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 LSM 510 meta is a Zeiss laser scanning confocal system attached to an inverted motorized microscope (Axiovert 200M). It has three PMTs and the Meta detector which allows simultaneous acquisition of up to 8 channels of the emission spectrum to separate fluorophores with overlapping emission spectra or as an additional PMT.
|Laser line||Wavelength||Power||Suitable fluorophores|
|Multiline Argon||458, 477, 488, 514||30mW||Cy2, GFP, Mitotracker Green, JC1|
|Diode-pumped solid-state||561||10mW||Cy3, TAMRA, PI, Texas Red, PKH26|
|Helium-Neon||633||5mW||Cy5, APC, Alexa 647, TO-PRO-3Cy2, GFP, Mitotracker Green, JC1|
|UV Diode||405||30mW||DAPI, Hoechst|
|Objective lens||Magnification||NA||coverslip||Working distance||Immersion|
|EC Plan Neofluar||x5||0.15||0.17 mm||18.5 mm||Air|
|EC Plan Neofluar||x10||0.3||–||5.2 mm||Air|
|LCI Plan Neofluar||x25||0.8||0-0.17 mm||0.21-0.17 mm||Oil Glycerin Water|
|EC Plan Neofluar||x40||1.3||0.17 mm||0.21 mm||Oil|
|EC Plan Apochromat||x63||1.4||0.17 mm||0.19 mm||Oil|
|C Apochromat||x40||1.2||0.14-0.19 mm||0.28 mm @ cover glass 0.17||Water|
Acquisition is performed with the Zeiss proprietary ZEN 2009 software.
Three PMT detectors and the META detector for simultaneous detection 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: