Nanbiosis

U28-S019. Fluorescence microscopy

Fluorescence microscopy

Conventional widefield fluorescence microscopy is still the best choice for many applications. The CCD and CMOS-based sensors used for conventional microscopy are typically much more sensitive than the photomultiplier tubes used in confocal microscopes and flow cytometers. As the camera captures the whole field of view at the same time, it also allows for faster imaging in many cases. Examples where conventional microscopy may be advantageous include the visualization of individual molecules, receptors, or small organisms such as bacteria and yeast.

Customer benefits

We have SOPs and ISO9001 certification. We also have specialist technicians for the use of the equipment.

This service is essential to:

• Nanoscale imaging of fixed samples
• Dynamic imaging of cytoskeletal structures, focal adhesion formation, as well as endocytosis and vesicle dynamics in live cells.
• Fluorescent analysis of histological samples.

Target customer

Any company or research group interested in:

• Long-term (>4 days) imaging of bacterial colonies and bacterial interactions using DIC, temperature control, autofocus, and multi-field imaging.
• Routine multi-color fluorescent analysis of histological samples where confocal or other more advanced methods are not necessary.
• Imaging of bone, muscle, or connective tissues in histological samples using circularly-polarised light.

References

Carrillo P, Bernal M, Téllez-Quijorna C, Marrero AD, Vidal I, Castilla L, Caro C, Domínguez A, García-Martín ML, Quesada AR, Medina MA, Martínez-Poveda B. The synthetic molecule stauprimide impairs cell growth and migration in triple-negative breast cancer. Biomed Pharmacother. 2023 Feb;158:114070. doi: 10.1016/j.biopha.2022.114070. Epub 2022 Dec 14. PMID: 36526536.

U28-S018. Super-resolution microscopy

Super-resolution microscopy

Total Internal Reflection Fluorescence (or TIRF) is a powerful technique that combines the sensitivity of conventional fluorescence with selective illumination to improve the contrast of features very close to the sample coverslip. TIRF is often used for studies related to membrane dynamics, receptor-ligand interactions, and vesicular transport.
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. The Nikon N-Storm system is capable of localizing molecules with a resolution of up to 20nm and is compatible with all most current localization protocols and fluorophores (including Alexafluor 647 and Atto488).

Customer benefits

We have SOPs and ISO9001 certification. We also have specialist technicians for the use of the equipment.

This service is essential to:

• Nanoscale imaging of fixed samples
• Dynamic imaging of cytoskeletal structures, focal adhesion formation, as well as endocytosis and vesicle dynamics in live cells.
• Single-molecule studies and localization microscopy modalities including N-STORM, Direct STORM, and PALM.
• Fluorescent analysis of histological samples.

Target customer

Any company or research group interested in:

• STORM image processing: we can convert localization imaging data into SR images using both proprietary and open source software packages.
• TIRF imaging of membrane and/or cytoskeletal dynamics in live adherent cells using commercial fluorescent labels such as DiR, FM4-64, and Cell-Light or user-supplied reagents.

U28-S017. Multiphoton Microscopy

Multiphoton Microscopy

Multi- or two-photon (2P) 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 all the photons. In practice, this means that lower energy infrared (IR) light can be used to see fluorescent molecules that are normally excited by high energy ultraviolet and visible wavelength. As IR light is less susceptible to diffusion and absorption, we can visualise fluorescent molecules at greater depths than conventional microscopy. Infrared light also tends to beless damaging to live tissues than UV or blue excitation, making it ideal for timelapse imaging of model organisms or tissue explants.

Customer benefits

We have SOPs and ISO9001 certification. We also have specialist technicians for the use of the equipment.

This service is essential to:

• 3D imaging of fixed multi-color immunofluorescence preparations
• Long-term and high-speed live cell imaging.
• FRAP (Fluorescence Recovery after Photobleaching) and photoactivation methods for studying molecular dynamics.
• Foster Resonance Energy Transfer (FRET) for studying molecular interactions at sub–nanometric distances.
• Characterization of single (1P) and two (2P) photon fluorescence properties of novel materials in vitro and in vivo.
• Two-photon deep tissue imaging (>100 microns) of fluorescent proteins.

