Technologies

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Deconvolution is a computational technique for improving the contrast and resolution of digital images. It includes a suite of methods that seek to remove or reverse the blurring present in microscopes images caused by the limited aperture of the microscope objective lens. Nearly any image acquired on a digital fluorescence microscope can be deconvolved. In addition, new applications to transmitted light images are now available. Three-dimensional images made up of a series of optical sections are particularly well suited for improvement by deconvolution.

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Image Scanning microscopy is a method for increasing resolution of a confocal microscope. This is achieved by replacing the point detector of a classical confocal with an array detector. Each pixel of the array detector records its own scan image when the sample is scanned with the laser spot and the individual images are then combined into a single image. The final ISM image corresponds theoretically to an image with a confocal taken with an entirely closed pinhole, which results in the increased resolution observed. ISM has the potential to nearly double the resolution of a confocal imaging system. Multiple commercial implementations of Image Scanning Microscopy exist both on laser scanning confocals as well as spinning disk systems, such as AiryScan, rescan confocal microscope and SoRa. Non-commercial ISM solutions are also available and in general ISM can be applied to 2-photon imaging and many other light microscopy modalities. Please contact the Node offering the technology to find out more about the specific implementation offered.

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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. Images are acquired point-by-point and reconstructed with a computer. This allows three-dimensional reconstructions of topologically complex objects. However, CLSM is significantly slower than widefield or spinning-disk confocal microscopy, because images are acquired pixel-by-pixel and the laser power is concentrated on small diffraction limited spots which increases the phototoxicity load on the samples. CLSM typically requires high-laser power excitation, which can lead to phototoxicity and limited observation of live cells.

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Spinning disk 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. Since a series of pinholes scans an area in parallel, each pinhole is allowed to hover over a specific area for a longer amount of time than on laser scanning confocals (CLSM) thereby reducing the excitation energy needed to illuminate a sample . Furthermore, cameras (often EMCCDs or sCMOS), typically have quantum efficiencies 2-3x higher than PMTs, so much less laser excitation energy is necessary and that reduces photo-toxicity and photo-bleaching of a sample, making it the preferred system for imaging live cells or organisms when optical slicing is necessary. However, optical sectioning in depth is more limited than in CLSM. This technology can be improved with image scanning mic/pixel reassignment to allow non-diffraction limited images which provide resolutions 100-200nm.

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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. The evanescent wave is generated only when the incident light is totally internally reflected at the glass-water interface. The evanescent electromagnetic field decays exponentially from the interface, and thus penetrates to a depth of only approximately 100 nm into the sample medium. Thus the TIRF microscope enables a selective visualization of surface regions such as the basal plasma membrane (which are about 7.5 nm thick) of cells. This technique is often used also for observing molecular dynamics in vitro, or study the details of cell locomotion or adhesion to substrata.

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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. However, for each excitation, two (or three) photons of low energy infrared light are absorbed “simultaneously” to provide the required energy for electrons in the fluorophore to reach the excited state. Using infrared light minimizes scattering in the tissue and allows imaging deeper than is possible with common confocal microscopes. Due to the localized multiphoton absorption effect, the background signal is strongly suppressed and a pinhole is not required in front of the detector (PMTs). Both effects lead to an increased penetration depth for these microscopes. Two-photon microscopy is often used to image intravitally, and it may facilitate imaging of unlabelled tissues, using autofluorescence or SHG (second harmonic generation).

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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. This sparse nature of the signal allows the computational calculation of the exact position of each of the emitting fluorophores. By combining the large number of images with precise locations, an image with very high resolution can be formed. Below are descriptions of some of the common SMLM techniques

The fundamental principle behind stochastic optical reconstruction microscopy (STORM) is that the activated state of a photo-switchable molecule must lead to the consecutive emission of sufficient photons to enable precise localization before it enters a dark state or becomes deactivated by photobleaching. Additionally, the sparsely activated fluorescent molecules must be separated by a distance that exceeds the Abbe diffraction limit (in effect, greater than approximately 250 nanometers) to enable the parallel recording of many individual emitters, each having a distinct set of coordinates in the lateral image plane.

