There are a few critical components should be included in a Materials & Methods section describing your microscopy experiments:
- Make and model of microscope (e.g., Nikon Ti-E)
- Make and model of any advanced imaging modality – confocal, TIRF, super-resolution, etc. (e.g., Nikon TIRF illuminator)
- Hardware or software auto-focusing, if used (e.g., Nikon Perfect Focus System)
- Type, magnification, and numerical aperture of the objective lenses (e.g., Nikon Plan Apo 60x 1.4 NA)
- Imaging environmental conditions – chamber, media, temperature, buffering method, etc.
- Specific fluorophores. For fluorescent proteins, be sure to indicate the specific variant – mEmerald is a specific variant, GFP is NOT.
- Fluorescent filters, including peak transmission and bandwidth (e.g., Chroma 490/30 excitation filter and 525/50 emission filter)
- Laser type, line, and the selection method used (e.g., the 488nm line from an argon laser, selected with a 488/10nm filter OR the 488nm line from an argon laser, selected with an ATOF)
- Camera make and model (e.g., Hamamatsu Flash 4.0 sCMOS camera)
- Other motorized components used (e.g., a Mad City piezo stage z-motor was used to collect z-series)
- Acquisition software make and version (e.g., NIS Elements 4.11)
- Any subsequent software used for image processing, with details about types of operations involved (e.g., type of deconvolution, 3D reconstructions, surface or volume rendering, gamma adjustments, etc.)
For NIC microscopes, you can find all of these specifications in the Equipment section of this website. Please feel to send a draft of your Materials & Methods section to Jennifer – she will be happy to edit your work for accuracy. And a reminder: If you collected your images in the NIC@HMS, you must acknowledge the Nikon Imaging Center at Harvard Medical School in your manuscript.
Below is an example of a complete Materials & Methods. Do not use this exact text – it doesn’t correspond to any microscope in the NIC. It’s just an example of how you might word your Materials & Methods section.
Cells were grown on No. 1.5 coverslips and mounted in a Tokai Hit INU microscope stage heated chamber warmed to 37°C. DMEM media with 25mM Hepes (pH 7.2) and without phenol red was used during image acquisition, with a layer of mineral oil on top of the media to prevent evaporation. All images were collected with a Yokogawa CSU-X1 spinning disk confocal on a Nikon Ti-E inverted microscope equipped with Plan Apo 100x NA 1.4 objective lens and the Perfect Focus System for maintenance of focus over time. Histone-EGFP fluorescence was excited with the 488nm line from a 100mW Melles Griot argon krypton laser (selected with an AOTF) and collected with a triple band pass dichroic mirror (Chroma # 53055) and a 525/50 emission filter. Images were acquired with a Hamamatsu ORCA R2 cooled-CCD camera controlled with MetaMorph 7.2 software. For time-lapse experiments, images were collected every 1 min, using an exposure time of 500 ms and 2×2 camera binning. At each time-point, 6 z-series optical sections were collected with a step-size of 0.5 microns, using the Nikon Ti-E internal focus motor. Multiple stage positions were collected using a Prior ProScan motorized stage. Z-series are displayed as maximum z-projections, and gamma, brightness, and contrast were adjusted (identically for compared image sets) using Image J software.