Confocal LSM 700

Introduction

In confocal microscopy, placement of a variable spatial pinhole at the confocal plane of the lens eliminates out of focus light thus reducing background interference.

The Zeiss LSM700 is a laser scanning confocal system attached to an inverted motorized microscope (Axio Observer). Its variable secondary dichroic beamsplitter splits emission signals between the two PMTs and the cutoff can be freely selected in 1nm steps. For short term live experiments, it can be optionally equipped with a heating stage insert for 35mm plates.

Laser line	Wavelength	Power	Suitable fluorophores
Solid state laser cassette	633 nm	5mW	Cy5, APC, Alexa 647, TO-PRO-3
Solid state laser cassette	555 nm	10mW	Cy3, TAMRA, PI, Texas Red, PKH26
Solid state laser cassette	488 nm	10mW	Cy2, GFP, Mitotracker Green, JC1
Solid state laser cassette	405 nm	5mW	DAPI, Hoechst

Objective lens	Magnification	NA	coverslip	Working distance	Immersion
EC Plan Neofluar	x10	0.3	0.17 mm	5.2 mm	Air
Plan Apochromat	x20	0.8	0.17 mm	0.55 mm	Air
LD LCI Plan Apochromat	x25	0.8	0-0.17 mm	0.57 mm	Oil Glycerin Water
LCI Plan Neofluar (optional)	x25	0.8	0-0.17 mm	0.21 mm	Oil Glycerin Silicone Oil Water
EC Plan Neofluar	x40	1.3	0.17 mm	0.21 mm	Oil
Plan Apochromat	x63	1.4	0.17 mm	0.19 mm	Oil

Acquisition

Acquisition is performed with the Zeiss proprietary ZEN software.

Two PMT detectors for simultaneous detection and up to four sequential tracks and one transmitted-light PMT allow superimposing fluorescence images on brightfield or DIC images.
The software allows 360° XY scanning field rotation, and freely definable regions of interest (ROI).
Acquisition modes: Spot, line/spline, frame, lambda stack.
Resolution up to 2048 x 2048 pixels, scanning speed up to 5 frames/sec at 512 x 512 pixels.

Special applications

Photobleaching, short term live cell imaging, ROI, positions, tiling, time series and combination (XYZ|T), increased intensity over Z.

Analysis

The Zen software provides a wide range of 2D/3D image processing tools: export to TIFF, maximum projection, 3D reconstruction, 2D colocalization, spectral unmixing, tile stitching and more. For basic visualization and processing you can download the free ZEN lite edition software. An offline full version is available for analysis at Computer IMARIS software. Output files can be opened by Imaris and ImageJ/FIJI for analysis.

Instructions for specific applications