Confocal microscopy

Facility offers both infrastructure and scientific- technical support for the achievement of microscopy confocal studies. We can study different samples: fixed, living, cell cultures and tissues both from animal and plant origin; as well as diverse biomaterials.
Equipment:
Confocal Laser Scanning Microscope (CLSM) LEICA TCS SP8 STED 3X fitted witth UV Laser 405 nm and White Light Laser freely tunable excitation from 470-670 nm (1 nm steps). AOTF (Acousto-Optical Tunable Filter) and AOBS (Acousto-Optical Beam Splitter) System. Scanning stage for motorized stage applications. Control temperature, CO2 and humidity system for p35, p60 petri-dish and standard slide
Confocal Laser Scanning Microscope (CLSM) Leica TCS SP5 with motorized stage that allows to take samples of multipositions and to take images reconstructing an area into the sample. System avalaible temperature control (thermostatically controlled stage). Axis control Z by means galvanometer stage. Laser lines at 488nm, 561nm and 633nm. Diverse speed of scanning, resolutions and modes of acquisition: XY, Z, T and lambda (spectral analysis). Applications: FRET, FRAP, colocalization analysis, cuantification and image tridimensional processing.

Confocal microscope Leica DM IRB TCS SP2 (100-630x)

Imaging of fluorescent molecules in biological samples located in the same focal plane.
A piece called pinhole prevents the passage of light planes unfocused obtaining a higher quality image and sharpness than that obtained with conventional fluorescence microscopy.
A scan system moves the laser beam in a raster across the sample.The wavelengths of these lasers in the visible spectrum ranging from blue to far red.
Fluorescent molecules can be observed are: autofluorescent, fluorescent dyes, fluorophores, fluorescent proteins ...
The acquisition series of images of the entire depth of the sample allows three-dimensional reconstruction.
Description of equipment:
1º LEICA TCS SP5 CONFOCAL MICROSCOPE ( UPRIGHT )
Acquisition date:2009
Excitation laser lines 405, 458, 476, 488, 496, 514, 561 y 633.
Motorized stage
Objectives:
10X 0.40 DRY; 20X 0.70 DRY; 40X 1.25 IMM OIL; 63X 1.40 IMM OIL; 100X 1.30 IMM OIL
Acquisition software: LEICA LAS AF
1º LEICA TCS SP5 CONFOCAL MICROSCOPE ( INVERTED )
Acquisition date:2005
Excitation laser lines 405, 458, 476, 488, 496, 514, 561, 594 y 633.
Motorized stage
Objectives:
10X 0.40 DRY, 20X 0.70 MULTI IMM, 40X 1.25 IMM OIL, 63X 1.40 IMM OIL, 63X 1.30 IMM GLYC
Hybrid detector
Acquisition software: LEICA LAS AF
Additional applications: FRET, FRAP, LIVE DATA MODE
Applications or techniques:
- Immunohistochemistry with fluorescent labels: Alexa or cyanine fluorophores
- Expression of fluorescent proteins
- Colocalization analyze: observation of the spatial overlap between two (or more) different fluorescent labels, each having a separate emission wavelength, to see if the different "targets" are located in the same area of the cell or very near to one another.
- Z-sequences of optical slices (3D image stacks) are sources for 3D reconstruction
-time-lapse movies of biological processes
-mosaic or acdquire separed images with motorized stage
-lambda scan: Imaging method that acquires the emission spectrum of the fluorescence molecules in the sample
-FRET: ( FÖRSTER RESONANCE ENERGY TRANSFER ): is a mechanism describing energy transfer between two fluorophores. It can be used to determine if two fluorophores are within a certain distance of each other.
-FRAP: FLUORESCENCE RECOVERY AFTER PHOTOBLEACHING.
is a method of determining the kinetics of diffusion in living cells (usually) using fluorescence microscopy. The general method is to label a specific cell component with a fluorescent molecule, image that cell, photobleach a small portion of the cell, then image the recovery of fluorescence over time.
- Reflection: study of the surface of some materials

Application fields:
Cell biology, histology, genetics, physiology, microbiology...

