Flow Cytometry and Advanced Optical Microscopy
The Flow Cytometry and Advanced Optical Microscopy Unit of IMIBIC comprises two technological areas that are of great importance in the field of biomedical research: Flow Cytometry and Confocal-fluorescence Microscopy.
Its mission is to offer access to equipment that covers a broad spectrum of applications in these areas to IMIBIC researchers and to both public and private institutions, as well as to provide technical expertise and training. The Unit also provides scientific and technical advice and support on experimental design, sample preparation, cytometry data analysis, image processing-analysis and data interpretation.
Equipment and facilities
FLOW CYTOMETRY AREA
What is Flow Cytometry?
Flow cytometry is a powerful technique that simultaneously and rapidly measures multiple physical and chemical characteristics of single particles in suspension, usually cells, as they flow in a fluid stream through a beam of laser light. The properties measured include particle’s relative size, relative granularity or internal complexity, and relative fluorescence intensity. These characteristics are determined using an optical-to-electronic coupling system that records how the cell or particle scatters incident laser light and emits fluorescence.
Cell Sorting is a specialised type of flow cytometry. It provides a method for separating or isolating subpopulations of cells from heterogeneous mixtures, one cell at a time, based on the specific light scattering and fluorescent characteristics of each cell.
- LSRFortessa SORP (Becton Dickinson) (Last update: 2017): equipped with 4 lasers (405 nm, 488 nm, 561 nm and 640 nm) and capable of analysing up to 20 parameters simultaneously, 18 fluorescence parameters plus forward and side scatter. (optical configuration)
- Cytomics FC500 (Beckman Coulter): equipped with 2 lasers (488 nm and 633 nm). It allows detection of 7 parameters simultaneously, 5 fluorescence parameters plus forward and side scatter. Equipped with Multi Carousel Loader (up to 32 tubes). (optical configuration)
- FACSCalibur (Becton Dickinson): equipped with 1 Argon laser (488 nm). It allows detection of 5 parameters simultaneously, 3 fluorescence parameters plus forward and side scatter. (optical configuration)
- FACSAria III (Becton Dickinson) (optical configuration) (Last update: 2014)
- Equipped with 3 lasers (407 nm, 488 nm and 633 nm), it allows to analyse up to 13 parameters simultaneously, 11 fluorescence parameters plus forward and side scatter.
- Nozzles available: 70µm, 85µm and 100µm.
- Four-way and single-cell sorting capabilities. Sorted cells can be collected into tubes (microtubes, 12 x 75 mm and 15 mL), plates (6, 24, 48, 96 and 384 wells), or onto slides (equipped with ACDU).
- Temperature Control: sample input (software-adjustable) and sample output for sort collection (water recirculation unit).
- Equipped with Aerosol Management Option (AMO).
There is a wide range of flow cytometry applications, including, but not limited to:
- Multicolour analysis of cell surface and intracellular antigens (Immunophenotyping)
- Cell viability
- Apoptosis assays (plasma membrane integrity, mitochondrial function, caspase activity, DNA fragmentation, etc.)
- DNA content analysis and cell cycle
- Functional assays, such as cell proliferation (CFSE), Calcium flux, intracellular pH, ROS production, etc
- Gene expression analysis by using reporter genes
- PrimeFlow RNA Assay
- Microvesicles analysis
- Microorganism analysis
- Cell sorting and cloning
CONFOCAL-FLUORESCENCE MICROSCOPY AREA
Confocal Microscopy principle
Confocal microscopy offers several advantages over conventional widefield fluorescence microscopy, including the ability to control depth of field, elimination or reduction of background information away from the focal plane, which increases resolution and contrast of image, and the capability to collect serial optical sections from thick specimens, which can be processed to produce a 3D view of specimen. The confocal principle is the use of spatial filtering techniques to eliminate out-of-focus light or glare in specimens whose thickness exceeds the immediate plane of focus. Thus, a beam of laser light is focused onto a scanning spot at a specific depth within the sample. This leads to the emission of fluorescent light at exactly this point. A pinhole conjugated to the focal plane cuts off signals that are out of focus, thus allowing only the fluorescence signals from the illuminated spot to enter the light detector. By scanning the specimen in a raster pattern, images of one single optical plane are created.
- LSM 710 Spectral Confocal Microscope (Carl Zeiss) (Last update: 2017)
- Stand: Axio Observer.Z1 motorized inverted microscope. Illumination: brightfield, differential interference contrast (DIC) and epifluorescence.
- Plan-Apochromat 25x/0.8 Imm DIC
- C-Apochromat 40x/1.1 W DIC
- Plan-Apochromat 40x/1.3 Oil DIC
- Plan-Neofluar 63x/1.3 Imm DIC
- Plan-Apochromat 63x/1.46 Oil
- Excitation lasers: Ar laser (458, 488, 514 nm), HeNe laser (543 nm), HeNe laser (594 nm), HeNe laser (633 nm) and violet diode laser (405 nm).
- Detection: 3 spectral fluorescence detection channels with PMTs and 1 transmitted light channel with PMT (DIC-capable).
- Integrated large incubation chamber.
- LSM 5 EXCITER Confocal Microscope (Carl Zeiss).
- Stand: Axio Observer.Z1 motorized inverted microscope. Illumination: brightfield and epifluorescence.
- Plan-Neofluar 10x/0.3
- Plan-Neofluar 20x/0.5
- Plan-Neofluar 40x/1.3 Oil
- Plan-Apochromat 63x/1.4 Oil DIC
- Excitation lasers: Ar laser (458, 488, 514 nm), HeNe laser (543 nm), HeNe laser (633 nm) and violet diode laser (405 nm).
- Detection: 2 fluorescence detection channels with PMTs.
- Stage top incubator.
- Axio Vert.A1 Fluorescence Microscopy (Carl Zeiss).
Inverted microscope with brightfield and epifluorescence illumination. Objectives: A-Plan 20x/0.35, Plan-Neofluar 40x/0.9 and Plan-Neofluar 63x/1.25 oil. Filter cubes for DAPI, GFP/FITC and Cy3/Texas Red. It is also equipped with an AxioCam ICm1-1.4 Mpx monochrome CCD camera and ZEN 2 software (Zeiss) for image acquisition.
The main applications of confocal microscopy include:
- Analysis and tracking of subcellular structures
- Colocalization studies
- Z-Series and 3D Imaging
- Multidimensional imaging (x, y, z, t, lambda stack)
- Tiled image acquisitions
- FRET analysis for the study of molecular interactions
- Analysis of Molecular Dynamics in live cells by FRAP and FLIP
- Live cell imaging
- Self-use (Operator-unassisted) of flow cytometry analysers and microscopy equipment (only for internal users properly trained in using the equipment).
- Operator-assisted use of the instruments.
- Cell sorting: only provided as an operator-assisted service.
- Processing and analysis of Image. Flow cytometry data analysis.
- Advice and support on experimental design, sample preparation methods, interpretation and presentation of data.
- Developing/implementing flow cytometry and microscopy assays/applications, and image analysis tools responding to the user needs.