Preclinical validation bioimaging - Services

U28-S021. Nuclear Molecular Imaging (PET/SPECT/CT)(Onsite&Remote)

Applications:

U28-S020.

High Content Screening

High content screening (HCS) attempts to bridge the gap between microscopy and cytometry, providing both large quantities of statically useful and phenotypically rich data. HCS combines automated microscopy with customizable algorithms able to detect and quantify many different types of phenotypic information in both fixed and living cultured cells. The Perkin Elmer Operetta HCS system combines automated imaging with advanced Harmony HCS software, which contains many common algorithms such as nuclei and cell detection, as well as the ability to automatically detect complex phenotypes through machine learning.

Customer benefits

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

This service is essential to:

• Fast and statistically robust toxicity assays using adherent cells with the advantage of being able to visually confirm cell morphology.
• Drug dose-response curve calculation: On a single plate, multiple concentrations or combinations of compounds can be easily and quickly tested.
• Screening for specific phenotypes in cultured cells using libraries of small molecules, siRNAs, or other biologically-active collections.
• Screening for complex phenotypes: many cytological effects cannot be detected easily by cytometry Operetta allows complex phenotypes (eg organelle distribution or cell organization changes) to be detected automatically and reliably.
• Long-term cell culture studies: Operetta is equipped with temperature and CO2 control, making it suitable for maintaining live cultured cells in optimal conditions during image acquisition.

Target customer

Any company or research group interested in:

• High content screening design and setup advice The most important step in performing a successful HCS screen is good experimental design and planning.
• Experimental validation and pilot study testing For more complex projects, it is essential to perform preliminary tests to identify possible problems with labeling, marker expression, cell morphology, and other sources of variability before expending valuable time and reagents.
• Custom experiments and HCS analysis design: we can design both assays and custom automatic image analysis pipelines using the Harmony platform based on your requirements.
• Integrated cytotoxicity testing service We have developed robust protocols from the incubation of cultured cells with test compounds to measuring cytotoxic responses at different time points Different criteria such as plasma membrane integrity, metabolic activity, or intracellular esterase activity can be chosen depending on experimental requirements.

References

1. E. Gómez-González, C. Caro, M.L. García-Martín, A.I. Becerro,* M Ocaña. Outstanding MRI contrast with Dysprosium phosphate Nanoparticles of tuneable size. Nanoscale 2022, 14, 11461-11470. doi: 10.1039/D2NR02630A
2. Caro C, Guzzi C, Moral-Sánchez I, Urbano-Gámez JD, Beltrán AM, García-Martín ML. Smart Design of ZnFe and ZnFe@Fe Nanoparticles for MRI-Tracked Magnetic Hyperthermia Therapy: Challenging Classical Theories of Nanoparticles Growth and Nanomagnetism. Adv Healthc Mater. 2024 Feb 2:e2304044. doi: 10.1002/adhm.202304044. Epub ahead of print. PMID: 38303644.

U28-S019.

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

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

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

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