CLSM - Leica SP8 inverse STED 3X (Irchel) | Center for Microscopy and Image Analysis

The gSTED super-resolution microscope is based on a Leica SP8 inverted confocal microscope outfitted with a very flexible white light laser source and additional depletion laser lines. This allows using the STED (STimulated Emission Depletion) principle to perform imaging beyond the diffraction limit. Under optimal conditions a resolution of 50 nm laterally (xy) and 130 nm axially (z) can be achieved. In contrast to other super-resolution techniques, STED microscopy can directly be applied to samples suitable for standard confocal microscopy. This also includes imaging living cells.

Location

University Zurich, Irchel Campus, Room Y42-H-81.

Training Request

Follow this link to apply for an introduction to the microscope.

Technical Specifications

Microscope

inverted confocal microscope (Leica DMI6000B, Model SP8)
5 freely selectable emission detection windows
motorized x/y stage
3D capability (confocal, STED)
time lapse capability

Light Sources and Lasers

Halogen lamp for transmitted light
External fluorescence lamp LQ-HXP 120
Diode laser 405 nm (50mW)
White light laser (WLL): continuous laser output in the range of 470-670nm; AOTF allows to choose up to 8 different wavelengths simultaneously; pulsed laser system with a repetition rate of 80 MHz
Continuous wave depletion lasers at 592 nm and 660 nm
Pulsed depletion laser at 775 nm

Scanners (tandem scanner system)

confocal point-scanning - optical sectioning
zoom function
Regular scanner (field-of-view scanner): 1 Hz to 1800 Hz
Resonant scanner: 12 kHz

Detectors

5 confocal fluorescence channels: three Photomultipliertubes (PMTs) and two super-sensitive Hybrid detectors (HyD, suitable for time gated detection)
one transmission light-channel (PMT-Trans)
time gated single photon detection with the two HyD detectors: allows to improve the STED resolution using the time domain (gated STED) as well as for suppression of short lived effects such as scattering of excitation light

Objectives

Name	Magnification	NA	Immersion	WD (mm)
HCX PL FLUOTAR	10x	0.3	Air	11.0
HC PL APO CS2	20x	0.75	IMM	0.68
HC PL APO CS2	63x	1.4	oil	0.14
HC PL APO STED WHITE - motCORR	93x	1.3	glycerol	0.3
HC PL APO STED WHITE	100x	1.4	oil	0.1

^{CS2 objectives: improved color correction, perfect VIS-405; WHITE: optimal color correction for full spectrum; IMM = multi immersion (either water, glycerol or oil)}

Fluorescence Filters

Name	Excitation Range	Excitation Filter	Dichroic	Emission Filter
A4	UV	BP 360/40	400	BP 470/40
I3	blue	BP 470/40	510	LP 515
N2.1	green	BP 515-560	580	LP 590
CFP/YFP	violet/blue	BP 436/12; 500/20	445; 515	BP 467/37; 545/45

Accessories

controlled environmental conditions (CO₂, temperature, humidity)
Ludin chambers for 12 mm and 18 mm coverslips (reservation needed)
Matrix screening software (reservation needed)

Make sure to acknowledge the Center for Microscopy in your publication to support us.

How to acknowledge contributions of the Center for Microscopy

Remarks

Using a depletion laser is an obligate requirement for performing STED super-resolution imaging. Choosing the appropriate dyes or fluorescent proteins suitable for depletion by one of the different depletion lasers is crucial for performing successful STED experiments. We highly encourage you to contact us for suitable candidates before starting your experiment.