2 photons excitation + photo-ablation

Two-photon excitation microscopy is an alternative to confocal microscopy. It allows observation of fluorescence at a greater depth which limits photobleaching and phototoxicity. It is particularly suitable for the observation of thick, living samples over a long period of time, but also for transparent samples up to 1mm. The straight microscope stand allows intravital observation. This microscope also allows the detection of second (SHG) and third harmonics (THG).

  • Principle

Like confocal microscopy, multi-photon (or bi-photon) microscopy uses a pulsed laser to excite a fluorescent marker on a sample and a detector to acquire the emission light.
However, the laser used in two-photon microscopy allows the excitation of a fluorescent marker by its simultaneous absorption of the energy of two photons instead of just one in conventional microscopy. The laser is pulsed in order to send highly concentrated photon bundles (high peak powers) and increase the chances of simultaneous photon absorption. The two photons absorbed almost simultaneously are only absorbed at the focal point where the light is most concentrated and must be of close wavelength. The two absorbed photons are of low energy and have the same effect as an absorbed photon of higher energy.
These characteristics lead to several advantages:
- As the photons are of lower energy, their wavelength is longer (near infrared for fluorophores absorbing in the visible). The longer wavelengths used are less damaging to the sample and penetrate more into the tissue than those of shorter wavelengths.
- The absorption of the energy of several photons will be confined to the focal region. Fluorescence is therefore emitted only from this region, there is no light coming from outside the focus that could blur the image. This creates the conditions for a confocal image that does not require a pinhole at emission.
- Photodynamic processes such as the irreversible destruction of fluorescent molecules or the production of free radicals only occur at the focal region. Often minimizing the invasiveness of bi-photon microscopy compared to single-photon confocal microscopy.
- The wavelength of bi-photon excitation is typically twice the wavelength of single-photon excitation. The separation between the two excitation and emission spectra ensures that the excitation light will be well filtered and the emitted light will be detected.

Equipements disponibles

Lasers femtosecond :
Insight Spectra Physics (690-1300nm), puissance moyenne 1W partout
MaiTai Spectra Physics, 690-1060nm, puissance max 3W
Le laser Mai-Tai peut être utilisé indépendamment du laser Insight, comme un laser d’ablation, avec la possibilité d’ablater et de faire l’imagerie en même temps, en utilisant 2 scanners indépendants.

Balayage :
2 scanners galvanométriques pour le bras de l’imagerie et pour le bras de l’ablation, 1 scanner résonant pour l’imagerie
Objectifs : Zeiss 20x 1.0 (eau), Zeiss 40x 1.3 (eau), Zeiss 63x 1.3 (eau)

Emission :
Détection non déscannée en réflexion 4 PMTs GaAsp
405/15 SHG, 460/60 DAPI, 525/50 GFP, 560/40, 620/60, 670/30, 610/10 THG
Détection non descanée en reflexion 2 PMTs GaAsp couplés au scanner résonant

Innovation and specificity of the microscope:

  • 2 IR pulsed lasers (Insight 700-1300nm and MaiTai 700-1060nm)
  • 4 GaAsp detectors in reflection – 2 GaAsp detecors for resonant scanner
  • Possibility of ablating and imaging at the same time (with 2 lasers – 2 different excitation paths-scanners)
  • Classic or resonant scanner

Applications:

  • Label-free imaging (SHG-THG)
  • In depth imaging of brain slices/tissues/organisms
  • Embryon imaging and development
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