There are a few critical components should be included in a Materials & Methods section describing your microscopy experiments listed below. You can find the microscope equipment information for our scopes on our Equipment page, except objective lenses, which are listed on the desktop of each computer. If you are unsure or have any questions, PLEASE ASK!
- 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, cell culture (for live samples) or mounting (for fixed cells) 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 or Prior Proscan linear encoded stage was used to collect multiple stage positions)
- 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.)
The best way to confirm your equipment is to look at the equipment (e.g. camera, objective lens) on the microscope. You are encouraged to send a draft of your Materials & Methods section to the NIC staff – we 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.
NOTE: All microscope related parts in [brackets] below can be found on our Equipment page for the microscope you worked on, except the objective lens. The objective lens details can be found on the desktop of the microscope computer.
Cells were grown on No. 1.5 coverslips and mounted in a [INSERT MODEL AND TYPE (stage top or enclosure) OF HEATED CHAMBER] heated chamber warmed to X°C. [INSERT TISSUE CULTURE MEDIA and METHOD OF BUFFERING] 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 [INSERT CONFOCAL OR TIRF MODEL] on a Nikon Ti-E inverted OR Nikon 80i upright microscope equipped with [INSERT OBJECTIVE LENS Correction Mag/NA] and the Perfect Focus System for maintenance of focus over time. [INSERT FLUOROPHORE – Be specific!! If you used a fluorescent protein, state the exact variant – e.g., mEGFP not GFP, or TagRFP2 not RFP] fluorescence was excited with the [INSERT LASER LINE] line from a [INSERT LASER POWER] mW [INSERT LASER TYPE] (selected with an AOTF) and collected with a [INSERT DICHROIC MIRROR MODEL] (Chroma) and a [INSERT EMISSION FILTER Peak wavelength/bandwidth]. Images were acquired with a [INSERT CAMERA MODEL] [cooled-CCD or EM-CCD or sCMOS camera] controlled with [INSERT ACQUISITION SOFTWARE] software. For time-lapse experiments, images were collected every X min, using an exposure time of X ms [include exposure time only if performing a timelapse as it is a hard limit on how fast you were able to acquire images – exposure time for fix imaging is meaningless to the reader (as us why!)] and 2×2 [e.g] camera binning. At each time-point, X z-series optical sections were collected with a step-size of X microns, using the [MODEL FOCUS MOTOR]. Multiple stage positions were collected using a [MODEL MOTORIZED STAGE]. Z-series are displayed as maximum z-projections, and gamma, brightness, and contrast were adjusted (identically for compared image sets) using [IMAGE ANALYSIS SOFTWARE; if you worked with IDAC, ask them for help writing this section] software.