Our confocal laser scanning microscope (LSM710 Zeiss) allows the collection of optical sections from samples stained with fluorescent markers. This microscope is particularly well-suited for spectral separation of different fluorophores, analysis of three-dimensional structures, colocalization studies, as well as for acquisition of reflection images from materials.
The microscope is composed by:
Microscope: inverted Observer.Z1
Objectives: Plan-APOCHROMAT (10x/0.45 DIC, 25x/0.8 Imm Korr DIC, 40x/1.3 Oil DIC, 63x/1.40 Oil DIC).
Laser lines: 405, 488, 458, 514, 561, 633.
Detectors: 3 fluorescence detectors with spectral detection and 1 transmission detector for DIC imaging.
Software: Zen200

3) Nikon C-1 Modular Confocal Laser Scanning Microscope System, integrated by the following units:
-Nikon inverted microscope Eclipse TE2000-U. Motor operated stage PROSCAN NIKON TE2000. Brightfield, Nomarski DIC observation and epifluorescence (standard mercury lamp, standard block filters: blue, green, ultraviolet).
-Objective CFI PL APO 4x AN 0,2 WD 15,7 mm
-Objective CFI PL APO 10x AN 0,45 WD 4,0 mm
-Objective CFI PL FLUOR 20x AN 0,75 WD 0,35 mm multi-immersion
-Objective CFI PL FLUOR 40x AN 1,30 WD 0,2 mm oil
-Objective CFI PL APO 60x AN 1,4 WD 0,21 mm oil
-Objetive CFI PL APO VC 100x AN 1,40 WD 0,13 mm oil
-Confocal module, non-spectral, non-fully motorized, four-channels simultaneous detection (three lasers plus transmission diascopic channel). Variable motorized pinhole (three sizes + open). Scanning resolution up to 2048x2048 pixels (12 bits). Piezo-electric z-axis motor. Laser unit (4 lasers + AOTF)
Lasers available:
-Blue diode (36 mW) (405 nm)
-Ar (50 mW) (457 nm, 477 nm, 488 nm, 514 nm)
-He-Ne (2 mW) (543 nm)
-Red diode (10 mW) (638 nm)

1- Adquisition of topographies (200 augmentations, surfaces of 600x400 microns)
2- Analysis of topographies through software of texture analysis
3- Intepertation of results

We have seven confocal laser scanning systems, one confocal spinning disk system and super-resolution confocal (STED) and Fluorescence Half-Life Imaging Microscopy (FLIM)
This fluorescence technique increases the XY and Z optical resolution reached by Widefield microscopes. Applications which can be run on these systems: FLIM, FRET, FLIM-FRET, FRAP, Photoactivation/Photoconversion, 3D imaging, Image Stitching, spectral characterization of fluorochromes.
Four of our confocal systems have an incubation chamber for temperature, humidity and CO2 control to develop long-term in vivo experiments.
To get details about the equipment features go to our Web page: http://www.cbm.csic.es/confocal
To use them contact with the SMOC personnel:
Phone number: 34-91 196 4643
E-mail: confocal-cbm@listas.csic.es

Cytometry and Confocal Microscopy Service has a Leica TCS-SP2 to perform confocal microscopy analysis with the following specifications:
* Type: laser scanning confocal microscope (LSCM).
* Techniques: confocal, epifluorescencia, DIC, bright field, 3D reconstructions.
* Tecnical information:
- Leica DMRE microscope.
- Software: Leica Lite.
- Epifluorescence: 50W mercury lamp. Fluorescence filter systems: A (DAPI), N2.1 (FITC) y I3 (Rodamina).
- Transmitted light: 12V- 100W hologen lamp.
- Lasers:
1) He-Cd: 442 nm excitation line.
2) Ion Ar: 458, 476, 488, 514 nm excitation lines.
3) He-Ne (green): 543 nm excitation line.
4) He-Ne (orange): 633 nm excitation line.
- Acousto-optical tunable filter (AOTF).
- Dichroic mirror: RSP 455, RSP 500, DD 458/514, TD 488/543/633, Substrat, RT 30/70
- Objetives:
1) HC PL APO CS 10x/0.40.
2) HC PL APO CS 20x/0.70 IMM/CORR.
3) HCX PL APO CS 40x/1.25-0.75 OIL.
4) HCX PL APO CS 63x/1.40-0.6 OIL.
5) HCX APO L U-V-I 40x/0.80 W.
6) HCX APO L U-V-I 63x/0.90 W.

Spectral Confocal Microscopy allows the study of samples with fluorescent labeling, making optical sections thereof. About the point sample is excited by a laser. The emission wavelength of that sample is higher than excitation, and is the latter when passing through a small aperture (pinhole) allows detection of a single focal plane.
This advance in the field of microscopy enables us to study the three-dimensional samples, including its interior, in particular materials and allows imaging of the surface by reflection.
The system also allows laser plots of surface microtopography.
- Spectral Confocal Microscope Leica TCS SPE, with 3 lines Laser 488nm, 532 nm and 635 nm fully automated. Has a program MetaMorph image analysis available to the user.
Confocal microscopy sessions may have technical assistance or not have it.
Sessions with technical assistance:
Lab technicians try to adjust the schedule and needs of the various working groups. This has been made sessions and different time durations to allow greater flexibility and adaptation to user needs with the lab.
The available technical assistance sessions have the following times:
There are four sessions: 8: 30-12: 00 // 9: 30-13: 00 // 13: 00-14: 30 // 14: 30 to 16:00
For more effective, except in special cases operation, please avoid booking more than 2 sessions per week.
To schedule the job to do, is to get on initial telephone contact with the laboratory; depending on the timing of the users and the need for new work a series of initial sessions will be scheduled. The number and frequency of these will take place trying to complete the work of each user without accumulation of equal work sessions or one person takes place, in order to avoid too long waiting time for users. The sessions are to be provided well in advance to avoid long waiting lists and agglomerations in certain periods of greatest assistance due to foreign visitors, conferences or routine checks. Due to the long-term programming that takes place in some cases, it may be the fact of having to change and / or cancel any already scheduled session. In these cases we will try, where possible, to minimize impacts users trying to be fair with them.
The reservation can be canceled at any time by notifying the technical manager of laboratory time to change shifts to another user. If cancellations consecutive repetition without giving notice, ie arriving at the day of the appointment, and consecutively the person concerned does not show, you can choose to collect the session although it has not enjoyed.
Sessions WITHOUT technical assistance:
NO technical assistance sessions are:
8:10 to 11: 30 // 11: 30-14: 30 // 14: 30 16:50
For more efficient operation, it should not exceed 6 hours of use per week per person. If necessary to use the service for longer time may be extended after it known to service technicians who report the availability of equipment. In case of saturation of the service, users will be notified in advance of a reduced maximum time of booking.
The reservation can be canceled at any time by notifying the technical manager of laboratory time to change shifts to another user.
Users who will manage the team by themselves, will have the first meeting attended and the following will be advised and assisted by technical staff assigned to the service, to gain autonomy in the development work. Should any difficulties arise during the session unattended equipment, lab technicians can advise and seek to resolve the problem. These users communicate technical staff incidents that may have occurred during handling equipment, in particular those that may require repair or maintenance. This will avoid inconvenience to users who want to use the computer in the next session.
Routines and more delicate operations (the calibrated laser alignments and changes) MUST be done by lab technicians). The third training by users is not advisable or desirable therefore is not allowed.

Confocal Leica SP5, with 4 different lasers:
-UV diode
-Argon laser, frequencies 458, 476, 488, 496 y 514nm
-HeNe laser, 543 nm
-HeNe laser, 633 nm
Capabilities:
Multidimensional imaging (xyzt)
High speed acquisition of optical sections
Optimized for colocalization and quantification
Mosaic-type image generation
3D reconstructions
FRAP, FRET, FLIP
Personalized assistance during image acquisition and quantification provided by the Service.

