Preclinical validation bioimaging - Services

U28-S015. Sample vitrification (Onsite&Remote) OUTSTANDING

The Vitrobot is an automated device for vitrification (plunge freezing) of aqueous samples.
Conventional TEM requires high vacuum inside the microscope column, therefore all samples need to be dried out before observation. Cryo-TEM is a good alternative for the direct observation of liquid samples in its original state: specimens are vitrified in liquid ethane or propane and analyzed in the microscope at low temperature. The vitrification method is based in a very fast sample cooling that prevents the formation of crystalline ice. Moreover, the thin layer of amorphous ice formed during the vitrification process protects the material from electron beam damage.
The Vitrobot Mark IV provides precise but flexible control of all critical parameters in the plunge-freezing process.

Applications:
The new Vitrobot Mark IV is an unit that offers great value to the demanding scientific areas of cell biology and molecular imaging as well as being very suitable for food, industrial, pharmaceutical and nanotechnological applications. Fundamental research in cellular and structural biology is increasingly focused

U28-S014. Freeze substitution (Onsite&Remote) OUTSTANDING

Freeze Substitution (FS) of specimens in methanol, acetone or any other FS media at low temperatures is the next step following high pressure freezing and other cryo-fixation methods.
Progressive Lowering of Temperature (PLT) allows substitution and resin infiltration of chemically fixed specimens. Finally, the sample is polymerized under UV light in the EM AFS2 and can be cut and immunolabelled.

Applications:
Fundamental research in cellular and structural biology, and studies involving soft materials that cannot be resin-embedded due to their composition.

U28-S013. High-pressure freezing (Onsite&Remote) OUTSTANDING

High pressure freezing is the most significant sample preparation method for morphological and immunocytochemical high resolution studies for electron microscopy.
High pressure freezing has made it possible to observe aqueous biological and industrial samples near to native state.
The 2100 bar of high pressure applied to the sample during high pressure freezing using the Leica EM HPM100 suppresses ice crystal formation and growth, while cryo-immobilization immediately after pressurization prevents structural damage to the sample.
High pressure frozen samples can be completely vitrified up to a thickness of 200 µm, a 10 to 40-fold increase in the depth of amorphous ice.

Applications:
Once frozen, samples can be placed into the cryo chamber of an ultramicrotome for frozen hydrated sectioning. In combination with freeze substitution, high pressure freezing is an excellent alternative to chemical fixation for immunoelectron microscopy as the antigenicity and ultrastructure are both well preserved. The 6 mm carrier opens up new perspectives for correlative microscopy, as it allows a true pre-selection of a region of interest within large areas and the EM investigation of the same sample without the drawback of artefacts caused by chemical fixation.

U28-S012. Cryo-ultramicrotomy (Onsite&Remote) OUTSTANDING

Sectioning of specimens for cryo-electron microscopy examination, using a cryo-chamber accessory to keep low temperature of samples previously cryo-fixed with the high-pressure freezing system.

Applications:
High quality sectioning of specimens for cryo-electron microscopy examination.
Applied in a cryo-electron microscopy workflow, after the high pressure freezing of samples, mainly for structural biology, immunohistochemistry for electron microscopy (Tokuyashu technique for thermal- sensible epitopes), or ultramicrotomy of soft materials without resin embedding process.

U28-S011. Environmental SEM (ESEM) (Onsite&Remote) OUTSTANDING

Environmental SEM allows the observation of samples in a vacuum-free atmosphere. Water vapor is insufflated inside the chamber during the pumping process (trough purge cycles) what will allow to work at chamber pressures as high as 2600 Pa. The combination of this mode with a peltier stage allow us to work at 100% humidity in the chamber what will facilitate the visualization of fully hydrated samples and perform dynamic experiments through pressure changes, including hydration – dehydration sequences.

Applications:
>> Cellular characterization
>> Tissues characterization
>> Biomaterials characterization
>> Macromolecular complexes characterization

U28-S010. Scanning Electron Microscopy (SEM) (Onsite&Remote)

SEM comprises a set of techniques related to the interaction of an electron beam that raster the surface of the sample with it. Secondary electron, backscattered electrons, X rays, Auger electrons among others are generated and provides information about surface topopgraphy, composition and other properties of the sample characterized. A Field Emission Gun (FEG) source will allow resolutions below 2 nm.

Applications:
>> Cellular characterization
>> Tissues characterization
>> Biomaterials characterization
>> Macromolecular complexes characterization

U28-S08. Cryo-TEM (Onsite&Remote) OUTSTANDING

Cryo-TEM is the technique of choice for the visualization of biological suspensions and any other sub-micron particles in solution. Cryo-immobilization is performed through plunge freezing where samples are transferred to the cryo-holder and to the microscope at temperatures close to liquid nitrogen. Visualization with Low Dose mode allows beam damage and re-crystallization risks to be minimized.

Applications:
>> Cellular characterization
>> Tissues characterization
>> Biomaterials characterization
>> Macromolecular complexes characterization

U28-S06. Time Domain NMR (TD-NMR) (Onsite&Remote)

NMR relaxometry refers to the measurement of relaxation times, typically T1 and T2, as a function of magnetic field. This is crucial in the development of new contrast agent for MRI.

Applications:
>> Measurement of Relaxation Times (T1, T2, T2*)

U28-S05. High Resolution Magic Angle Spinning (HR-MAS) NMR (Onsite&Remote)

HR-MAS NMR is based on solid-state NMR, which uses magic-angle spinning at high speeds to average anisotropic interactions, leading to narrower widths. The moderate spin rates used in HR-MAS allows high resolution spectra to be obtained from intact tissue, i.e., without the need of any metabolite extraction procedure. HR-MAS NMR is widely used for metabolomic studies, especially in cancer research, where the metabolic profile (metaboloma) of tumor biopsies can be characterized using just few milligrams of intact tissue (tissue biopsy). HR-MAS can also be used for the structure characterization and elucidation of partially insoluble compounds.

Applications:
>> Metabolomics of intact tissue samples (tumor biopsies, …)
>> Structure Characterization and Elucidation of partially insoluble compounds)

U28-S04. High Resolution Liquid NMR (Onsite&Remote) OUTSTANDING

Liquid NMR is a fundamental tool both in chemistry and biomedicine research. Its applications to chemistry range from structure elucidation of small molecules to studies of ligand-protein interactions. In biomedicine, the use of liquid NMR has expanded from the classical metabolic studies using enriched stable isotope tracers to the more recent NMR-based metabolomics of biofluids (urine, plasma, CSF).

Applications:
>> Structure verification and elucidation
>> Molecular mobility: determination of conformation in solution
>> Determination of enantiomeric excess
>> Kinetic and temperature studies of reaction mixtures
>> Metabolomics of liquid samples (CSF, plasma, urine and tissue extracts)
>> Metabolomics of liquid samples (CSF, plasma, urine and tissue extracts)