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Microscopy (Oxford, England) Apr 2022Differential phase contrast (DPC) scanning transmission electron microscopy can directly visualize electromagnetic fields inside a specimen. However, their image...
Differential phase contrast (DPC) scanning transmission electron microscopy can directly visualize electromagnetic fields inside a specimen. However, their image contrast is not only sensitive to the electromagnetic fields in the sample, but also the changes in diffraction conditions such as sample bends or thickness changes. These additional contrasts are called diffraction contrasts, and sometimes make it difficult to extract pure electromagnetic field information from the experimental DPC images. In this study, we developed a beam scan system that can acquire many DPC images from the same sample region with arbitrarily varying incident beam tilt angles to the sample. Then, these images are precisely averaged to form tilt-scan averaged DPC images. It is shown that the diffraction contrast can be effectively reduced in the tilt-scan averaged DPC images.
Topics: Microscopy, Electron, Scanning Transmission; Microscopy, Phase-Contrast
PubMed: 35032164
DOI: 10.1093/jmicro/dfac002 -
Optics Letters May 2005Broadband interferometry is an attractive technique for the detection of cellular motions because it provides depth-resolved phase information via coherence gating. We...
Broadband interferometry is an attractive technique for the detection of cellular motions because it provides depth-resolved phase information via coherence gating. We present a phase-sensitive technique called spectral-domain phase microscopy (SDPM). SDPM is a functional extension of spectral-domain optical coherence tomography that allows for the detection of nanometer-scale motions in living cells. The sensitivity of the technique is demonstrated, and its calibration is verified. A shot-noise limit to the displacement sensitivity of this technique is derived. Measurement of cellular dynamics was performed on spontaneously beating cardiomyocytes isolated from chick embryos.
Topics: Animals; Cell Movement; Cells, Cultured; Chick Embryo; Equipment Design; Equipment Failure Analysis; Microscopy, Interference; Microscopy, Phase-Contrast; Myocardial Contraction; Myocytes, Cardiac; Tomography, Optical Coherence
PubMed: 15945141
DOI: 10.1364/ol.30.001162 -
Analytical Chemistry Nov 2021Vibrational microscopy methods based on Raman scattering or infrared absorption provide a label-free approach for chemical-contrast imaging, but employ point-by-point...
Vibrational microscopy methods based on Raman scattering or infrared absorption provide a label-free approach for chemical-contrast imaging, but employ point-by-point scanning and impose a compromise between the imaging speed and field-of-view (FOV). Optothermal microscopy has been proposed as a promising imaging modality to avoid this compromise, although at restrictively small FOVs capable of imaging only few cells. Here, we present wide-field optothermal mid-infrared microscopy (WOMiM) for wide-field chemical-contrast imaging based on snapshot pump-probe detection of optothermal signal, using a custom-made condenser-free phase contrast microscopy to capture the phase change of samples after mid-infrared irradiation. We achieved chemical contrast for FOVs up to 180 μm in diameter, yielding 10-fold larger imaging areas than the state-of-the-art, at imaging speeds of 1 ms/frame. The maximum possible imaging speed of WOMiM was determined by the relaxation time of optothermal heat, measured to be 32.8 μs in water, corresponding to a frame rate of ∼30 kHz. This proof-of-concept demonstrates that vibrational imaging can be achieved at an unprecedented imaging speed and large FOV with the potential to significantly facilitate label-free imaging of cellular dynamics.
Topics: Hyperspectral Imaging; Microscopy; Microscopy, Phase-Contrast; Spectrum Analysis, Raman; Vibration
PubMed: 34766751
DOI: 10.1021/acs.analchem.1c02805 -
Scientific Reports 2013Cell imaging often relies on synthetic or genetic fluorescent labels, to provide contrast which can be far from ideal for imaging cells in their in vivo state. We report...
