Preclinical validation bioimaging - Services

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.

U28-S08. Cryo-TEM

Cryo-TEM

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:

• Cellular characterization: cell structures and ultrastructures visualization and its relationship to function are the most important contributions of cryo-EM to cell biology.
• Tissue characterization: cryo-electron microscopy has a role in the characterization of interactions between cells and with other components inside tissues.
• Biomaterials characterization: cryo-electron microscopy plays a double role in biomaterial characterization On one hand, it provides structural and compositional information on the materials engineered to be used inside biological systems, favouring its development On the other hand, it allows us to visualize their interactions 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 cryo-EM.
• Macromolecular complexes characterization: structural biology can benefit from cryo-electron microscopy to determine native 3D structures of macromolecular complexes.

Target customer

Any company or research group interested in:

• Electron Microscopy of frozen-hydrated samples.
• Plunge-freezing of samples in water solution (vitrification) for cryo-transmission electron microscopy.
• Cryo-fixation of samples at high pressure (High-Pressure Freezing).
• Glow-discharge of film-coated transmission electron microscopy grids.
• Data and image processing.
• Technical advice for users regarding the selection of protocols and procedures for experiments involving electron microscopy.

References

1. Galisteo-González F, Molina-Bolívar JA, Navarro SA, Boulaiz H, Aguilera-Garrido A, Ramírez A, Marchal JA. Albumin-covered lipid nanocapsules exhibit enhanced uptake performance by breast-tumor cells. Colloids Surf B Biointerfaces. 2018 May 1;165:103-110. doi: 10.1016/j.colsurfb.2018.02.024. Epub 2018 Feb 13. PMID: 29471218.
2. Molina-Bolívar J.A., Galisteo-González F. Olive-oil nanocapsules stabilized by HSA: Influence of processing variables on particle properties. J. Nanoparticle Res. 2015;17 doi: 10.1007/s11051-015-3192-1

U28-S06. Time Domain NMR (TD-NMR)

Time Domain NMR (TD-NMR)

The NMR service is equipped with a TD-NMR analyzer for relaxivity measurements at low magnetic field (1.44 T).

Customer benefits

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

This service is essential for relaxivity measurement of magnetic compounds, such as magnetic nanoparticles or compounds containing lanthanides (Gd, Dy, etc.)

Target customer

Any company or research group interested in relaxivity measurements (r1 and r2) at low magnetic field (1.44 T).

References

1. Caro C., Paez-Muñoz J.M., Beltrán A.M., Leal M.P., García-Martín M.L. PEGylated Terbium-Based Nanorods as Multimodal Bioimaging Contrast Agents. ACS Appl. Nano Mater. 2021;4:4199–4207. doi: 10.1021/acsanm.1c00569.
2. Ashish Avasthi, Carlos Caro, María Luisa Garcia-Martin, Manuel Pernia Leal. Deciphering the parameters to produce highly reproducible and scalable iron oxide nanoparticles. Reaction Chemistry & Engineering 2023, 8 (7) , 1638-1653. doi: 10.1039/D2RE00516F

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

High Resolution Magic Angle Spinning (HR-MAS) NMR

The NMR service is equipped with a High resolution NMR Spectrometers: a 600 MHz, spectrometer dedicated NMR studies that are more demanding in terms of sensitivity. This system is also equipped with a high resolution magic angle spinning (HR-MAS) NMR to analyze heterogeneous samples (tissue biopsies or partially insoluble compounds).
Applications of NMR spectroscopy to chemistry range from structure elucidation of small molecules to studies of ligand-protein interactions.

Customer benefits

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

This service is essential for metabolomic analysis of intact tissue (biopsies)

Target customer

Any company or research group interested in:

• Acquisition of 1 H and 31P HR-MAS NMR spectra.
• Preparation of tissue samples for HR-MAS NMR spectroscopy.
• Basic processing of NMR spectra.

References

Kawashima Y, Nagai H, Konno R, Ishikawa M, Nakajima D, Sato H, Nakamura R, Furuyashiki T, Ohara O. Single-Shot 10K Proteome Approach: Over 10,000 Protein Identifications by Data-Independent Acquisition-Based Single-Shot Proteomics with Ion Mobility Spectrometry. J Proteome Res. 2022 Jun 3;21(6):1418-1427. doi: 10.1021/acs.jproteome.2c00023. Epub 2022 May 6. PMID: 35522919; PMCID: PMC9171847.

U28-S04. High Resolution Liquid NMR

High Resolution Liquid NMR

The NMR service is equipped with a High resolution NMR Spectrometers :a 400MHz (NMR spectrometer for routine experiments and a higher filed) y 600MHz (spectrometer dedicated NMR studies that are more demanding in terms of sensitivity)
Applications of NMR spectroscopy to chemistry range from structure elucidation of small molecules to studies of ligand-protein interactions. In biomedicine, the use of NMR spectroscopy has expanded from the classical metabolic studies using enriched stable isotope tracers to the more recent NMR-based metabolomics of biofluids (urine, plasma, CSF).

Customer benefits

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

This service is essential to:

• Structure verification and elucidation
• Molecular mobility: determination of conformation in solution
• Determination of enantiomeric excess
• Kinetic and temperature studies of reaction mixtures
• Metabolomic analysis of biofluids (urine, blood serum, etc.)

Target customer

Any company or research group interested in:

• Acquisition of 1D NMR spectra at 400 MHz and 600MHz (1 H, 13C, 31P, etc.)
• Acquisition of basic 2D experiments: COSY, TOCSY, HSQC, HMBC, J-RES
• Nuclear Overhauser Effect experiments (NOE and ROE), Diffusion experiments (DOSY), selective excitation experiments.
• Preparation of biofluid samples (urine, blood serum/plasma) for high resolution NMR spectroscopy following standardized protocols.
• Basic processing of NMR spectra.

References

1. Pomary PK, Eichau S, Amigó N, Barrios L, Matesanz F, García-Valdecasas M, Hrom I, García Sánchez MI, Garcia-Martin ML. Multifaceted Analysis of Cerebrospinal Fluid and Serum from Progressive Multiple Sclerosis Patients: Potential Role of Vitamin C and Metal Ion Imbalance in the Divergence of Primary Progressive Multiple Sclerosis and Secondary Progressive Multiple Sclerosis. J Proteome Res. 2023 Mar 3;22(3):743-757. doi: 10.1021/acs.jproteome.2c00460. Epub 2023 Jan 31. PMID: 36720471; PMCID: PMC9990127.
2. Molina N, Martin-Serrano A, Fernandez TD, Tesfaye A, Najera F, Torres MJ, Mayorga C, Vida Y, Montañez MI, Perez-Inestrosa E. Dendrimeric Antigens for Drug Allergy Diagnosis: A New Approach for Basophil Activation Tests. Molecules. 2018 Apr 24;23(5):997. doi: 10.3390/molecules23050997. PMID: 29695102; PMCID: PMC6100007.
3. Fernández-Palacios S, Matamoros E, Morato Rojas I, López Navarrete JT, Ruiz Delgado MC, Vida Y, Perez-Inestrosa E. New Insights into Acylhydrazones E/Z Isomerization: An Experimental and Theoretical Approach. Int J Mol Sci. 2023 Sep 29;24(19):14739. doi: 10.3390/ijms241914739. PMID: 37834186; PMCID: PMC10648745.