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Sensors (Basel, Switzerland) Feb 2022In this work, the design, building, and testing of the most portable, easy-to-build, robust, handheld, and cost-effective Fourier Lightfield Microscope (FLMic) to date...
In this work, the design, building, and testing of the most portable, easy-to-build, robust, handheld, and cost-effective Fourier Lightfield Microscope (FLMic) to date is reported. The FLMic is built by means of a surveillance camera lens and additional off-the-shelf optical elements, resulting in a cost-effective FLMic exhibiting all the regular sought features in lightfield microscopy, such as refocusing and gathering 3D information of samples by means of a single-shot approach. The proposed FLMic features reduced dimensions and light weight, which, combined with its low cost, turn the presented FLMic into a strong candidate for in-field application where 3D imaging capabilities are pursued. The use of cost-effective optical elements has a relatively low impact on the optical performance, regarding the figures dictated by the theory, while its price can be at least 100 times lower than that of a regular FLMic. The system operability is tested in both bright-field and fluorescent modes by imaging a resolution target, a honeybee wing, and a knot of dyed cotton fibers.
Topics: Cost-Benefit Analysis; Imaging, Three-Dimensional; Microscopy
PubMed: 35214359
DOI: 10.3390/s22041459 -
Journal of Biomedical Optics Jun 2024Full-field optical coherence microscopy (FF-OCM) is a prevalent technique for backscattering and phase imaging with epi-detection. Traditional methods have two...
SIGNIFICANCE
Full-field optical coherence microscopy (FF-OCM) is a prevalent technique for backscattering and phase imaging with epi-detection. Traditional methods have two limitations: suboptimal utilization of functional information about the sample and complicated optical design with several moving parts for phase contrast.
AIM
We report an OCM setup capable of generating dynamic intensity, phase, and pseudo-spectroscopic contrast with single-shot full-field video-rate imaging called bichromatic tetraphasic (BiTe) full-field OCM with no moving parts.
APPROACH
BiTe OCM resourcefully uses the phase-shifting properties of anti-reflection (AR) coatings outside the rated bandwidths to create four unique phase shifts, which are detected with two emission filters for spectroscopic contrast.
RESULTS
BiTe OCM overcomes the disadvantages of previous FF-OCM setup techniques by capturing both the intensity and phase profiles without any artifacts or speckle noise for imaging scattering samples in three-dimensional (3D). BiTe OCM also utilizes the raw data effectively to generate three complementary contrasts: intensity, phase, and color. We demonstrate BiTe OCM to observe cellular dynamics, image live, and moving micro-animals in 3D, capture the spectroscopic hemodynamics of scattering tissues along with dynamic intensity and phase profiles, and image the microstructure of fall foliage with two different colors.
CONCLUSIONS
BiTe OCM can maximize the information efficiency of FF-OCM while maintaining overall simplicity in design for quantitative, dynamic, and spectroscopic characterization of biological samples.
Topics: Animals; Microscopy; Tomography, Optical Coherence; Microscopy, Phase-Contrast
PubMed: 38584966
DOI: 10.1117/1.JBO.29.S2.S22704 -
Communications Biology May 2023The ability to image cell chemistry at the nanoscale is key for understanding cell biology, but many optical microscopies are restricted by the ~(200-250)nm diffraction...
The ability to image cell chemistry at the nanoscale is key for understanding cell biology, but many optical microscopies are restricted by the ~(200-250)nm diffraction limit. Electron microscopy and super-resolution fluorescence techniques beat this limit, but rely on staining and specialised labelling to generate image contrast. It is challenging, therefore, to obtain information about the functional chemistry of intracellular components. Here we demonstrate a technique for intracellular label-free chemical mapping with nanoscale (~30 nm) resolution. We use a probe-based optical microscope illuminated with a mid-infrared laser whose wavelengths excite vibrational modes of functional groups occurring within biological molecules. As a demonstration, we chemically map intracellular structures in human multiple myeloma cells and compare the morphologies with electron micrographs of the same cell line. We also demonstrate label-free mapping at wavelengths chosen to target the chemical signatures of proteins and nucleic acids, in a way that can be used to identify biochemical markers in the study of disease and pharmacology.
Topics: Humans; Microscopy; Light; Proteins; Organelles
PubMed: 37258606
DOI: 10.1038/s42003-023-04943-7 -
Critical Reviews in Biomedical... 2015Light microscopy plays a key role in biological studies and medical diagnosis. The spatial resolution of conventional optical microscopes is limited to approximately... (Review)
Review
Light microscopy plays a key role in biological studies and medical diagnosis. The spatial resolution of conventional optical microscopes is limited to approximately half the wavelength of the illumination light as a result of the diffraction limit. Several approaches-including confocal microscopy, stimulated emission depletion microscopy, stochastic optical reconstruction microscopy, photoactivated localization microscopy, and structured illumination microscopy-have been established to achieve super-resolution imaging. However, none of these methods is suitable for the super-resolution ophthalmoscopy of retinal structures because of laser safety issues and inevitable eye movements. We recently experimentally validated virtually structured detection (VSD) as an alternative strategy to extend the diffraction limit. Without the complexity of structured illumination, VSD provides an easy, low-cost, and phase artifact-free strategy to achieve super-resolution in scanning laser microscopy. In this article we summarize the basic principles of the VSD method, review our demonstrated single-point and line-scan super-resolution systems, and discuss both technical challenges and the potential of VSD-based instrumentation for super-resolution ophthalmoscopy of the retina.
Topics: Fourier Analysis; Microscopy; Microscopy, Confocal; Microscopy, Electron, Scanning; Retina; Retinal Diseases; Virtual Reality
PubMed: 27480461
DOI: 10.1615/CritRevBiomedEng.2016016445 -
Lab on a Chip May 2018This paper reports a novel method, opto-acousto-fluidic microscopy, for label-free detection of droplets and cells in microfluidic networks. Leveraging the optoacoustic...
This paper reports a novel method, opto-acousto-fluidic microscopy, for label-free detection of droplets and cells in microfluidic networks. Leveraging the optoacoustic effect, the microscopic system possesses capabilities of visualizing flowing droplets, analyzing droplet contents, and detecting cell populations encapsulated in droplets via the sensing of acoustic waves induced by the intrinsic light-absorbance of matter.
Topics: Animals; Equipment Design; Erythrocytes; Microfluidic Analytical Techniques; Microscopy; Photoacoustic Techniques; Rats
PubMed: 29619468
DOI: 10.1039/c8lc00106e -
Neurobiology of Disease Jul 2021One of the biggest unsolved questions in neuroscience is how molecules and neuronal circuitry create behaviors, and how their misregulation or dysfunction results in... (Review)
Review
One of the biggest unsolved questions in neuroscience is how molecules and neuronal circuitry create behaviors, and how their misregulation or dysfunction results in neurological disease. Light microscopy is a vital tool for the study of neural molecules and circuits. However, the fundamental optical diffraction limit precludes the use of conventional light microscopy for sufficient characterization of critical signaling compartments and nanoscopic organizations of synapse-associated molecules. We have witnessed rapid development of super-resolution microscopy methods that circumvent the resolution limit by controlling the number of emitting molecules in specific imaging volumes and allow highly resolved imaging in the 10-100 nm range. Most recently, Expansion Microscopy (ExM) emerged as an alternative solution to overcome the diffraction limit by physically magnifying biological specimens, including nervous systems. Here, we discuss how ExM works in general and currently available ExM methods. We then review ExM imaging in a wide range of nervous systems, including Caenorhabditis elegans, Drosophila, zebrafish, mouse, and human, and their applications to synaptic imaging, neuronal tracing, and the study of neurological disease. Finally, we provide our prospects for expansion microscopy as a powerful nanoscale imaging tool in the neurosciences.
