To accurately describe the way in which you did your imaging you should include the following information (where relevant):
- Type of system/microscope (eg Leica SP8 confocal attached to a DMi8 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 emission set to 490nm-550nm)
- Camera (eg Coolsnap HQ2 from Photometrics or Andor iXon 888 EMCCD)
- Software for acquisition (eg MetaMorph 7.10 or Zeiss Zen blue) 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. Please feel free to ask us questions. We have been involved in promoting improved microscopy methods reporting: Montero Llopis et al 2021 Nature Methods - "Best practices and tools for reporting reproducible fluorescence microscopy methods".
Zeiss 880 laser scanning confocal
Fixed (or live) (xxx – sample) were imaged on a Zeiss 880 inverted confocal microscope with a Märzhäuser linearly encoded x,y stage attached to a Zeiss Observer Z.1 inverted stand and a 63x 1.4 NA oil-immersion plan-Apochromat objective. Laser illumination was via Argon for 488nm, diode 405nm and 561nm and HeNe 633nm. Fluorescence signal was collected with 2 photomultiplier tubes and one GaAsP detector in the following emission ranges: for DAPI - 415-487nm, for AlexaFluor 488 - 490-570nm GaAsP, for mCherry 570-633nm (GaAsP) and for AlexaFluor 647 - 633-670nm. 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. Images were acquired sequentially by line scanning at 0.52 microseconds per pixel with line averaging of 4 and a size of 0.044 μm x 0.044 μm with pinhole set to 63 microns (calculated to be 1 airy unit for far red emission) using Zeiss Zen software (version xx ) and saved as Carl Zeiss Image files (.czi).
Spinning Disk
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 solid state lasers for excitation and Andor iXon 888 EMCCD camera, controlled by MetaMorph software.
Zeiss Axio Imager
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 Zeiss Monochrome 505 CCD camera using Zeiss Zen blue software.
Leica SP8 confocal
Cells were imaged on a Leica SP8 confocal microscope on an upright DM6 stand using a 40x/1.3 NA oil objective using laser illumination via Argon for 488nm, diode 405nm and 561nm and HeNe 633nm. Fluorescence signal was collected with 2 photomultiplier tubes and 2 HyD detectors in the following emission ranges: for DAPI - 415-480nm, for AlexaFluor 488 - 490-550nm, for mCherry (or red Alexa) 570-630nm and for AlexaFluor 647 - 633-670nm (HyD).
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.
Acknowledgements
Acknowledgement of the Light Microscopy Core Facility in publications is appreciated and helps to demonstrate our utility to funding sources and Duke administration and researchers. We welcome general acknowlegement of the the LMCF or personal if one or more of the LMCF staff has helped you. Thank you.
The LMCF currently has four microscopy systems funded by an NIH Shared Instrument Grant. Please cite the following grant numbers in your publications and presentations (this is required by NIH). Please only site these grant numbers for these four specific pieces of equipment and not any others in the LMCF. Please let us know if you have questions.