Photo activated localization microscopy (PALM) is a widefield fluorescence microscopy imaging methods that allow obtaining images with a resolution beyond the diffraction limit. PALM is based on collecting a large number of images each containing just a few active isolated fluorophores. The imaging sequence allows for the many emission cycles necessary to stochastically activate each fluorophore from a non-emissive state to a bright state, and back to a bleached state. During each cycle, the density of activated molecules is kept low enough that the molecular images of individual fluorophores do not typically overlap.

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Single-Particle Tracking involves tracking the motion of individual particles over time to obtain information on their dynamic behavior. These particles can range from molecules and nanoparticles to cellular components like proteins and vesicles. The tracking process typically begins by labeling the particle of interest with a fluorescent marker making it detectable under a microscope.

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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. STED can provide 3D datasets of samples, often with resolutions down to 50 nm or less.

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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. Alternatively the light sheet can be moved through the specimen. In multiple-view imaging, the same volume inside the specimen or even the entire specimen is recorded along several angles. The resulting multiple-view information can be combined into a single image stack by data post-processing using a fusion algorithm.

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Raman spectroscopy is a spectroscopic technique based on inelastic scattering of monochromatic light, usually from a laser source. Inelastic scattering means that the frequency of photons in monochromatic light changes upon interaction with a sample.

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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. SHG and THG are not fluorescence, but coherent scattering processes instead, so there is no photobleaching and triplet state induced phototoxicity, and they are well suited especially for live sample imaging. SHG is also a very sensitive method to detect trace amounts of crystallinity e.g. in pharmaceutical materials. SHG/THG are often available in multi-photon systems (2PM using pulsed NIR lasers) which allows imaging of unlabelled samples containing aligned fibrous. Some of the systems available at Euro-BioImaging Nodes can also perform polarized second harmonic microscopy and third harmonic microscopy.

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Expansion microscopy (ExM) is a super-resolution approach that achieves sub-diffraction information by physically expanding the specimen. This allows it to seamlessly obtain resolutions of ~50 nm (up to 10 nm) in four colors in any widefield or confocal microscope.

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Biological systems are inherently complex, and the response of even the more stable model can be highly heterogeneous. Therefore, many samples must be imaged to achieve statistically meaningful results. In microscopy, obtaining sufficiently large quantitative datasets during manual operation can become very time-consuming because the operator must first identify the targets of interest and then acquire each with experiment-specific settings. This workflow imposes limits on the number of events that can be recorded and the reproducibility of the results, especially when the phenotypes of interest are rare or occur only during specific biological stages. Feedback microscopy or smart microscopy allows automation of the image acquisition process, often in combination with responsive image analysis to find the sites of interest. This method can also play a key role in driving unbiased imaging of specimens. In many cases, AI or deep learning are used in the image analysis and object recognition parts of the feedback microscopy process.

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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. By following changes on these parameters it is possible to study binding events of the molecules or even conformational changes on them.

Fluorescence cross-correlation spectroscopy (FCCS) extends the FCS procedure in that it looks at the correlation between different colors (cross-correlation) rather than just the same color (auto-correlation). In other words, coincident green and red intensity fluctuations correlate if green and red labeled particles are moving together. As a result, FCCS provides a highly sensitive measurement of molecular interactions independent of diffusion rate.

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Fluorescence-lifetime imaging microscopy (FLIM) is an imaging technique for producing an image based on differences in the fluorescence-lifetime rather than its intensity. By quantifying variations in the exponential decay rate of the fluorescence from a fluorescent sample (fluorescence-lifetime) it is possible to report on molecule proximity, pH changes and even polarity. It can be used as an imaging technique in confocal microscopy, two-photon excitation microscopy and multiphoton tomography. Since the fluorescence-lifetime is insensitive to changes in fluorophore intensity or concentration, it is the most quantitatively precise technique to report on fluoresce resonance energy transfer (FRET).

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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. This technique is very useful in biological studies of cell membrane diffusion and protein binding as it not only reports on the diffusion rates of mobile fractions of molecules but also provides information about the proportion of immobile molecules. In addition, surface deposition of a fluorescent phospholipid bilayer (or monolayer) allows the characterization of hydrophilic (or hydrophobic) surfaces in terms of surface structure and free energy.

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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. The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, making FRET extremely sensitive to small changes in distance and therefore an excellent reporter on molecule proximity and interaction.

