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 upright motorized microscope (Axio Imager.Z2). Its innovative variable secondary dichroic beamsplitter splits emission signals between the two PMTs and the cutoff can be freely selected in 1nm steps.
|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|
|EC Plan Neofluar||x40||1.3||0.17 mm||0.21 mm||Oil|
|W Plan Apochromat||x40||1.0||2.5 mm||Water dipping|
|Plan Apochromat||x63||1.4||0.17 mm||0.19 mm||Oil|
|C Apochromat (optional)||X40||1.2||0.14-0.19||0.28 mm||Water|
Acquisition is performed with the Zeiss proprietary ZEN 2010 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.
Photobleaching, live imaging, ROI, positions, tiling, time series and combination (XYZ|T), increased intensity over Z.
The Zen software provides a wide range of 2D/3D image processing tools: export to TIFF, maximum projection, 3D reconstruction, 2D colocalization, spectral unmixing 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 Analysis 4. Output files can be opened by Imaris and ImageJ/FIJI for analysis.
Please contact Edith Suss-Toby, tel. +972-4-8295347/61 to coordinate a meeting.
Tips for immunofluorescence experiments.
Please prepare controls before starting a new model.
Reading materials prior to the first instruction session: