Widefield-TIRF - Leica SR GSD 3D (Irchel) | Center for Microscopy and Image Analysis

The Leica GSDIM (Ground State Depletion followed by individual molecule return) microscope consists of an inverted widefield fluorescence microscope combined with a very sensitive EMCCD camera for single molecule detection. Fluorescent molecules can be excited using 3 different laser lines. This single molecule excitation and detection scheme allows the acquisition of super resolved images using the STORM (STochastic Optical Reconstruction Microscopy) principle. Compared to standard widefield fluorescence microscopy, this technique offers a dramatically increased lateral (20nm) and axial (50 nm) resolution. For experiments in membranes, and in the presence of high fluorescence background, TIRF (Total Internal Reflection Fluorescence) imaging is also available.

Location

University Zurich, Irchel Campus, Room Y42-H-93.

Training Request

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

Technical Specifications

Microscope

inverted widefield microscope (Leica DMI6000B / AM TIRF MC)
SuMo Stage - manual sample stage
"online" super-resolution image projection - see the emerging image during aquisition
PIFOC piezo nanofocusing system for 160x objective

Light Sources and Lasers

Halogen lamp for transmitted light
External fluorescence lamp LQ-HXP 120
solid state diode lasers: 405 nm (30 mW), 488 nm (300 mW), 532 nm (500 mW), 642 nm (500 mW)

Objectives

Name	Magnification	NA	Immersion	WD (mm)
HCX PL APO	10x	0.4	Air	2.2
HCX PL APO CS	20x	0.7	IMM	0.26-0.17
HCX PL APO CS2	63x	1.3	glycerol	0.3
HCX PL APO (GSD/TIRF)	160x	1.43	oil	0.07

IMM = multi immersion (either water, glycerol or oil)

Fluorescence Filters

Name	Mode	Excitation Range	Excitation Filter	Dichroic	Emission Filter
488 HP-T	GSD/TIRF	blue	BP 488/10 *	496	BP 555/100
532 HP-T	GSD/TIRF	yellow	BP 532/10 *	541	BP 600/100
642 HP-T	GSD/TIRF	red	BP 642/10 *	649	BP 710/100
488T	TIRF	blue	BP 488/10	500	BP 525/50
QUADHP-T	GSD/TIRF	violet/blue/yellow/red	BP 405/10; BP 488/10; BP 532/10; BP 642/10	417; 496; 544; 655	BP 449/56; BP 508/22; BP 584/74; BP 699/66

* additionally show an excitation transmission window for 405nm laser (BP 405/10) to allow for back-pumping of fluorophores during super resolution measurements

Camera Systems

Andor iXon Ultra 897 EMCCD camera
cooled monochrome camera
512 x 512 pixel (16 x 16 um) back-illuminated CCD
max. frame rate at 512 x 512 pixel: 56 fps

Accessories

sample holder for 75 x 25 mm glass slides
special inset for 35 mm petri dishes
custom made sample holder for 75 x 25 mm ibidi chambers

Remarks

Single molecular techniques such as GSD have special requirements in terms of fluorescence dyes and buffer systems. Be sure to contact the responsible persons before planning your experiments.