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High-throughput microscopy has created new opportunities for studying biological phenomena in an unbiased manner. Using automated cell manipulations and microscopy platforms, it is possible to easily screen entire genomes for genes that affect any cellular process that can be visualized. HTM techniques rely on fully automated widefield or confocal microscopy systems which facilitate the capture of thousands of images, often in multi-well plates. Labs supporting HCS also have the expertise to automatically deliver drugs to multiwell plates, and to handle, process and analyse thousands of images using high-content screening analysis methodologies. HTM may also refer to “slide scanner technology” or microscopes capable of tile & stitching large areas of tissue at high-resolution, thus producing multi-megapixel 2D datasets.

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High-throughput microscopy has created new opportunities for studying biological phenomena in an unbiased manner. Using automated cell manipulations and microscopy platforms, it is possible to easily screen entire genomes for genes that affect any cellular process that can be visualized. HTM techniques rely on fully automated widefield or confocal microscopy systems which facilitate the capture of thousands of images, often in multi-well plates. Labs supporting HCS also have the expertise to automatically deliver drugs to multiwell plates, and to handle, process and analyse thousands of images using high-content screening analysis methodologies. HTM may also refer to “slide scanner technology” or microscopes capable of tile & stitching large areas of tissue at high-resolution, thus producing multi-megapixel 2D datasets.

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Intravital imaging covers a range of microscopy modalities used for the long term imaging of living animal models (e.g. zebrafish larvae, mice, rats), in various organs or tissues, or as a whole for smaller models. Intravital Imaging can be performed using 2-photon microscopy, light-sheet microscopy or others. The choice is usually made based on the required penetration depth and resolution. These techniques can be combined with optical manipulation systems, such as laser ablation or optogenetic manipulation systems, and also with a range of e.g. neurological or behavioral stimuli especially when awake, immobilized animals are imaged. Intravital imaging allows the real-time visualization of cellular processes in their native environment in the living organism. It can be performed as repetitive imaging on the same animal, allowing biological processes to be tracked over long periods of time, or as acute experiments.

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Microdissection is an established method for a large number of applications, mainly in molecular biology, particularly nucleic acid research, neurosciences, developmental biology, cancer research, forensics, proteomics, plant research, for cutting cell cultures and for single cell isolation.

Microdissection offers a precise and contamination-free solution for the isolation and selection of single cells or tissue. The tissue samples can be embedded, sectioned and stained according to conventional methods of preparation. Paraffin sections, frozen sections, smear preparations, chromosome specimens and cell cultures are all suitable for laser microdissection. The area selected for dissection is drawn on the PC screen and automatically separated from the surrounding tissue with a laser beam.

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Photomanipulation includes a variety of techniques in which lasers (or other light sources) are used to interact with the sample in a targeted fashion. This includes laser ablation to induce cellular injuries and measure physical properties, photoconversion or photobleaching of fluorescent proteins for tracking of protein movements, uncaging of probes or different compounds in the cell etc. Photomanipulation is key in using optogenetics approaches.

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Single molecule FRET allows the measurement of distances and distance changes between two fluorescent molecules in a biomolecular complexes, down to the Angstrom and the microsecond timescales. smFRET measurement can be performed on diffusing molecules, in a confocal geometry, to measure distances and fast conformational changes or on immobilized molecules to measure association dissociation kinetics and conformational changes in the seconds timescales.

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The restitution of 3D images from samples becomes a major challenge for visualizing, exploring, analyzing and quantifying. The major problem with samples is their intrinsic composition which prevents correct observation. Tissue clearing is a set of chemical procedures to render biological tissues transparent (as opposed to translucent), reducing light scattering and thereby allowing imaging in depths which would otherwise be impossible with fresh or live tissues. Tissue clearing is most commonly used on large “mesoscopic” samples (>1mm) which are then imaged with lightsheet microscopy, but can also be used to great advantage for smaller samples, such as those used in confocal or two-photon microscopy.

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The restitution of 3D images from samples becomes a major challenge for visualizing, exploring, analyzing and quantifying. The major problem with samples is their intrinsic composition which prevents correct observation. Tissue clearing is a set of chemical procedures to render biological tissues transparent (as opposed to translucent), reducing light scattering and thereby allowing imaging in depths which would otherwise be impossible with fresh or live tissues. Tissue clearing is most commonly used on large “mesoscopic” samples (>1mm) which are then imaged with lightsheet microscopy, but can also be used to great advantage for smaller samples, such as those used in confocal or two-photon microscopy.

