Confocal Laser Scanning Microscope (CLSM)

Principle

The confocal microscope is comparable to an optic microtome. Its principle is based on the focalization of a laser beam through an objective, which will excite dyes in one point of the sample, then a photomultiplier will collect signals emitted from this point. A diaphragm (pinhole) is positioned in front of the detector in order to stop signals coming from planes out of focus. These signals are amplified in the detector, treated for improving signal to noise ratio and digitized. The final image is a point-to-point construction resulting from the scanning of the laser beam thanks to deflective mirrors. A motorized plate moves the sample in z allowing the acquisition of several optic planes in the thickness of the object.

Available equipments

The facility offers 3 laser scanning confocal microscopes:

  • TCS SP5 (Leica) :

Lasers : Laser diode 405 nm (25mW) ; Argon 458, 476, 488 nm (40mW) 496 and 514 nm ; laser diode 561 nm (15mW) ; Helium neon 633 nm (10mW)
4 PMTs detectors for fluorescence (spectral acquisition available)
1 PMT detector in transmission for brightfield acquisitions and Nomarski interferential contrast (DIC)
"Resonant Scanner" system available for fast acquisitions.
Motorised plate in XY for multi-position and tile scan acquisitions.
Motorization in Z and galvanometric plate
Chamber controlled for temperature and CO2

Avalaible Objectives on the TCS SP5 confocal microscope

  • LSM 710 (Zeiss) :

Statif Axio Observer Z.1
Lasers : Laser diode 405 nm (30 mW) ; Argon 458, 476, 488 nm (25mW) 496 and 514 nm ; DPSS 561 nm (15mW) ; Helium neon 633 nm (5mW)
2 PMTs detectors for fluorescence
Spectral detector for real-time spectral acquisitions
1 PMT detector in transmission for brightfield acquisitions and Nomarski interferential contrast (DIC)
Motorized plate in XY for multi-position and tile scan acquisitions
Motorization in Z
Chamber controlled in temperature and CO2
Available objectives on the LSM710 confocal microscope

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[Translate to Anglais:]

  • LSM 780 (Zeiss) :

- Statif Axio Observer Z.1
- Lasers: Diode Laser raie à 405nm(30mW); Argon raies à 458, 476, 488 (25mW) 496 et 514nm; DPSS raie à 561nm(15mW); Hélium néon raie à 633nm (5mW)
- 2 détecteurs PMTs pour la fluorescence
- 1 détecteur spectral GaAsP plus sensible pour des acquisitions spectrales en temps réel
- 1 détecteur PMT en transmission pour des acquisitions en brightfield et contraste interférentiel de Nomarski (DIC)
- Platine motorisée en XY pour des acquisitions multi-position et mosaïque
- Motorisation en Z
- Enceinte contrôlée en température et CO2
- Système d’Autofocus intégré (Definite Focus) pour éliminer la dérive en Z
Objectifs disponibles sur le microscope confocal LSM780

Tutorial

Online tutorial on ibiology.org.

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