Accurate surface analysis is essential in industry and research to help ensure optimal performance of materials and components. But surfaces can be made up of intricate structures with highly sloping areas demanding lateral resolution of a few microns, or critical micro peaks and valleys requiring vertical analysis on the sub-nanometer scale
Sensofar PL 2300 is a perfect tool to evaluate roughness and texture parameters of material surfaces. It allows three-dimensional measurements of surfaces of samples using the high repeatability of interferometrical techniques. It is a non contact fast optical profiler, avoiding scratching, flexing and dumping typical of contact profilers.
The profiler provides Confocal and Vertical Scanning Interferometry measurement techniques. The choice of the technology being used is as easy as selecting the appropriate objective in the software.
The PL 2300 can use three different types of confocal objectives. The EPI objectives are designed to have the highest numerical apertures, up to 0.95, working very close to the surface. These objectives give the highest repeatability of the system and are ideal for flat and thin samples. The ELWD and SLWD series are objectives of Extra Long Working Distance and Super Long Working Distance, up to 21 mm. They have low numerical aperture and are used in samples where the objective can collide physically. The PL 2300 can also use interferential objectives.
The equipment is located in the laboratories of the area of Materials Science and Metallurgical Engineering at the School of Engineering and Architecture ( Torres Quevedo Building, Campus Rio Ebro ) .

Multispectral confocal microscopy studies with:
* Leica TCS SP5 system. Detection: 5 PMTs + PMT transmitted light. Excitation: laser lines at 405, 458, 476, 488, 514, 561, 594 and 633 nm. Plan Apo objectives: 10x/0.4, 20x/0.75,40x/1.25 oil, 63x/1.20 water, 63x/1.40 oil and 100x/1.4 oil.
* Leica TCS SP8 system: Detection: 3 gating hybrid detectors (gHyD) + 2 PMTS + PMT transmitted light. Excitation: 405 nm laser lines and WLL2 white laser (from 470 to 670 nm). Plan Apo CS2 objectives: 10x/0.4, 20x/0.75, 63x/1.20 water, 63x/1.40 oil and 100x/1.4 oil.
STED (STimulated Emission Depletion) nanoscopy with the TCS SP8 system. Depletion laser lines at 592 and 660 nm.
Dynamic and time-lapse studies with the TCS SP5 confocal microscope (incubation system with CO2 and temperature control).

- Confocal microscope Leica TCS SPE with lasers of wavelengths 488, 532 and 635.
- Andor Dragonfly Spinning disk microscope with three lasers (405 nm , 488 nm y 561nm). Andor Dragonfly Spining disk is a multi-point confocal
for high-speed and high-sensitivity imaging. Dragonfly is the optimal solution for live cell imaging, providing low phototoxicity and photobleaching, or perfect for fast volume acquisition of fixed samples.
The use may be self-service or by specialized staff of the Institute (training)
See general conditions of use duties of users.
USER DUTIES:
1) Read and respect the rules and instructions of microscopes usage, both placed in the Web site (https://www.iibb.csic.es/ ) and arranged on the notice board of the instruments.
2) Book the instruments through the IIBB intranet (internal users) or by contacting the persons in charge of the service (external users).
3) Receive the necessary training from the technical staff until necessary. Once the user can manage the equipment without technical assistance, he shall be regarded as "advanced user" of such a system.
4) Leave the equipment and material clean and protected after use.
5) Notify the technical staff about any incident or problem observed.
6) Respect safety rules.

In particular the following technologies are offered:
1.Confocal and two-photon microscopy
2.TIRF microscopy
3.Functional Imaging. With emphasis upon quantification of molecular interactions by FRET-FLIM and FCCS
3.1.Fluorescence Lifetime Imaging (FLIM), including living cell FLIM, focused on quantitative inter- and intramolecular FRET quantification
3.2.Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Cross Correlation Spectroscopy (FCCS): measurements of molecular diffusion, affinity constants and intermolecular interactions in solution
3.3.Scanning FCS and scanning FCCS: measurements of molecular diffusion, lipid-protein and protein-protein affinity constants and molecular interaction in GUV (giant unilamellar vesicles) and living cells.
3.4.Generalised Polarisation (GP): quantitative measurements of lipid order in GUV
Technical description of the instrumentation
¿Multiphoton scanning microscope: Leica SP5, Spectra-Physics Mai-Tai (pulsed Ti:sapphire laser tunable from 690 to 1040nm) and Coherent Mira (pulsed Ti:sapphire)
¿Incubator and chamber for living cell imaging during extended periods
¿High quantum efficiency GaAsP hybrid detectors and APDs for sensitive detection
¿TCSPC card 1: Becker & Hickl SPC 830 (FLIM, FCS and FCCS)
¿Analysis software: Imaris and Huygens. In-house developed analysis software