Microscopy and Image Analysis Core Facility (SMAI)

Grupo Microscopía

Microscopy and Image Analysis Core Facility (SMAI)

 

Heads of service: Dr. José Ángel Rodríguez Alfaro
                               Dr. Javier Mazarío Torrijos

Tel.: 925 396 830
E-mail: microscopia.hnp@sescam.jccm.es

 

 

The Microscopy and Image Analysis Core Facility (SMAI) is a facility of the Hospital Nacional de Parapléjicos (HNP), under SESCAM. The SMAI provides HNP research groups with access to state-of-the-art equipment and the expertise of its staff for acquiring and analyzing images of biological and material samples. Under certain conditions, it can also offer support to other public institutions and private companies in the area.

 

The SMAI at HNP was established in 2007 following the acquisition of a Leica SP5 confocal microscope. Since then, its capacity has expanded, and it now features a wide variety of cutting-edge equipment, including multiple epifluorescence and brightfield microscopes, two inverted microscopes for Video Time-Lapse capture, a laser microdissector, two confocal microscopes (one of them also equipped for live sample imaging), and a robotic microscope for High Content Screening studies. Additionally, SMAI holds licenses for commercial image analysis software such as NewCAST and Neurolucida.

 

The SMAI routinely performs large-area super-image acquisition (brightfield and fluorescence), fluorescence imaging of samples stained with up to five fluorochromes, cell migration studies, colocalization studies, selective dissection of specific tissue regions for RNA or protein analysis, as well as digital image processing and analysis. Additionally, SMAI is equipped for FRAP, FRET, and stereology studies.

 

 

 

Service Equipment

 

LEICA TCS SP5 CONFOCAL MICROSCOPE

The Leica TCS SP5 confocal microscope is equipped with nine excitation lines, four simultaneous detection channels (three detectors for reflection and/or fluorescence and one for transmitted light), and an AOBS (Acousto-Optical Beam Splitter) system.

Resolution: 200 nm

Lasers:
- UV Diode 405 nm  
- Multiline Argon 458, 476, 488, 496, and 514 nm  
- Helium-Neon 543 nm  
- Helium-Neon 594 nm  
- Helium-Neon 633 nm  

Objectives:
- 10x PH1; HC PL APO 20x/0.70 CS; HCX PL APO 40x/1.25-0.75 oil CS; HCX PL APO 63x/1.4-0.6 oil HCX; PL APO 63x/1.3 glycerol Corr CS

LEICA TCS SP5 FAST SCANNING CONFOCAL MICROSCOPE

The Leica TCS SP5 confocal microscope is equipped with eight excitation lines, four simultaneous detection channels (three detectors for reflection and/or fluorescence and one for transmitted light), and an AOBS (Acousto-Optical Beam Splitter) system. It features a fully motorized stage that allows for mosaic imaging and position recording for scanning at different times. This confocal microscope is equipped with a fast scanning module (resonant scanner), specially designed for in vivo imaging and high-speed dynamics studies.

Resolution: 200 nm

Lasers:
- UV Diode 405 nm  
- DPSS 561 nm  
- Helium-Neon 633 nm  
- White laser (supercontinuum). Tunable between 470 and 670 nm

Objectives:
- FL PLAN 5x/0.12; PL APO 10x/0.4; PL APO 20x/0.70 CS; PL APO 40x/1.25-0.75 oil CS; PL APO 63x/1.4-0.6 oil CS; PL APO 63x/1.3 glycerol Corr CS

LEICA LMD 6000 LASER MICRODISSECTOR

The LMD 6000 is a microdissection system consisting of a fully automated Leica microscope coupled to a high-power solid-state UV diode laser at 355 nm, with a gravity-based sample collection system to avoid additional forces that could damage the dissected samples. It allows for the dissection of microscopic specimens or tissues to isolate homogeneous and smaller fragments of the sample for further analysis or culture (in the case of live samples).

Equipped with fluorescence illumination and filters for DAPI, FITC, and TEXAS RED.

Objectives:
- PL FLUO 1.25x/0.04; UVI MICRODIS 6.3x/0.3; PL FLUO 20x/0.4; PL FLUO 40x/0.6 air; PL FLUO 63x/0.7 air.

OLYMPUS IX83 INVERTED MICROSCOPE FOR HIGH CONTENT ANALYSIS AND LARGE AREA IMAGE MOSAICS

The IX83 microscope is fully motorized and equipped with a 7-channel LED fluorescence illumination source covering the ultraviolet to infrared range. It also features six emission filters in a fast filter wheel. The combination of excitation lines and emission filters allows for the use of a wide variety of fluorescent markers that can be sampled at high speed. Images are acquired with a Hamamatsu Orca 4.0 camera.

The ScanR software turns this equipment into a high-content analysis platform, enabling multiple images at different wavelengths in culture plates from 6 to 384 wells as well as slides, quickly and automatically, while simultaneously analyzing these images.

With the CellSens Dimensions software, we can automatically capture large-area high-resolution image mosaics on up to four slides simultaneously.

