or structural changes of the object’s phase in response to external stimulus. This approach will open many new possibilities in imaging neuronal activity and, overall, in light sheet imaging. In this paper, we present IHLLS-2L as a well-suited technique for quantifying cell membrane deformation in neurons without the actuation of a sample stage or detection microscope objective. Full article

Color digital holography (DH) has been researched in various fields such as the holographic camera and holographic microscope because it acquires a realistic color object wave by measuring both amplitude and phase. Among the methods for color DH, the phase-shifting DH has an advantage of obtaining a signal wave of objects without the autocorrelation and conjugate noises. However, this method usually requires many interferograms to obtain signals for all wavelengths. In addition, the phase-shift algorithm is sensitive to the phase-shift error caused by the instability or hysteresis of the phase shifter. In this paper, we propose a new method of color phase-shifting digital holography with monitoring the phase-shift. The color interferograms are recorded by using a focal plane array (FPA) with a Bayer color filter. In order to obtain the color signal wave from the interferograms with unexpected phase-shift values, we devise a generalized phase-shifting DH algorithm. The proposed method enables the robust measurement in the interferograms. Experimentally, we demonstrate the proposed algorithm to reconstruct the object image with negligibly small conjugate noises. Full article

The nanoscale magnetic configuration of self-assembled groups of magnetite 40 nm cubic nanoparticles has been investigated by means of electron holography in the transmission electron microscope (TEM). The arrangement of the cubes in the form of chains driven by the alignment of their dipoles of single nanocubes is assessed by the measured in-plane magnetic induction maps, in good agreement with theoretical calculations. Full article