The Microscopy and Imaging Facility consists of a fully equipped laboratory with advanced instrumentation for optical microscopy and microanalysis, and a laboratory to work with photodocumentation, photography and different techniques related to the light detection.
The Microscopy service covers complete processes, from sample preparation and sectioning to observation, as well as the application of immunotechniques and in situ-hybridization techniques. The Service also has a laser microdissection platform that allows recovery of cells or cell groups, selected under microscopic control from complex tissues, for the specific molecular analysis of those cells.
The Microscopy and Imaging Facility provides advice on experimental design, and applications and support for image data analysis to internal and external customers. Training to use the Facility equipment is also provided to CRAG users.
The available microscopy and histology techniques comprise:
- Confocal microscopy
- Laser microdissection
- Widefield microscopy
- Thin tissue sectioning with ultramicrotom.
- Image processing and analysis
Additionally, CRAG users have access to a fully equipped Imaging Lab, covering form scientific photography to chemiluminiscence and measurement of photosynthetic efficiency.
CONFOCAL Zeiss 980 Elyra 7
The Zeiss LSM 980 confocal microscope is the Service´s most recent acquisition. Due to high sensitivity, it is highly suitable for observation of samples with very low fluorescence and demanding structures in terms of resolution, as well as for high resolution three-dimensional observation of biological structures and live cell studies. For samples that exhibit a high level of autofluorescence, a highly specific detector (32-channel spectral detector) enables the separation of multiple fluorochromes within a single scan.
Additionally, the microscope is equipped with a GaAsP Fast Multiplex Airyscan 2 detector, which is comprised of 32 detection units that collect light to generate a final image of high resolution and contrast (1.7x increase in spatial resolution). The Airyscan Multiplex Mode also allows for extraction of more spatial information, improves image acquisition speed by 8 times compared to standard confocal equipment, and reduces the illumination dosage for the sample.
Elyra 7 with Lattice SIM 2 is a wide-field based super-resolution system that uses a specialized 3D Lattice illumination pattern for 3D structured-illumination (SIM) imaging - the image reconstruction algorithm from Zeiss, that increases the resolution and sectioning quality. Importantly, it enables to obtain a better signal to noise ratio, combining the acquisition of very dynamic images at high speed and sensitivity with super-resolution, without the need to prepare specialized samples.
Overall, our Zeiss Elyra 7 system combined with confocal microscopy (LSM980) offers three modalities of super resolution: Image Scanning Microscopy (Airyscan2), Structured Illumination Microscopy (SIM), and Single Molecule Localization Microscopy (SMLM : STORM, PALM and variants). The system allows to achieve the resolution of up to 120-60 nanometres and incorporates an incubation chamber to control temperature, humidity and CO2/O2.
Leica Confocal SP5
The TCS SP5 confocal II (Leica) allows tridimensional, spectral and time captures (xyztλ). It is equipped with six laser excitation lines and four simultaneous detection channels (two fluorescence and reflection PMT detectors, a HyD fluorescence detector and one for transmitted light).
The pinhole excludes out of focus light resulting in increased image resolution (200nm). It allows us to capture high color resolution images (16 bits) for fluorescence intensity quantification.
Confocal Spectral Microscope: Olympus FV1000. With inverted microscope
This confocal microscope is equipped with six laser excitation lines. The signal emitted by the sample can be captured by three photomultipliers (PMT) for fluorescence and reflection, and other detector for transmitted light, this allows us to acquire images on four channels simultaneously. We can detect a wide spectral range from 400 nm to 800 nm. The pinhole, the aperture that gives the confocality, excludes the light from out of focus planes increasing the resolution of the image. This equipment allows us to perform three-dimensional, spectral and time catchments. The confocal FV 1000 incorporates two fully synchronized independent scanners that allow the excitation of the sample and observation of it in high resolution. It also has a laser configuration with a diode green laser with a high intensity to excite fluorophores in the red spectral band.