Cell imaging often relies on synthetic or genetic fluorescent labels, to provide contrast which can be far from ideal for imaging cells in their in vivo state. We report on the biological application of a, label-free, high contrast microscopy technique known as ptychography, in which the image producing step is transferred from the microscope lens to a high-speed phase retrieval algorithm. We demonstrate that this technology is appropriate for label-free imaging of adherent cells and is particularly suitable for reporting cellular changes such as mitosis, apoptosis and cell differentiation. The high contrast, artefact-free, focus-free information rich images allow dividing cells to be distinguished from non-dividing cells by a greater than two-fold increase in cell contrast, and we demonstrate this technique is suitable for downstream automated cell segmentation and analysis.
Topics: Animals; Cells, Cultured; Equipment Design; Equipment Failure Analysis; Humans; Image Enhancement; Lighting; Microscopy, Phase-Contrast; Reproducibility of Results; Sensitivity and Specificity
PubMed: 23917865
DOI: 10.1038/srep02369 -
Optics Express Dec 2011We report on a compact twin-beam interferometer that can be adopted as a flexible diagnostic tool in microfluidic platforms with twofold functionality. The novel...
We report on a compact twin-beam interferometer that can be adopted as a flexible diagnostic tool in microfluidic platforms with twofold functionality. The novel configuration allows 3D tracking of micro-particles and, at same time, can simultaneously furnish Quantitative Phase-contrast maps of tracked micro-objects by interference microscopy, without changing the configuration. Experimental demonstration is given on for in vitro cells in a microfluidic environment.
Topics: Equipment Design; Equipment Failure Analysis; Holography; Image Enhancement; Lasers; Microfluidic Analytical Techniques; Microscopy, Phase-Contrast; Signal Processing, Computer-Assisted
PubMed: 22273976
DOI: 10.1364/OE.19.025833 -
Journal of Biomedical Optics Feb 2024The imaging depth of microscopy techniques is limited by the ability of light to penetrate biological tissue. Recent research has addressed this limitation by combining...
SIGNIFICANCE
The imaging depth of microscopy techniques is limited by the ability of light to penetrate biological tissue. Recent research has addressed this limitation by combining a reflectance confocal microscope with the NIR-II (or shortwave infrared) spectrum. This approach offers significant imaging depth, is straightforward in design, and remains cost-effective. However, the imaging system, which relies on intrinsic signals, could benefit from adjustments in its optical design and post-processing methods to differentiate cortical cells, such as neurons and small blood vessels.
AIM
We implemented a phase contrast detection scheme to a reflectance confocal microscope using NIR-II spectral range as illumination.
APPROACH
We analyzed the features retrieved in the images while testing the imaging depth. Moreover, we introduce an acquisition method for distinguishing dynamic signals from the background, allowing the creation of vascular maps similar to those produced by optical coherence tomography.
RESULTS
The phase contrast implementation is successful to retrieve deep images in the cortex up to using a cranial window. Vascular maps were retrieved at similar cortical depth and the possibility of combining multiple images can provide a vessel network.
CONCLUSIONS
Phase contrast reflectance confocal microscopy can improve the outlining of cortical cell bodies. With the presented framework, angiograms can be retrieved from the dynamic signal in the biological tissue. Our work presents an optical implementation and analysis techniques from a former microscope design.
Topics: Microscopy; Tomography, Optical Coherence; Microscopy, Phase-Contrast; Neuroimaging; Microscopy, Confocal
PubMed: 38414657
DOI: 10.1117/1.JBO.29.2.026501 -
Scientific Reports Sep 2019Phase imaging techniques are an invaluable tool in microscopy for quickly examining thin transparent specimens. Existing methods are limited to either simple and...
Phase imaging techniques are an invaluable tool in microscopy for quickly examining thin transparent specimens. Existing methods are limited to either simple and inexpensive methods that produce only qualitative phase information (e.g. phase contrast microscopy, DIC), or significantly more elaborate and expensive quantitative methods. Here we demonstrate a low-cost, easy to implement microscopy setup for quantitative imaging of phase and bright field amplitude using collimated white light illumination.