Topics: Animals; Brain; Brain Chemistry; Humans; Microscopy; Microscopy, Fluorescence; Nanotechnology; Neurosciences; Synapses
PubMed: 33813047
DOI: 10.1016/j.nbd.2021.105362 -
Nature Protocols May 2017Linear 2D- or 3D-structured illumination microscopy (SIM or3D-SIM, respectively) enables multicolor volumetric imaging of fixed and live specimens with subdiffraction... (Review)
Review
Linear 2D- or 3D-structured illumination microscopy (SIM or3D-SIM, respectively) enables multicolor volumetric imaging of fixed and live specimens with subdiffraction resolution in all spatial dimensions. However, the reliance of SIM on algorithmic post-processing renders it particularly sensitive to artifacts that may reduce resolution, compromise data and its interpretations, and drain resources in terms of money and time spent. Here we present a protocol that allows users to generate high-quality SIM data while accounting and correcting for common artifacts. The protocol details preparation of calibration bead slides designed for SIM-based experiments, the acquisition of calibration data, the documentation of typically encountered SIM artifacts and corrective measures that should be taken to reduce them. It also includes a conceptual overview and checklist for experimental design and calibration decisions, and is applicable to any commercially available or custom platform. This protocol, plus accompanying guidelines, allows researchers from students to imaging professionals to create an optimal SIM imaging environment regardless of specimen type or structure of interest. The calibration sample preparation and system calibration protocol can be executed within 1-2 d.
Topics: Animals; Artifacts; Lighting; Microscopy; Quality Control
PubMed: 28406496
DOI: 10.1038/nprot.2017.019 -
Histochemistry and Cell Biology Sep 2022A major aim in structural cell biology is to analyze intact cells in three dimensions, visualize subcellular structures, and even localize proteins at the best possible... (Review)
Review
A major aim in structural cell biology is to analyze intact cells in three dimensions, visualize subcellular structures, and even localize proteins at the best possible resolution in three dimensions. Though recently developed electron microscopy tools such as electron tomography, or three-dimensional (3D) scanning electron microscopy, offer great resolution in three dimensions, the challenge is that, the better the resolution, usually the smaller the volume under investigation. Several different approaches to overcome this challenge were presented at the Microscopy Conference in Vienna in 2021. These tools include array tomography, batch tomography, or scanning transmission electron tomography, all of which can nowadays be extended toward correlative light and electron tomography, with greatly increased 3D information. Here, we review these tools, describe the underlying procedures, and discuss their advantages and limits.
Topics: Electron Microscope Tomography; Imaging, Three-Dimensional; Microscopy, Electron, Scanning; Microscopy, Electron, Scanning Transmission
PubMed: 35829815
DOI: 10.1007/s00418-022-02117-w -
Annual Review of Neuroscience 2009Since the work of Golgi and Cajal, light microscopy has remained a key tool for neuroscientists to observe cellular properties. Ongoing advances have enabled new... (Review)
Review
Since the work of Golgi and Cajal, light microscopy has remained a key tool for neuroscientists to observe cellular properties. Ongoing advances have enabled new experimental capabilities using light to inspect the nervous system across multiple spatial scales, including ultrastructural scales finer than the optical diffraction limit. Other progress permits functional imaging at faster speeds, at greater depths in brain tissue, and over larger tissue volumes than previously possible. Portable, miniaturized fluorescence microscopes now allow brain imaging in freely behaving mice. Complementary progress on animal preparations has enabled imaging in head-restrained behaving animals, as well as time-lapse microscopy studies in the brains of live subjects. Mouse genetic approaches permit mosaic and inducible fluorescence-labeling strategies, whereas intrinsic contrast mechanisms allow in vivo imaging of animals and humans without use of exogenous markers. This review surveys such advances and highlights emerging capabilities of particular interest to neuroscientists.
Topics: Animals; Humans; Image Cytometry; Mice; Mice, Transgenic; Microscopy; Microscopy, Confocal; Microscopy, Fluorescence; Molecular Biology; Nervous System; Neurons; Neurosciences
PubMed: 19555292
DOI: 10.1146/annurev.neuro.051508.135540 -
ELife Jan 2021We introduce a random-access parallel (RAP) imaging modality that uses a novel design inspired by a Newtonian telescope to image multiple spatially separated samples...
We introduce a random-access parallel (RAP) imaging modality that uses a novel design inspired by a Newtonian telescope to image multiple spatially separated samples without moving parts or robotics. This scheme enables near-simultaneous image capture of multiple petri dishes and random-access imaging with sub-millisecond switching times at the full resolution of the camera. This enables the RAP system to capture long-duration records from different samples in parallel, which is not possible using conventional automated microscopes. The system is demonstrated by continuously imaging multiple cardiac monolayer and preparations.
Topics: Animals; Caenorhabditis elegans; Heart; Microscopy; Myocardium
PubMed: 33432922
DOI: 10.7554/eLife.56426