91˿Ƶ

CIAN-SP8 – Confocal Laser Scanning

91˿Ƶ

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

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