Topics: Image Processing, Computer-Assisted; Light; Microscopy, Interference; Microscopy, Phase-Contrast
PubMed: 31551461
DOI: 10.1038/s41598-019-50264-3 -
Applied Microbiology Sep 1969The three-dimensional immages of free and intrasporangial spores produced by scanning electron microscopy show surface structures not visible by phase-contrast... (Comparative Study)
Comparative Study
The three-dimensional immages of free and intrasporangial spores produced by scanning electron microscopy show surface structures not visible by phase-contrast microscopy. Although fine surface detail is not elucidated by scanning electron microscopy, this technique does afford a definitive picture of the general shape of spores. Spores of Bacillus popilliae, B. lentimorbus, B. thuringiensis, B. alvei, B. cereus, and Sarcina ureae have varying patterns of surface ridge formation, whereas spores of B. larvae, B. subtilis, and B. licheniformis have relatively smooth surfaces.
Topics: Bacillus; Microscopy, Electron; Microscopy, Phase-Contrast; Sarcina; Spores
PubMed: 4907010
DOI: 10.1128/am.18.3.490-495.1969 -
Physics in Medicine and Biology Dec 2018X-ray phase imaging has the potential to dramatically improve soft tissue contrast sensitivity, which is a crucial requirement in many diagnostic applications such as...
X-ray phase imaging has the potential to dramatically improve soft tissue contrast sensitivity, which is a crucial requirement in many diagnostic applications such as breast imaging. In this context, a program devoted to perform in vivo phase-contrast synchrotron radiation breast computed tomography is ongoing at the Elettra facility (Trieste, Italy). The used phase-contrast technique is the propagation-based configuration, which requires a spatially coherent source and a sufficient object-to-detector distance. In this work the effect of this distance on image quality is quantitatively investigated scanning a large breast surgical specimen at three object-to-detector distances (1.6, 3, 9 m) and comparing the images both before and after applying the phase-retrieval procedure. The sample is imaged at 30 keV with a [Formula: see text] pixel pitch CdTe single-photon-counting detector, positioned at a fixed distance of 31.6 m from the source. The detector fluence is kept constant for all acquisitions. The study shows that, at the largest distance, a 20-fold SNR increase can be obtained by applying the phase-retrieval procedure. Moreover, it is shown that, for phase-retrieved images, changing the object-to-detector distance does not affect spatial resolution while boosting SNR (four-fold increase going from the shortest to the largest distance). The experimental results are supported by a theoretical model proposed by other authors, whose salient results are presented in this paper.
Topics: Breast; Breast Neoplasms; Female; Humans; Hypertrophy; Image Processing, Computer-Assisted; Microscopy, Phase-Contrast; Models, Theoretical; Quantum Dots; Synchrotrons; Tomography, X-Ray Computed
PubMed: 30524112
DOI: 10.1088/1361-6560/aaf2e1 -
Optics Express Mar 2018The observation of retinal cellular structures is fundamental to the understanding of eye pathologies. However, except for rods and cones, most of the retinal...
The observation of retinal cellular structures is fundamental to the understanding of eye pathologies. However, except for rods and cones, most of the retinal microstructures are weakly reflective and thus difficult to image with state of the art reflective optical imaging techniques such as optical coherence tomography. Recently, we demonstrated the possibility of obtaining the phase contrast of retinal cells in the eye using oblique illumination of the retina. Indeed, by illuminating the eye with incoherent oblique illumination, we obtain a secondary oblique illumination from the backscattered light which can then be used to obtain phase contrast in an effective transmission-like configuration. In this technique, a weak phase signal is modulated over an intense background. Maximizing this phase contrast is thus crucial for the image quality. Here, we investigate the parameters that affect phase contrast by modelling image formation with the backscattered light. We find that the key parameter for maximizing contrast is the intensity profile of the backscattered light. Specifically, the gradient of the profile is found to be proportional to the phase contrast. We validate the model by comparing simulations with experimental results on ex-vivo retina samples.
Topics: Animals; Humans; Light; Microscopy, Phase-Contrast; Retina; Scattering, Radiation; Swine
PubMed: 29609366
DOI: 10.1364/OE.26.006785