Phiv, plateau d'histologie végétale

Multiphoton microscope coupled to a vibratome for imaging 3D

Principe

Confocal: Light microscopy uses transmitted light to directly observe the object. Confocal microscopy uses exciting laser beam focused with a convergent lens that scans surface by positioning the pinhole in front of the detector, in a focal plan combined in the object focal plan (confocal plans). As a consequence, only the photons coming from the focal plan pass through the pinhole and build the image, from where the name “confocal”.
Scanning by the laser is done using two orthogonal mirrors. The detectors used are tubes photomultipliers (PMT). The light intensity is measured and digitized according to the position of the laser beam in the sample: one obtains digital images directly.
The most frequently used lasers are as follows:
* argon-ion (wavelengths: 457nm, 488nm, 514nm)
* helium-neon (543nm)
* helium-neon (633nm)

Multiphoton:The low field depth is obtained when emitted fluorescence is coming from a small volume submitted to an excitation by 2 or more photons (multiphoton) using pulsed lasers (100 fs) in infra-red (700-1300 nm). The fluorescence emission is only reached with a quasi synchronous excitation of a molecule by more than one photon with a wavelength close to a multiple of the optimal excitation wavelength with a unique photon. There is no need of a pinhole and the totality of the fluorescence reaches the detector.
Advantages : a better penetration in the sample (up to 500 µm depth), the cellular photo-bleaching restricted to the only focal area, improving signal-to-noise ratio.

Use

Access to in situ localisation of molecules and biological activities.
3D Imagery: 3D reconstruction from serial optical sections

Examples