Microscope: Leica SP8 point-scanning confocal system, on a Leica DMI6000B inverted microscope, fully motorized, with Leica “SuperZ” Galvo stage, four spectral fluorescent light detectors (three PMT, one HyD high sensitivity detector), one transmitted light detector (PMT)
Objectives:
Position | Objective | DIC |
1 | 10x/0.4 dry HC PL APO 1.x/0.40 CS | (prism not installed) |
2 | 20x/0.7 dry HC PL APO 20x/0.70 CS | DIC |
3 | 40x/0.85 dry HCX PL APO 40X/0.85 CORR CS,0.11- | DIC |
4 | 40x/1.1 water HC PL APO 40x/1.10 W CORR CS2 | DIC |
5 | 40x/1.3 oil HC PL APO 40x/1.30 Oil CS2 | DIC |
6 | 63x/1.4 oil HC PL APO 63x/1.40 OIL CS2 | DIC |
Confocal mode: Lasers, no fluorescence emission filters (all spectral detectors):
Lasers (all diode) | Typical fluorophore |
405nm, 50mW, DMOD | DAPI |
448nm, 40mW, AOTF | CFP |
488nm, 20mW, AOTF | AF488, GFP, YFP |
552nm, 20mW, AOTF | TRITC, Cy3 Texas Red, RFP |
638nm, 30mW, AOTF | AF633, Cy5, FarRedFP |
Laser modulation by AOTF (acusto-optic tunable filters), or DMDO (direct modulation of laser intensity)
Wide-field fluorescence:
Light source: Leica EL6000 metal-halide lamp
Fluorescence filter cubes, Leica:
Filter set | Excitation filter | Dichroic | Emission filter | Typical fluorophore |
I3 | BP450-490 | 510 | LP515 | AF488, GFP |
N2.1 | BP515-560 | 580 | LP590 | TRITC, RFP |
A | BP340-380 | 400 | LP425 | DAPI |