To accurately describe the way in which you did your imaging you should include the following information (where relevant):
- Type of system/microscope (eg Zeiss 510 confocal mounted on an Axio Observer microscope stand)
- Objective you used (eg 63x 1.4 NA oil Plan-Apochromat)
- Wavelengths of excitation and emission (eg 488 nm line of Argon laser with a longpass 500nm filter)
- Camera (eg Coolsnap ES2 from Photometrics)
- Software for acquisition (eg MetaMorph 7.5) and general typical settings (eg ND, exposure time, binning, interval in t and z)
- Details of any image processing or analysis procedures
Below are example starting points for preparing methods sections of papers, which should be expanded with the relevant information outlined above. Further technical details of the microscopes are on their respective pages.
Zeiss 510 confocal
Samples were examined using a Zeiss LSM 510 confocal on an Axio Observer microscope using a Zeiss Plan-Apochromat 63x/1.4 oil objective. Images were collected using 405, 488 and 561 nm laser lines for excitation and BP420-480, BP505-550, and LP575 emission filters for DAPI, Alexa488, and Alexa568 fluorophores, respectively, with pinholes set to 1 airy unit for each channel, line averaging of 8, 1024x1024 image format, and 2X optical zoom. Z-stacks were acquired with an interval of 0.3 um and the gain and offset optimized for the brightest central planes of the stack.
Cells were imaged on a spinning disk confocal (Yokogawa CSU10 scanhead) on an olympus IX-70 inverted microscope using 100x/1.35 NA UPlanApo oil objective, 488 nm and 568 nm Kr-Ar laser lines for excitation and ORCA ER CCD camera, the system was controlled by MetaMorph.
Cells were imaged on a Zeiss Axio Imager upright widefield fluorescence microscope using a 40x/0.75 Plan-NeoFluar objective and green and red filtercubes (chroma). Images were captured on a Hamamatsu ORCA ER CCD camera, the system was controlled by MetaMorph.
Cells were imaged on a Leica SP5 confocal microscope using a 40x/1.25 oil objective using 488, 561 and 633 laser lines for excitation. The settings were optimized and the final images scanned with line-averaing of 4.
Live cell station
Cells were imaged at 37C on a Zeiss Axio Observer inverted microscope with stage incubator, CO2 buffering and outer environmental chamber. A Coolsnap ES2 OR QUANTEM EMCCD camera was used for acquisition and the system was controlled by Metamorph.
Acknowledgement of LMCF in publications is appreciated and helps to demonstrate our utility to funding sources. Shared instrumentation grant 1S10RR027528-01 funded the DeltaVision and 1S10RR027867-01 funded the intravital confocal systems.