Multi-photon microscope uses an ultra-fast pulsing laser to excite fluorescent molecules. Briefly, 2 (multi)-photon microscopy is based on the principle that a given fluorescent molecules which normally would only be excited by absorbing the energy of a single photon of certain wavelength can also be excited by combination of the energies produced by simultaneous absorption of 2 (or multi-) lower-energy photons of double (multiple) wavelength.
The emission of the fluorescence is quadratically dependent on the excitation intensity. This steep dependence of absorption rate on photon concentration gives multi-photon Laser Scanning microscope the intrinsic three-dimensional resolution with the depth of field defined by a) intensity of the excitation light, b) numerical aperture of the lens used and c) the wavelengths of the lights. This z-resolution is comparable to conventional Laser Scanning confocal microscope.
Multi-photon offers some distinct advantages over confocal microscope:
- Longer wavelength used for excitation allows imaging deeper into thick specimens due to less scattering of longer wavelength (IR) light.
- Photo-bleaching and damaging are confined to the focus points only.
- It is possible to image UV dyes with infra-red light. This is particularly useful for live cell imaging where UV light is highly damaging to the cells
Therefore, multi-photon microscopy is highly suitable for imaging of live, thick specimens.
The Facility possesses a Zeiss NLO510 system with a Coherent Mira 900 femto-second laser pumped with a 5W Verdi laser (Coherent Inc.). The system is easy to use and the user interface is virtually the same as Zeiss LSM710. Anyone used Zeiss LSM 710 can use it with a short training. However, tuning of the femto-second laser is accomplished by dedicated person only.
Here Hela cells were stained with Hoechst dye and imaged with 2-photon at wavelength of 720nm using a short pass filter of 680. The nucleus was reconstructed from 38 optical sections of the cells using Zeiss LSM software.
LSM 510 NLO info:
Microscope: Zeiss Axiovert 200M (inverted)
10x 0.45NA EC Plan-Neofluar
20x 0.8NA Multi immersion Fluar
40x 1.3NA oil Plan Neofluar
40x 1.2NA Water C-Apochromat
63x 1.4NA Oil DIC Plan-Apochromat
Argon: 458, 488, 514nm
Detectors: 2 fluorescence PMTs, 1 transmission PMT, and FCS
Location: Room 2322