Leica Microdissector LMD6000
Laser Microdissection (LMD) is an automated technique that enables isolation of specific cells from a mixed population or a tissue under microscopic visualization. LMD technique offers an extremely precise, highly selective microdissection for a broad range of applications.
LMD6000 Microdissector, with upright microscope, allows us precise and specific sample collection by gravity without any manipulation of the sample. It performs sample preparation for molecular biology analysis directly from the tissue section using a UV laser. The LMD does not alter the morphology and the chemistry of the collected samples and therefore, is a useful method for collecting selected cells for molecular biology analysis.
Leica DM6. Epifluorescence Vertical Microscope
Leica DM6 is a completely automatized xyz epifluorescent upright microscope with two transmitted light techniques: brightfield and DIC in all objectives. One of the objectives is a multiimmersion objective (glycerol/water) to match the refraction index of our mounting medium (aqueous medium). It has different filtercubes to detect many fluorescents dyes.
The system has two cameras: a CMOS digital color camera with 10 million pixels and a high sensitivity monochrome camera to take the fluorescence images. This monochrome microscope camera with newest scientific CMOS (sCMOS) sensor technology for fluorescence imaging enables imaging cells under near-native conditions: protect the cells with short exposure times, capture highly dynamic processes at high speed and get more information in one image due to large field of view.
Upright Widefield Fluorescence Microscope: Axiophot Zeiss
Axiophot is an epifluorescent upright microscope. It has different types of transmitted light: brightfield, DIC, phase contrast and dark field. It has different fluorescence filters configuration to observe many different fluorocroms, from UV to Red.
It is connected to a cooled CCD digital color camera with 1.45 million pixels. Digital color camera DP70 Olympus (1.5 million pixels).
Vertical Microscope: Leica DMLB
This epifluorescence microscope allows us to visualize the sample with brightfield and fluorescence, has three dichroic standard filters to display fluorochromes from blue to red spectrum.
Stereoscope Microscope: Olympus SZX16
It is a stereoscopic microscope that can reach about 200 ×. It has transmitted and incident light. Also has fluorescence lamp with different filters settings from UV to Red spectra (LP and BP). It is fitted with a digital color camera DP71 Olympus (12.5 megapixels).
“In situ” Laboratory
The CRAG Microscopy Service also offers "in situ" laboratory with the necessary equipment for sample preparation and sectioning for “in situ” or immunohistochemistry protocols.
Shandon Histocenter 3. Thermo scientific. Paraffin inclusion center.
Shandon Varistain 24-4. Thermo Scientific. A rotational benchtop automated staining unit.
Vibratome Series 1000. Tissue sectioning system.
Microtome 2050, Reichert-Jung. Sectioning of paraffin embedded tissue.
Ultramicrotome RM 2265, Leica. Thin sectioning of paraffin or plastic embedded tissue.
The Microscopy Core Facility also offers computers for image processing and treatment. Available software includes:
LAS X. 3.1.5. LEICA MICROSYSTEMS.
FLUOVIEW. FV10-ASW VIEWER. OLYMPUS.
PHOTOSHOP SC5 V12.1
IMARIS V.9.1 (SPOTS, VOLUME, SURFACE, 3D MODULE)
The Laboratory is equipped for:
Photography (camera Nikon D7000 + Camera Nikon D80 fitted with different objectives, a REPRODUCTION TABLE KAISER and Lights Led RB 5020 DS KAISER with diffuser).
Absorbance, colorimetry and fluorescence (SPECTRAMAX M3).
Chemifluorescence and Chemiluminescence (LUMINOMETER CENTRO LB 960 and CHEMI-DOC TOUCN BIO-RAD, LAS 4000 FUJIFILM).
Blotting and Protein gels analysis (CHEMI-DOC TOUCN BIO-RAD, LAS 4000 FUJIFILM, PERSONAL MOLECULAR IMAGER BIO RAD).
Measurement of Photosynthetic efficiency (Imaging PAM MAXI).
You can contact Ángel Sánchez directly, if you need to use the equipment in the Imaging Laboratory.