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Current Opinion in Structural Biology Jun 2024Although defocus can be used to generate partial phase contrast in transmission electron microscope images, cryo-electron microscopy (cryo-EM) can be further improved by... (Review)
Review
Although defocus can be used to generate partial phase contrast in transmission electron microscope images, cryo-electron microscopy (cryo-EM) can be further improved by the development of phase plates which increase contrast by applying a phase shift to the unscattered part of the electron beam. Many approaches have been investigated, including the ponderomotive interaction between light and electrons. We review the recent successes achieved with this method in high-resolution, single-particle cryo-EM. We also review the status of using pulsed or near-field enhanced laser light as alternatives, along with approaches that use scanning transmission electron microscopy (STEM) with a segmented detector rather than a phase plate.
Topics: Cryoelectron Microscopy; Microscopy, Phase-Contrast
PubMed: 38531188
DOI: 10.1016/j.sbi.2024.102805 -
Optics Letters Jul 2023Quantitative differential phase-contrast (DPC) microscopy produces phase images of transparent objects based on a number of intensity images. To reconstruct the phase,...
Quantitative differential phase-contrast (DPC) microscopy produces phase images of transparent objects based on a number of intensity images. To reconstruct the phase, in DPC microscopy, a linearized model for weakly scattering objects is considered; this limits the range of objects to be imaged, and requires additional measurements and complicated algorithms to correct for system aberrations. Here, we present a self-calibrated DPC microscope using an untrained neural network (UNN), which incorporates the nonlinear image formation model. Our method alleviates the restrictions on the object to be imaged and simultaneously reconstructs the complex object information and aberrations, without any training dataset. We demonstrate the viability of UNN-DPC microscopy through both numerical simulations and LED microscope-based experiments.
Topics: Microscopy, Phase-Contrast; Deep Learning; Algorithms; Neural Networks, Computer
PubMed: 37390192
DOI: 10.1364/OL.493391 -
Applied Optics May 2022Quantitative phase microscopy (QPM) is a label-free microscopic technique that exploits the phase of a wave passing through a sample; hence, it has been applied to many...
Quantitative phase microscopy (QPM) is a label-free microscopic technique that exploits the phase of a wave passing through a sample; hence, it has been applied to many fields, including biomedical research and industrial inspection. However, the high spatiotemporal resolution imaging of reflective samples still challenges conventional transmission QPM. In this paper, we propose reflectional quantitative phase-contrast microscopy based on annular epi-illumination of light-emitting diodes. The unscattered wave from the sample is successively phase-retarded by 0, /2, , and 3/2 through a spatial light modulator, and high-resolution phase-contrast images are obtained, revealing the finer structure or three-dimensional tomography of reflective samples. With this system, we have quantitatively obtained the contour of tissue slices and silicon semiconductor wafers. We believe that the proposed system will be very helpful for the high-resolution imaging of industrial devices and biomedical dynamics.
Topics: Microscopy, Phase-Contrast; Silicon; Lighting; Microscopy; Semiconductors
PubMed: 36256403
DOI: 10.1364/AO.451761 -
Methods in Molecular Biology (Clifton,... 2014Here, I review the principles and applications of phase-contrast electron microscopy using phase plates. First, I develop the principle of phase contrast based on a... (Review)
Review
Here, I review the principles and applications of phase-contrast electron microscopy using phase plates. First, I develop the principle of phase contrast based on a minimal model of microscopy, introducing a double Fourier-transform process to mathematically formulate the image formation. Next, I explain four phase-contrast (PC) schemes, defocus PC, Zernike PC, Hilbert differential contrast, and schlieren optics, as image-filtering processes in the context of the minimal model, with particular emphases on the Zernike PC and corresponding Zernike phase plates. Finally, I review applications of Zernike PC cryo-electron microscopy to biological systems such as protein molecules, virus particles, and cells, including single-particle analysis to delineate three-dimensional (3D) structures of protein and virus particles and cryo-electron tomography to reconstruct 3D images of complex protein systems and cells.
Topics: Cryoelectron Microscopy; Microscopy, Phase-Contrast
PubMed: 24357373
DOI: 10.1007/978-1-62703-776-1_18 -
Journal of Biophotonics Dec 2021We present a robust, low-cost single-shot implementation of differential phase microscopy utilising a polarisation-sensitive camera to simultaneously acquire four images...
We present a robust, low-cost single-shot implementation of differential phase microscopy utilising a polarisation-sensitive camera to simultaneously acquire four images from which phase contrast images can be calculated. This polarisation-resolved differential phase contrast (pDPC) microscopy technique can be easily integrated with fluorescence microscopy.
Topics: Microscopy; Microscopy, Phase-Contrast
PubMed: 34390220
DOI: 10.1002/jbio.202100144 -
Philosophical Transactions of the Royal... Jun 2008Phase contrast transmission electron microscopy (TEM) based on thin-film phase plates has been developed and applied to biological systems. Currently, development is... (Review)
Review
Phase contrast transmission electron microscopy (TEM) based on thin-film phase plates has been developed and applied to biological systems. Currently, development is focused on two techniques that employ two different types of phase plates. The first technique uses a Zernike phase plate, which is made of a uniform amorphous carbon film that completely covers the aperture of an objective lens and can retard the phase of electron waves by pi/2, except at the centre where a tiny hole is drilled. The other technique uses a Hilbert phase plate, which is made of an amorphous carbon film that is twice as thick as the Zernike phase plate, covers only half of the aperture and retards the electron wave phase by pi. By combining the power of efficient phase contrast detection with the accurate preservation achieved by a cryotechnique such as vitrification, macromolecular complexes and supermolecular structures inside intact bacterial or eukaryotic cells may be visualized without staining. Phase contrast cryo-TEM has the potential to bridge the gap between cellular and molecular biology in terms of high-resolution visualization. Examples using proteins, viruses, cyanobacteria and somatic cells are provided.