Target customer

Any company or research group interested in:

• Integrated cellular interaction analyses; we offer rapid assays analyzing uptake and subcellular localization of fluorescently-labeled molecules in standard cultured cell lines
• Quantitative, semi-quantitative, and comparative analyses of fluorescent expression/staining in different models. We can advise on the required controls or limitations of different methodologies
• Co-localisation studies comparing localization with standard sub-cellular markers, fluorescent proteins, and antibodies with rigorous statistical analysis performed using commercial (IMARIS) and open-source (FIJI) co-localization analysis tools.
• We specialize in the long-term (>4d) microscopic visualization of cell models, including primary cells, model organisms, and bacterial colonies
• 3D quantification and visualization using the advanced IMARIS 3D analysis package, including volume quantification, 3D object tracking, and cell type and subcellular organelle counting
• Two-photon emission and excitation spectra measurements and Quantum efficiency (QE) estimation by comparison with reference compounds.
• High-resolution intravital imaging of sub-surface tissues taking advantage of higher tissue penetration of two-excitation and the long working distance 25x water immersion objective

References

1. Caro C, Gámez F, Quaresma P, Páez-Muñoz JM, Domínguez A, Pearson JR, Pernía Leal M, Beltrán AM, Fernandez-Afonso Y, De la Fuente JM, Franco R, Pereira E, García-Martín ML. Fe3O4-Au Core-Shell Nanoparticles as a Multimodal Platform for In Vivo Imaging and Focused Photothermal Therapy. Pharmaceutics. 2021 Mar 20;13(3):416. doi: 10.3390/pharmaceutics13030416. PMID: 33804636; PMCID: PMC8003746.
2. Zanocco RP, Bresolí-Obach R, Nájera F, Pérez-Inestrosa E, Zanocco AL, Lemp E, Nonell S. NanoFN10: A High-Contrast Turn-On Fluorescence Nanoprobe for Multiphoton Singlet Oxygen Imaging. Sensors (Basel). 2023 May 9;23(10):4603. doi: 10.3390/s23104603. PMID: 37430516; PMCID: PMC10222627.

U28-S016. Confocal Microscopy

Confocal Microscopy

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 that can be used to build up rich 3D models. Confocal microscopy is one of the most versatile and widely used techniques in optical microscopy. We offer Leica SP5 confocal microscopes featuring HyD hybrid detectors for the best possible sensitivity, as well as full environmental control for live cells and high-speed resonant scanning.

Customer benefits

We have SOPs and ISO9001 certification. We also have specialist technicians for the use of the equipment.

This service is essential to:

• 3D imaging of fixed multi-color immunofluorescence preparations
• Long-term and high-speed live cell imaging
• FRAP (Fluorescence Recovery after Photobleaching) and photoactivation methods for studying molecular dynamics
• Foster Resonance Energy Transfer (FRET) for studying molecular interactions at sub–nanometric distances

Target customer

Any company or research group interested in:

• Integrated cellular interaction analyses; we offer rapid assays analyzing uptake and subcellular localization of fluorescently-labeled molecules in standard cultured cell lines
• Quantitative, semi-quantitative, and comparative analyses of fluorescent expression/staining in different models. We can advise on the required controls or limitations of different methodologies
• Co-localisation studies comparing localization with standard sub-cellular markers, fluorescent proteins, and antibodies with rigorous statistical analysis performed using commercial (IMARIS) and open-source (FIJI) co-localization analysis tools
• We specialize in the long-term (>4d) microscopic visualization of cell models, including primary cells, model organisms, and bacterial colonies
• 3D quantification and visualization using the advanced IMARIS 3D analysis package, including volume quantification, 3D object tracking, and cell type and subcellular organelle counting

U28-S015. Sample vitrification

Sample vitrification

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.

Customer benefits

We have SOPs and ISO9001 certification. We also have specialist technicians for the use of the equipment.

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.

Target customer

Any company or research group interested in plunge-freezing of samples in water solution (vitrification) for cryo-transmission electron microscopy.

Additional information

• Albumin-covered lipid nanocapsules exhibit enhanced uptake performance by breast-tumor cells. DOI: 10.1016/j.colsurfb.2018.02.024

U28-S014. Freeze substitution

Freeze substitution

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 with epoxy resins for conventional electron microscopy or tomography analysis, or with acrylic resins under UV light for immunolabelling studies.

Customer benefits

We have SOPs and ISO9001 certification. We also have specialist technicians for the use of the equipment.

Fundamental research in cellular and structural biology for electron tomography and immulabelling studies at room temperature, avoiding cryo-electron microscopy drawbacks.

