Nanbiosis

U28-S019. Fluorescence microscopy (Onsite&Remote)

For many applications conventional widefield fluorescence microscopy is still the best choice. The CCD detected used for conventional microscopy are often more sensitive that the photomultiplier tubes used in confocal microscopes and flow cytometers. As the camera capture the whole field of view at the same time – it also allows for faster imaging in many cases. Examples where conventional microscopy may be more appropriate include the visualization of individual molecules, receptors or small organisms such as bacteria and yeast. Total Internal Reflection Fluorescence (or TIRF) is a powerful technique which combines the sensitivity of conventional fluorescence without out of focus light. User have access to a dedicated conventional fluorescence system equipped for time-lapse microscopy and a laser-based multi-color TIRF system (also used as the basis for the N-STORM system)

Applications:
>> Endocytosis and vesicle dynamics: High contrast, speed and sensitivity makes it possible to follow live processes close to the membrane with TIRF
>> Single molecule studies: The advanced EM-CCD camera allows the fluorescence of individual molecules to be detected even with relatively short exposures
>> Actin cytoskeleton and focal contact dynamics in live cells: Using fluorescent protein markers these processes can be followed using high time-lapse imaging
>> Yeast and bacteria studies: It is often difficult to fluorescence from microorganism by confocal microscopy without bleaching, whereas the combination of a 100x 1.49NA objective and a very sensitive camera allows fluorescence to be de-tected without need high laser power or very low exposures.
>> High speed imaging of calcium dynamics

U28-S018. Super-resolution Microscopy (Onsite&Remote) OUTSTANDING

Fluorescence microscopy offers a wide array of tools for visualizing microscopic events. The intrinsic diffraction of light has historically made it difficult to use fluorescence to distinguish structures closer than 200nm apart. Super-resolution microscopy refers to techniques that selectively activate fluorescent molecules to map their position with up to 10 times more accuracy than conventional fluorescent microscopy. With the aim of promoting the use nanoscale imaging in biomedicine, Bionand has established one of Spain´s first N-STORM super-resolution platforms dedicated to practical applications. The Nikon N-Storm system is capable of localizing molecules with a resolution of up to 20nm.

Applications:
>> Structure determination of nanometric structures: e.g. nuclear pore receptors where individual subunits can be differentially labelled
>> High resolution neuron tracing: Super-resolution imaging has been shown to greatly improve the accuracy of neural maps given then many neural projects are below limit of conventional microscopy
>> Correlative microscopy: the higher resolution of super-resolution fluorescence makes it easier to associated with structures detected by electron microscopy
>> Single molecular studies: e.g. synapse neuroreceptor distribution
>> Nanoscale cytoskeletal and vesicle trafficking studies

U28-S017. Multiphoton Microscopy (Onsite&Remote) OUTSTANDING

Multi-photon (or two-photon microscopy) takes advantages of the near simultaneous absorption of two or more photons which act to excite a fluorescent molecule with the combined energy of both photons. In practice this means that low energy infrared light can be used to see fluorescent molecules that are normally excited by high energy ultraviolet and blue light. Using this method we can visualize fluorescent molecules at much greater depths than conventional microscopy. Infrared light is also much less damaging to live tissues than UV or blue excitation, making it ideal for visualizing model organisms or tissue explants. The Leica SP5 MP HyD combines both confocal and multi-photon modes in a single machine and can easily switch between different modalities. It is also fitted with the brand new, super-sensitive external HyD hybrid detectors dedicated for multi-photon microscopy.

Applications:
>> Deep tissue imaging: imaging thicker tissue sections (>100 microns) and complex models such as Zebrafish where GFP is used as a marker
>> Long term live imaging: where UV or Blue illumination would be toxic (IR light is much less damaging)
>> High resolution intravital imaging: Often used in near -surface tissues such as skin, retina and brain
>> Quantitative photoactivation: multiphoton-excitation only activates mole-cules in the focal plan unlike conventional fluorescence
>> Fluorescence properties of novel materials: For example nanoparticles often exhibit special absorbance and fluorescence properties when excited by two photon activation

U28-S016. Confocal Microscopy (Onsite&Remote) OUTSTANDING

Laser-scanning confocal microscopy is a very popular technique that uses a combination of laser illumination and a “pinhole” mask to ensure that only fluorescence from the focal plane reaches the detector. This avoids the characteristic blur typical of conventional fluorescence microscopy and allows images to captured as detailed optical slices and then used to build up rich 3D models. Confocal microscopy is one of the most versatile techniques available in a optical microscopy. The Advanced Fluorescence Service offers two extremely well specified Leica SP5 confocal microscopes. Their features include HyD hybrid detectors for the best possible sensitivity, as well as full environmental control for live cells and high-speed resonant scanning.

Applications:
>> High-speed live cell imaging: suitable for a wide range of samples including cultured cells and model organisms such as Drosophila Melanogaster
>> High quality imaging of fixed multi-color immunofluorescence preparations
>> Small to medium scale screening applications: useful where the highest possible imaging quality is required to detect a particular phenotype
>> Advanced fluorescence techniques: including FRAP for studying molecular dynamics, photoactivation, and FRET for studying molecular interactions at sub –nanometric distances

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