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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, metabolism etc), with high spatial and temporal resolution. MRI scanners use strong magnetic fields, radiofrequency pulses, and field gradients to generate images of soft tissues throughout the body.

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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, metabolism etc), with high spatial and temporal resolution. MRI scanners use strong magnetic fields, radiofrequency pulses, and field gradients to generate images of soft tissues throughout the body.

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Population Imaging relies on the application of advanced image analysis tools to large numbers of medical images related to population cohorts. The aim is the identification and monitoring of specific imaging biomarkers which can provide information about diseases at the population level, allowing early diagnosis, early identification of people at risk and preventive measures.

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Intravital imaging covers a range of microscopy modalities used for the long term imaging of living animal models (e.g. zebrafish larvae, mice, rats), in various organs or tissues, or as a whole for smaller models. Intravital Imaging can be performed using 2-photon microscopy, light-sheet microscopy or others. The choice is usually made based on the required penetration depth and resolution. These techniques can be combined with optical manipulation systems, such as laser ablation or optogenetic manipulation systems, and also with a range of e.g. neurological or behavioral stimuli especially when awake, immobilized animals are imaged. Intravital imaging allows the real-time visualization of cellular processes in their native environment in the living organism. It can be performed as repetitive imaging on the same animal, allowing biological processes to be tracked over long periods of time, or as acute experiments.

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Positron Emission Tomography–Magnetic Resonance Imaging (PET-MRI) is a hybrid imaging technology that incorporates Magnetic Resonance Imaging (MRI) soft tissue morphological imaging, and Positron Emission Tomography (PET) molecular imaging. The technology combines the exquisite structural and functional characterization of tissue provided by MRI with the extreme sensitivity of PET imaging for the determination of receptor density/biological reaction rates/detection of radiolabelled drugs or vectors.  MR and PET acquisition can be made either separately or inline on individual MR and PET scanners, or simultaneously on bimodal machines. MR and PET images are then superimposed to provide single images with the characteristics of both technologies.

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Nuclear Medicine is the field dedicated to the imaging of short-lived radiolabelled compounds injected into the body, and is thus true ‘molecular imaging’. Due to its exquisite sensitivity, very low amounts of compounds that bind to cellular targets (radioligands) or are substrates for biological processes (radiotracers) can be detected, providing quantitative information about the rate of biological processes or number of receptors within organs and tissues without altering the underlying biology (the ‘tracer’ principle). Nuclear medicine makes use of two major modalities depending on the type of radioisotope used for radiolabelling, namely PET (Positron Emission Tomography) and SPECT (Single Photon Emission Computed Tomography).

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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, metabolism etc), with high spatial and temporal resolution. MRI scanners use strong magnetic fields, radiofrequency pulses, and field gradients to generate images of soft tissues throughout the body.

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Imaging technologies record a specimen and produce a digital output. This digital data could then require multiple rounds of analysis steps to infer knowledge from the images. For large, complex, and multimodal datasets, the analysis process highly benefits from standardised and/or automated procedures, often supported by computational methods. Image analysts are experts who can support users with handling, processing, quantification, statistics as well as interpretation of image data. In a collaborative setting, Image analysts can also support projects with experimental design based on analysis of pilot datasets.

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Imaging technologies record a specimen and produce a digital output. This digital data could then require multiple rounds of analysis steps to infer knowledge from the images. For large, complex, and multimodal datasets, the analysis process highly benefits from standardised and/or automated procedures, often supported by computational methods. Image analysts are experts who can support users with handling, processing, quantification, statistics as well as interpretation of image data. In a collaborative setting, Image analysts can also support projects with experimental design based on analysis of pilot datasets.

🔬 Population Imaging Valencia

Population Imaging relies on the application of advanced image analysis tools to large numbers of medical images related to population cohorts. The aim is the identification and monitoring of specific imaging biomarkers which can provide information about diseases at the population level, allowing early diagnosis, early identification of people at risk and preventive measures.

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Atomic Force Microscopy (AFM) is a technique allowing the characterization of sample morphology at nanoscale. Images are acquired by raster scanning a nanometric tip in gentle contact or intermittent contact with the sample. The tip is positioned at the end of a micrometric force transducer, the AFM cantilever: it records the variation of sample topography due to changes of tip-sample interaction during scanning operation. In addition, by measuring the tip-sample interaction force as a function of the tip-sample distance, AFMs can evaluate sample elastic and viscous properties.