LSM510NLO
The system consists an Axiovert 100 inverted microscope
Lenses: 10x, 20x dry; 40x water (1.2 n.a., working distance 220 µm); 63 x oil (1.4 n.a.,working distance 90 µm); 100 x oil (1.3 n.a., working distance 60 µm)
3 detectors + transmission detector and monitor diode.
Scanning system:
Visual light lasers: Argon gas laser (458, 488, 514 nm); Helium Neon (543 nm); Helium Neon (633 nm)
Multi-photon lasers: Coherent Verdi pump laser (5W), Mira 900 multi-photon laser tunable (600-1000 nm). This laser is coupled to the microscope with a fibre. Usable wavelengths 750-920 nm.
Beam splitters: NFT 490, 515, 545, 635 VIS Filters: BP 390-465 IR, 480-520 IR, 505-530 IR, 500-550 IR, 535-590 IR, 565-615 IR, LP 475, 505, 530, 560, 650, KP 685 (IR filters are specially 'coated' to block the multi-photon infrarad light)
Confocal microscope
A pinhole in front of the detector is used to reduce the out of focus light dramatically. In this way very sharp 3D images can be made without fixation of the sample. We use a Pekon P-insert with a air CO2 flow in combination with lens heating to keep cells alive and follow them normally for 14-16h, but 48h is also possible.
Multi-photon microscope
The advantages of a multi-photon laser scanning microscopy are mainly found in the deeper penetration. Because of the confocal nature of multi-photon microscopy no pinhole is needed in front of the detector which leads to higher photon yields. This in combination of the longer wavelength used gives to ability to penetrate deeper into the specimen.
FCS
Zeiss FCS scanhead.
Ability to measure single fluorescent molecules that travel through the static laser beam. 2 avalange photo diodes (APD) are used to measure the signal. The software provides a diffusion time and the number of molecules in the confocal volume. These values can be recalculated to diffusion coefficients (µm2/s) and concentration