Topics: Cells; Cryoelectron Microscopy; Microscopy, Electron, Transmission; Microscopy, Phase-Contrast; Proteins; Viruses
PubMed: 18339604
DOI: 10.1098/rstb.2008.2268 -
Scientific Reports Mar 2017We present a new approach for retrieving halo-free phase contrast microscopy (hfPC) images by upgrading the conventional PC microscope with an external interferometric...
We present a new approach for retrieving halo-free phase contrast microscopy (hfPC) images by upgrading the conventional PC microscope with an external interferometric module, which generates sufficient data for reversing the halo artifact. Acquiring four independent intensity images, our approach first measures haloed phase maps of the sample. We solve for the halo-free sample transmission function by using a physical model of the image formation under partial spatial coherence. Using this halo-free sample transmission, we can numerically generate artifact-free PC images. Furthermore, this transmission can be further used to obtain quantitative information about the sample, e.g., the thickness with known refractive indices, dry mass of live cells during their cycles. We tested our hfPC method on various control samples, e.g., beads, pillars and validated its potential for biological investigation by imaging live HeLa cells, red blood cells, and neurons.
Topics: Artifacts; Erythrocytes; HeLa Cells; Humans; Image Processing, Computer-Assisted; Microscopy, Phase-Contrast; Models, Theoretical; Neurons; Refractometry
PubMed: 28338086
DOI: 10.1038/srep44034 -
Journal of Biophotonics Aug 2018Quantitative phase imaging (QPI) has been investigated to retrieve optical phase information of an object and applied to biological microscopy and related medical...
Quantitative phase imaging (QPI) has been investigated to retrieve optical phase information of an object and applied to biological microscopy and related medical studies. In recent examples, differential phase contrast (DPC) microscopy can recover phase image of thin sample under multi-axis intensity measurements in wide-field scheme. Unlike conventional DPC, based on theoretical approach under partially coherent condition, we propose a new method to achieve isotropic differential phase contrast (iDPC) with high accuracy and stability for phase recovery in simple and high-speed fashion. The iDPC is simply implemented with a partially coherent microscopy and a programmable thin-film transistor (TFT) shield to digitally modulate structured illumination patterns for QPI. In this article, simulation results show consistency of our theoretical approach for iDPC under partial coherence. In addition, we further demonstrate experiments of quantitative phase images of a standard micro-lens array, as well as label-free live human cell samples.
Topics: Algorithms; Animals; Cell Line, Tumor; Image Processing, Computer-Assisted; Lenses; Light; Mice; Microscopy, Phase-Contrast
PubMed: 29770615
DOI: 10.1002/jbio.201700364 -
Journal of Biomedical Optics May 2022Quantitative differential phase contrast (qDPC) microscopy enhances phase contrast by asymmetric illumination using partially coherent light and multiple intensity...
SIGNIFICANCE
Quantitative differential phase contrast (qDPC) microscopy enhances phase contrast by asymmetric illumination using partially coherent light and multiple intensity measurements. However, for live cell imaging, motion artifacts and image acquisition time are important issues. For live cell imaging, a large number of intensity measurements can limit the imaging quality and speed. The minimum number of intensity measurements in qDPC can greatly enhance performance for live imaging.
AIM
To obtain high-contrast, isotropic qDPC images with two intensity measurements and perform time-lapse imaging of biological samples.
APPROACH
Based on the color-coded design, a dual-color linear-gradient pupil is proposed to achieve isotropic phase contrast response with two intensity measurements. In our method, the purpose of designing a dual-color coded pupil is twofold: first, to obtain a linear amplitude gradient for asymmetric illumination, which is required to get a circular symmetry of transfer function, and second, to reduce the required number of frames for phase retrieval.
RESULTS
To demonstrate the imaging performance of our system, standard microlens arrays were used as samples. We performed time-lapse quantitative phase imaging of rat astrocytes under a low-oxygen environment. Detailed morphology and dynamic changes such as the apoptosis process and migration of cells were observed.
CONCLUSIONS
It is shown that dual-color linear-gradient pupils in qDPC can outperform half-circle and vortex pupils, and isotropic phase transfer function can be achieved with only two-axis measurements. The reduced number of frames helps in achieving faster imaging speed as compared to the typical qDPC system. The imaging performance of our system is evaluated by time-lapse imaging of rat astrocytes. Different morphological changes in cells during their life cycle were observed in terms of quantitative phase change values.
Topics: Animals; Lighting; Microscopy, Phase-Contrast; Rats; Time-Lapse Imaging
PubMed: 35578382
DOI: 10.1117/1.JBO.27.5.056002 -
Nature Aug 1948
Topics: Microscopy, Phase-Contrast; Viruses
PubMed: 18877096
DOI: 10.1038/162251a0