Target customer

Any company or research group interested in:

• Structrural biology.
• Cell and tissue samples processed after high pressure or not suitable for high temperature resin embedding.

U28-S013. High-pressure freezing

High-pressure freezing

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.

Customer benefits

We have SOPs and ISO9001 certification. We also have specialist technicians for the use of the equipment.

Cellular ultrastructure visualization using electron tomography to obtain a 3D reconstruction of cells and their internal structures close to a native state, improving the information provided by standard 2D analysis.

Target customer

Any company or research group interested in:

• Structural biology.
• Cell and tissue samples not suitable for 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)

Environmental SEM (ESEM)

The Electron Microscopy Service offers access to transmission electron microscopy (TEM), cryo-TEM and electron tomography, high resolution scanning electron microscopy (SEM), and environmental SEM. A special emphasis has been made on high-end sample preparation techniques through cryo-immobilization. All the equipment has been configured to provide its best for biological and nanomedicine applications.

Customer benefits

We have SOPs and ISO9001 certification. We also have specialist technicians for the use of the equipment.

This service is essential to:

Cells, tissues, and other biological systems: cells, tissues, leaves, flowers, insects, and microorganisms are among the structures that can be topographically characterized using SEM. For these systems, as for others with high water content, ESEM has become an important tool due to its ability for surface visualization even in a 100% humidity environment, reducing sample preparation and permitting dynamic analysis through hydration-dehydration cycles Dynamics experiments are useful in Materials Science, food industry, water-oil emulsions characterization, and pharmaceutical quality control.

Target customer

Any company or research group interested in:

• Environmental scanning electron microscopy of hydrated samples and dynamic experiments.
• Wet-scanning transmission electron microscopy of samples in thin-film water solution.
• Data and image processing.
• Technical advice for users regarding the selection of protocols and procedures for experiments involving electron microscopy.

References

1. Rico, V., Mora, J., García, P., Agüero, A., Borrás, A., González-Elipe, A. R., & López-Santos, C. (2020). Robust anti-icing superhydrophobic aluminum alloy surfaces by grafting fluorocarbon molecular chains. Applied Materials Today, 21. doi: 10.1016/j.apmt.2020.100815
2. Molina N, González A, Monopoli D, Mentado B, Becerra J, Santos-Ruiz L, Vida Y, Perez-Inestrosa E. Dendritic Scaffold onto Titanium Implants. A Versatile Strategy Increasing Biocompatibility. Polymers (Basel). 2020 Apr 1;12(4):770. doi: 10.3390/polym12040770. PMID: 32244665; PMCID: PMC7240519.

U28-S010. Scanning Electron Microscopy (SEM)

Scanning Electron Microscopy (SEM)

The Electron Microscopy Service offers access to transmission electron microscopy (TEM), cryo-TEM and electron tomography, high resolution scanning electron microscopy (SEM), and environmental SEM. A special emphasis has been made on high-end sample preparation techniques through cryo-immobilization. All the equipment has been configured to provide its best for biological and nanomedicine applications.

Customer benefits

We have SOPs and ISO9001 certification. We also have specialist technicians for the use of the equipment.

This service is essential to:

• Cells, tissues, and other biological systems: cells, tissues, leaves, flowers, insects, and microorganisms are among the structures that can be topographically characterized using SEM.
• Biomaterials characterization: Functionalized polymeric and metallic nanoparticles for drug delivery, dental implants, bone plates and cements, and artificial tissues are among the biomaterials that can benefit from the information provided by SEM.

Target customer

Any company or research group interested in:

• Scanning electron microscopy for structural studies of samples.
• Data and image processing.
• Technical advice for users regarding the selection of protocols and procedures for experiments involving electron microscopy.

References

1. Gofas, S., C. Salas, J.L. Rueda, J. Canoura, C. Farias and J. Gil 2014 Mollusca from a species-rich deep-water Leptometra community in the Alboran Sea. Sci. Mar. 78(4):537-553. doi: 10.3989/scimar.04097.27A
2. Paris JL, Monío C, Pérez-Moreno AM, Jurado-Escobar R, Bogas G, Fernández TD, Montañez MI, Mayorga C, Torres MJ. Influence of Pore Size in Protein G’-Grafted Mesoporous Silica Nanoparticles as a Serum Pretreatment System for In Vitro Allergy Diagnosis. Adv Healthc Mater. 2023 Jun;12(15):e2203321. doi: 10.1002/adhm.202203321. Epub 2023 Mar 8. PMID: 36847336.