Protocols
Information for Manuscript Preparation
Microscope: Leica DMIRBE
Confocal: Leica TCS-NT/SP (Leica Microsystems GmbH, Mannheim, Germany)
Old software version: 1.6.587
New software version: 2.770
Objectives typically used: 40x NA 1.25, 63x NA 1.32, 100x NA 1.4
Excitation wavelengths available: 364nm (UV, DAPI, Hoechst) 488nm (FITC, GFP, Cy3) 568nm (TR, Rhodamine, PI, Alexa 568), 633nm (Cy5)
Microscope: Leica DMIRBE
Confocal: Leica TCS-SP2 (Leica Microsystems GmbH, Mannheim, Germany)
Software version: 2.871
Objectives: 10x NA .4, 20x NA .7, 40x NA 1.25, 63x NA 1.32, 100x NA 1.4
Excitation Wavelengths: 364nm (UV, DAPI, Hoechst), 457nm (CFP), 476nm, 488nm (FITC, EGFP, Cy3), 514nm (YFP), 568nm (TR, Rhodamine, PI, Alexa 568), 633nm (Cy5)
Lasers: (both confocals) 364nm Argon laser, Enterprise 651, Coherent Inc, Santa Clara, CA
457, 476, 488, 514nm: Argon laser
568nm: Krypton laser
633nm: Helium-Neon laser (HeNe)
The zoom factor should be mentioned if confocal zoom was used. If Z stacks of images were collected (optical sections) the step size (increment) should be mentioned (e.g., .203um optical section thickness).
A typical paragraph might be as follows:
Images were collected on a Leica TCS-NT/SP confocal microscope (Leica Microsystems, Exton, PA USA) using a 63x oil immersion objective NA 1.32, zoom X. Fluorochromes were excited using an argon laser at 488nm for FITC and a krypton laser at 568nm for Alexa 568. DAPI was excited using an Argon laser (Enterprise model 651, Coherent Inc.) at 364 nm. Detector slits were configured to minimize any crosstalk between the channels. (If the two channels were collected separately, delete the previous line and indicate the two wavelengths were collected separately and later superimposed.) DIC (differential interference contrast) images were collected simultaneously with the fluorescence images using the transmitted light detector. Images were processed using Leica TCS-NT/SP software (version 1.6.587), Imaris 3.3.2 (Bitplane AG, Zurich Switzerland), and Adobe Photoshop 7.0 (Adobe systems).
If the detectors were configured specifically for your experiment, list the emission wavelengths collected when the minimization of crosstalk is mentioned. For example: FITC emission was collected between 504-551nm and Alexa 568 emission between 610-670nm.
Other software:
Adobe Illustrator 9.0
Autoaligner 2.0 (Bitplane AG)
Image Pro Plus 4.5 (Media Cybernetics)
back to top