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Light microscopy

Flagship methods

Advanced and unique imaging methods

Two-photon microscopy (2P)

Multiphoton microscopy – most commonly in the form of two-photon microscopy – is a fluorescence imaging technique that allows observation of living tissue up to about one millimeter in depth. It uses pulsed red-shifted excitation laser light, which can also excite visible fluorescent dyes.

Single molecule localisation microscopy (SMLM)

There are a variety of widely used implementations of single molecule localization microscopy. The unifying principle behind them is that a large series of individual images is acquired, in each of which only a small subset of the fluorescent labels in the sample are fluorescing.

Stimulated emission depletion microscopy (STED)

Stimulated emission depletion microscopy (STED) is one of the techniques that allows super-resolution microscopy. It is similar to confocal microscopy, in that is uses laser scanning imaging, however it creates super-resolved images by the selective deactivation/depletion of fluorophores in the peripheral area of the illumination PSF, minimizing the area of illumination at the focal point, and thus enhancing the achievable resolution for a given system.

Light-sheet mesoscopic imaging (SPIM or dSLSM)

Light sheet microscopy is a mesoscopic imaging technology that combines optical sectioning with multiple-view imaging to observe tissues and living organisms with impressive resolution. This method is often also referred to as single plane illumination microscopy (SPIM) and many different implementations are available. Three-dimensional imaging in light-sheet-based microscopy is performed by moving the specimen through the light sheet in small steps and recording a two-dimensional image at each step.

Optical projection tomography (OPT)

Optical projection tomography (OPT) is in many ways the optical equivalent of x-ray computed tomography (CT) or the medical CT scan. OPT differs in the way that it often uses ultraviolet, visible, and near-infrared photons as opposed to X-ray photons.

Coherent Anti-Stokes Raman Scattering microscopy* (CARS)

CARS microscopy is one of the label-free multiphoton imaging techniques. It allows for chemically specific imaging of biological materials without use of fluorescent labels. CARS is a three-photon nonlinear optical process where two synchronous and spatially overlapped optical beams are used to probe molecular vibrations in the sample material.

Quantitative Phase Imaging (QPI)

Quantitative Phase Imaging (QPI) has emerged as a valuable method for investigating cells and tissues. QPI operates on unlabelled specimens and, as such, is complementary to established fluorescence microscopy, exhibiting lower phototoxicity and no photobleaching. QPI is a label-free technique in which various methods (for example off-axis digital holography, wavefront sensing, spatial light interference, ptychography) are used to retrieve the phase information of light passing through the cell.

Second/Third Harmonics Generation (SHG/THG)

SHG is a label free technique to visualize specific structures e.g. fibrillar collagen, muscle myosin, and microtubules in vitro and in vivo. THG shows interfaces having local transitions of the refractive index e.g. between water-lipid molecules in cellular membranes and surroundings, and water-protein interfaces in protein-rich regions.

Standard methods

Commonly used and well-established methods

Laser scanning confocal microscopy (LSCM/CLSM)

Confocal laser scanning microscopy (CLSM) or laser scanning confocal [LSCM]), often colloquially referred to simply as “confocal”, is a technique for obtaining high resolution optical images with depth selectivity. The key feature of confocal microscopy is its ability to acquire in-focus images from selected depths, a process known as optical sectioning.

Spinning disc confocal microscopy (SDCM)

Spinning disc confocal microscopy is one of the solutions for routine and high-performance fluorescence live-cell imaging applications. SDCM uses a series of moving pinholes on a disc to scan spots of light, in combination with a high-sensitivity camera to acquire instantaneous optical slices.

Structured illumination microscopy (SIM)

SIM belongs to the family of super-resolution microscopy techniques that allow to acquire images with higher spatial resolution than with conventional fluorescence microscopy. SIM is a camera based widefield fluorescence microscope employing a patterned illumination, e.g. stripes, to excite the fluorescence in the sample.

Total internal reflection fluorescence microscopy (TIRF)

Total internal reflection fluorescence microscopy (TIRF) is a microscopy technique with which a thin region of the cell, usually less than 200nm can be observed. A TIRF microscope uses an evanescent wave to selectively illuminate and excite fluorophores in a restricted region of the specimen immediately adjacent to the glass-water interface.

Fluorescence (cross)-correlation spectroscopy (FCS/FCCS)

Fluorescence correlation spectroscopy (FCS) is a correlation analysis of fluctuations in the fluorescence intensity. The analysis provides information on physical parameters of the fluorescent particles (molecules) in solution, such as concentration, average fluorescence intensity and diffusion speed.

Fluorescence Resonance Energy Transfer (FRET)

Fluorescence resonance energy transfer (FRET) is a mechanism describing energy transfer between two light-sensitive molecules (chromophores). A donor chromophore, initially in its electronic excited state, may transfer energy to an acceptor chromophore through non-radiative dipole-dipole coupling.

Fluorescence Recovery after Photobleaching (FRAP)

Fluorescence recovery after photobleaching (FRAP) denotes an optical technique capable of quantifying the two dimensional lateral diffusion of a molecularly thin film containing fluorescently labeled probes.

Fluorescence Lifetime Imaging (FLIM)

Fluorescence-lifetime imaging microscopy (FLIM) is an imaging technique for producing an image based on differences in the fluorescence-lifetime rather than its intensity.

Electron microscopy

Conventional electron microscopy (SEM/TEM)

Conventional EM enables the visualisation of cell structures at the best resolution available. The specimen preparation techniques for TEM usually include chemical methods (fixation, postfixation, dehydration, resin infiltration, embedding, ultrathin sectioning, and contrasting) as well as advanced high-pressure freezing/freeze substitution techniques.

Volume electron microscopy (vEM)

vEM is used to image the spatial organisation of structures of biomolecules, cells, tissues and organisms. vEM includes both TEM and SEM-based technologies, in which a series of images are produced, combined and finally a digital representation of the sample volume is formed.

Cryo-electron microscopy

CryoEM is an essential tool for structural biology and increasingly also for cell biology. Vitrified samples are imaged using the direct electron detector in TEM (cryoTEM). Using subsequent image analysis, the structures of macromolecules can be studied in vitro for purified macromolecules (SPA) or in situ for assemblies in a cellular context, where lamellae produced by focused ion beam milling are observed cryo electron tomography (cryo-ET).

Immunoelectron microscopy

Immunoelectron microscopy is a powerful technique for detecting and locating antigens in their cellular context. Available methodologies combining immuno/affinity labelling are negative staining, Tokuyasu technique, embedding in acrylic resins, freeze-fractured replica labelling, array-tomography and pre-embedding labelling using different markers as well as correlative light electron microscopy techniques.

Optical projection tomography (OPT)

In addition to imaging the structure at high resolution, the composition of the sample can also be determined using EDS/EDXS, which relies on the X-rays emitted by the elements due to their interaction with the primary beam electrons. Elemental composition can be detected in TEM, SEM and FIB-SEM.

Correlative light and electron microscopy

Correlative light electron microscopy (CLEM) combines images obtained from the same areas of interest using both light and electron microscopy. The aims are to identify specific structures in EM sample volume size, locate rare structures, capture dynamic events, to get information of the cellular context surrounding the specific structure, etc.

Medical imaging

Preclinical imaging

Small animal Computed Tomography (CT)

X-rays Computed Tomography (CT) is based on reconstruction of projections (X-ray images taken from different angles), which provides tomographic anatomical information of measured tissue.

Small animal Positron Emission Tomography (PET)

A functional imaging technique that uses radioactive substances known as radiotracers to visualize and measure changes in metabolic processes, and in other physiological activities including blood flow, regional chemical composition, and absorption.

Small animal Single-Photon Emission Computed Tomography (SPECT)

o Single-photon emission computed tomography (SPECT) is a nuclear medicine tomographic imaging technique using gamma rays produced by radiotracer (gamma-emitting radioisotope) injected to observed tissue.

Small animal Single-Photon Emission Computed Tomography (SPECT)

Magnetic resonance imaging (MRI) is a non invasive imaging technique used to obtain 2D/3D in vivo images representing anatomy and physiological processes (e.g. dynamic perfusion or dynamic changes in blood oxygenation for functional brain mapping), with high spatial and temporal resolution. MRI scanners use strong magnetic fields (for animal studies typically 4.7T to 9.4T or even stronger), radiofrequency pulses, and field gradients to generate images of soft tissues throughout the body.

Small animal Magnetic Particle Imaging (MPI)

Magnetic particle imaging is a tomographic method based on detection of nonlinear response of superparamagnetic tracers (usually superparamagnetic Iron Oxide, SPIO) to alternating magnetic fields.

Small animal High-Intensity Focused Ultrasound (HIFU) with MRI Guidance

This method is typically useful as a specific extension to MRI. It enables opening of hematoencephalic barrier at selected regions of interest and thus study of specific metabolic and physiological processes.

Available at: ISI MR

Small animal in-vivo Optical Imaging

Optical Imaging uses non ionizing radiation such as visible, ultraviolet, and infrared light to obtain detailed images of organs and tissues as well as smaller structures including cells and even molecules.

Small animal Photoacoustic Imaging

Photoacoustic imaging is an imaging modality based on the non-ionizing laser pulses, which are delivered into biological tissues and part of the energy will be absorbed and converted into heat, leading to transient thermoelastic expansion and thus wideband (i.e. MHz) ultrasonic emission.

Human imaging

Human Magnetic Resonance Imaging (MRI) and Spectroscopy (MRS)

Magnetic resonance imaging (MRI) is a non invasive imaging technique used to obtain 2D/3D in vivo images representing anatomy and physiological processes (e.g. dynamic perfusion or dynamic changes in blood oxygenation for functional brain mapping), with high spatial and temporal resolution.

Human Electroencephalography (EEG)

The EEG is based on continuous measurement of electrical signals on the scalp over the time period (e.g. 10-30 minutes). These scalp signals represent the summation of electrical potentials in the brain and thus on the dynamic brain processes.

Human Non-invasive Brain Stimulation (NIBS)

The methods for non-invasive brain stimulation are very useful tools to study brain functions and complementary tools to standard imaging/mapping techniques like MRI, EEG, PET. It enables to modify the functional properties of specific brain regions (e.g. to excite or inhibit the region/function) or the properties of connections between brain regions.

Image data analysis

Image analysis and development of new methods

Image analysis and development of new methods – image analysis using freely available, commercially available as well as custom software packages; development of tailor-made image acquisition, processing and analysis algorithms for various types of imaging techniques; 3D reconstruction of imaged specimens with high spatial resolution; visualization of results; analysis of time series of images; development of custom-made software implementations.

Available at: BC LEM, CUNI IMCF BIOCEV, CUNI VMCF, MUNI CELLIM CEITEC, MAFIL, MUNI FI CBIA, IMG LM, IEM, BUT, IPHYS, ISI MR