Both software packages include licenses for 2D and 3D deconvolution and state-of-the-art artificial intelligence image analysis.

Objectives:
- UPLXAPO4X/0.16, UPLXAPO10X/0.40, UPLXAPO20X/0.80, UPLXAPO40X/0.95, UPLXAPO40XO/1.40, UPLXAPO60XO/1.42, LUCPLFLN20XPH/0.45, LUCPLFLN40XPH/0.60

Filters:
- Wide range of filters allowing the visualization of commonly used fluorescent markers in research.

COMPUTER FOR IMAGE ANALYSIS

HP ProDesk 400 G6 Micro Tower Computer, 1 x Core i7 9700 / 3 GHz, 32 GB RAM.

Image analysis software:
FIJI
QuPath
Olympus ScanR (licensed for 2D and 3D deconvolution and artificial intelligence)
Olympus CellSens Dimensions (licensed for 2D and 3D deconvolution and artificial intelligence)

LEICA DM5000B EPIFLUORESCENCE MICROSCOPE

This microscope is specially designed for fluorescence techniques, although it can also be used for transmitted light techniques (brightfield and phase contrast). It is equipped with a Leica DFC 350 FX ultra-high sensitivity monochrome digital camera.

Objectives: FL PLAN 5x; HCX PL S-APO 10x/0.30; HCX PL S-APO FLUOTAR 20x/0.50; HCX PL APO 40x/0.85 CORR; HCX PL APO 100x/1.40-0.70 oil.

Available filters: A wide range of filters allowing visualization of commonly used fluorescent markers in research.

LEICA DMI6000B INVERTED MICROSCOPE FOR LIVE CELL IMAGING (VTL)

Fully automated, inverted microscope with a motorized stage, designed for live-cell experiments (video time-lapse). It can capture transmitted light (brightfield, phase contrast, and darkfield) as well as incident fluorescence. It is equipped with a Leica DFC 350 FX ultra-high sensitivity monochrome digital camera.

Objectives: N PLAN 5x/0.12; HC PL FLUOTAR 10x/0.30 PH; HCX PL FLUOTAR 20x/0.40 CORR PH; HCX PL FLUOTAR 40x/0.60 CORR PH 2 XT.

Available filters: A wide range of filters allowing visualization of commonly used fluorescent markers in research.

OLYMPUS IX81 INVERTED MICROSCOPE FOR LIVE CELL IMAGING

Fully automated, inverted microscope with a motorized stage, designed for live-cell experiments (video time-lapse). It can capture transmitted light (brightfield, phase contrast, and darkfield) as well as incident fluorescence. It is equipped with a high-sensitivity Hamamatsu ORCA-ER monochrome CCD camera.

Objectives: Plan 4x/0.10; UPlanFL N 10x/0.3; LUCPlanFL N 20x/0.45; LUCPlan FL N 40x/0.6; PlanApo 60x/1.42 oil.

Filters: DAPI, FITC, Texas Red.

OLYMPUS BX61 MICROSCOPE FOR STEREOLOGY AND ANATOMICAL MAPPING

Microscope with motorized stage and focus. It is equipped with a 12-bit, 12.5-megapixel, cooled color digital camera (Olympus DP71) and an MBF CX9000 color camera. The system includes a license for the newCAST software for stereology studies and a license for the Neurolucida software for anatomical mapping and neuron tracing.

Objectives: PlanApo N 2x/0.08; UPlanSapo 4x/0.16; Plan N 10x/0.25; UPlanSapo 20x/0.75; UPlanSApo 40x/0.95; UPlanSApo 100x/1.40 oil.

NIKON AZ100 MULTIZOOM FLUORESCENCE MICROSCOPE

The AZ100 microscope combines the advantages of a stereoscopic microscope (large field of view and long working distances) with those of a traditional microscope, allowing high-resolution macro and micro imaging. It is equipped with a high-resolution Nikon DS-RI1 color CCD camera.

This microscope is particularly useful for medium-magnification imaging, such as transgenic verification, histological sections, and cultures.

Zoom: 10x-400x

Objectives: AZ-Plan Apo 1x, AZ-Plan Fluor 2x N. 0.2 WD 45, and AZ-Plan Fluor 5x NA 0.5 WD 15 (all long working distance).

Optical path zoom factor: Continuous from 1x to 8x with a stop mechanism at each zoom level (1, 2, 3, 4, 5, 6, 7, and 8) for reproducible magnifications.

Eyepieces: 10x

Illumination techniques:
The microscope is equipped with a mercury light source for fluorescence illumination and a turret with different filters: DAPI, CFP, FITC or GFP, Cy3 or TRITC, and Texas Red. It also features multiple transmitted light illumination options, including brightfield, polarized light, oblique light, and DIC.

OTHER EQUIPMENT

- Leica EM UC6 Ultramicrotome
Device for preparing ultrathin sections of biological or industrial samples for observation under an electron microscope. It is fully motorized and designed to use both glass and diamond blades.

- Microm HM 450 Sliding Microtome with Freezing Unit