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A frequent question we receive at MSL is what information to include about microscopy in the methods sections of your manuscripts.

The current state of affairs is a documented disaster, with very little proper reporting of microscopy methods, even in high-profile journals. The idea of a methods section is that an expert reader could fully understand and replicate the process by which data was obtained. Saying “a Zeiss confocal was used to acquire images” and leaving it at that is not enough. MSL users can and should do better. Below, we provide general and specific recommendations for MSL users. There is a lot to keep track of, so please contact us if you want us to review your methods before you submit a manuscript. In addition, we encourage you to look at the following publications on this important topic:


The following are some general recommendations for you to consider:

  • Always keep copies of the raw data in the format in which it was written when you acquired it on the microscope. This initial format typically contains a lot of metadata that includes many of the parameters used during image acquisition. By going back to this data you can often figure out how images were acquired.
  • You should make clear in your methods that all groups of images you are comparing to each other were acquired with the same settings. If they weren’t, you should explain how you are normalizing them to compare across different imaging conditions (ratiometric imaging? normalization to a reference standard?).
  • You should make clear in your methods that all groups of images that are compared side by side in figures had the same display adjustments made to them. If they didn’t, you should not be comparing them.
  • You should explain what steps you took to avoid saturation of pixels and how you dealt with images that had any saturation.
  • You should explain how you decided where in the sample to take images. Did you take any steps to randomize imaging locations within the sample? Did you have any explicit exclusion or search criteria? View more on sampling.
  • You should explain how you decided how many images to take. If applicable, you should explain any power analysis done before the experiment, and what kind of pilot data was used to perform the power analysis. Learn more on power analysis.
  • You should only present images acquired with same settings as those used for analysis. Avoid taking a lot of images with “fast” settings, doing analysis on those, then getting a gorgeous, low noise, high resolution image for the paper. This is not representative of what was used for analysis and is misleading, particularly if not disclosed.
  • If there is any graph with data on it, where each datapoint came from an image, show an image with behavior close to the mean or median of that graph. Ideally, if there is a dot plot, mark the particular dot the particular image shown corresponds to. For an example, see Fig 4.
  • If you have many images in your paper, acquired with many different settings, consider including a detailed table in a supplement with more details on how the data in each figure was acquired. For a great example of this in light-sheet microscopy, see theTable S2 in the supplement of this paper.
  • Avoid Matryoshka doll nested citations: “Microscopy was performed as previously described (citation 1)”, where citation 1 says “Microscopy was performed as previously described (citation 2)”, where citation 2 says “Microscopy was performed as previously described (citation 3)”, etc, etc. This makes it hard for readers to understand in detail what you did and likely does not capture changes in equipment and methods that took place in the lab over the course of many years. There are examples in the literature where the original methods are buried several papers deep, in manuscripts from over a decade ago that are very different from the starting point of the nested citation chain. It is almost impossible in these cases to figure out in detail what was done.

In addition, here are some specific recommendations for what is important to report when doing light microscopy, and what can be omitted.

Things you should always include:

  • Fluorophores: If you are using organic dyes, clearly state the name and source of the fluorophore (example: Alexa Fluor 488 (ThermoFisher)). If you are using fluorescent proteins, cite the paper or Addgene reference for where you got the plasmid encoding the protein. You can also consult FPbase, a fantastic resource with information on fluorescent proteins. As you can see on that site, saying you used “GFP” is not very informative, as there are dozens of variants with different properties.
  • Mounting media (if applicable): State the name and brand (example: ProlongDiamond (Thermofisher)). If using a mountant that changes its refractive index with curing time, state how long you cured for before sealing the sample with a coverslip, and provide an estimate of the refractive index. This information can be found in the manual for many mountants. If it is not, ask the manufacturer for it.
  • Type of microscope: For example: laser scanning confocal, widefield fluorescence, spinning disk confocal, light-sheet.
  • Brand and model: For example: Zeiss LSM710.
  • Light sources: For example: laser (state exact wavelength), Hg lamp, LED (what brand and model?).
  • Filters: On a widefield, for both excitation and emission state the type and wavelengths it allows (long pass, short pass, band pass, center, bandwith). On a confocal, state the wavelengths that are detected in each channel (example: 500-550nm). If there is a beamsplitter (like on the Zeiss LSM700), include information on its position. Explore this information, along with light source information.
  • Objective: Full information is essential. Saying 40x is not enough, saying 40x/1.4 is not enough; Zeiss Plan Apochromat 40/1.4 oil objective is the level of detail you need. If the objective has a correction collar, state what the position of that collar was when images were acquired. Learn more about the objectives on each of our microscopes.
  • Additional magnification on widefield microscopes: Report the use of optovars or magnification sliders if applicable (for example, on our spinning disk).
  • Detectors: Include:
    • Camera type, model and brand (example: Hamamatsu Flash4V2 sSCMOS)
    • PMT type (GAsP, conventional, etc)
  • Pixel size: This is absolutely critical and should always be included.
  • Z spacing: within stack, if applicable. Read this excellent paper to understand the effects of spherical aberration on the Z scaling of your images, and take actions before and after the imaging to account for it.
  • Time lapse settings:
    • Report the interval between images and total number of images (or time) taken.
    • Report the exposure time (in a camera-based system), or time to image an entire frame (in a point scanning confocal) so that a reader can understand how much of the time between images was used to acquire them, and how much was spent waiting.
    • Report the order in which channels were acquired, as this can affect bleaching and photodamage.
    • Report, if applicable, whether different channels were acquired at the same Z position, or whether Z stacks of each channel were acquired sequentially.
    • Report, on a confocal, whether images were acquired in line switching mode, or in frame switching mode.
  • Camera binning, if more than 1.
  • Microscope-specific settings:
    • Spinning disk: type of spinning disk head (example: Yokogawa CSU-W1)
    • Zeiss confocals:
      • Pinhole size (example: 1 AU, set on the longest wavelength fluorophore, and same size for all the rest)
      • Zoom, frame size
    • Light sheet:
      • Zoom
      • Sheet NA
      • Sheet width
      • Whether sample was imaged from both sides
      • Where the horizontal focus was set (middle, edge of sample, etc.)
      • How many sheets were used (1 or 3)
      • Whether there was cropping
      • If using dynamic focus, how many horizontal focus positions were used over what range
      • Any algorithms used to merge images acquired with different sheets or horizontal focus positions

Things that are optional or not necessary:

  • PMT gain, offset, digital offset: As long as there is no saturation, the exact gain does not provide a lot of information. The exact offset value is not important as long as it was set in a way that does not cut off a lot of the low signals (ie, it should be set as explained in our detailed instructions for Zeiss confocals). Digital gain should be assumed 1, but if not, mention it.
  • Scan speed, averaging, uni or bidirectional scanning in confocal: These fall into the category of nice to include, but not absolutely critical.
  • Advanced camera options: Mode (high speed, high precision), EM Gain, preamp settings, etc. These are useful to include if the imaging is very specialized: very few photons, very fast, etc.
  • Dichroics, exact filter model and manufacturer: I consider information on dichroics optional unless you have a lot of overlapping fluorophores and crosstalk is a major concern. Similarly, the exact filter model is not essential.
  • Stage manufacturer, air table: Can be omitted, unless you are making very precise measurements that require a careful analysis of vibration

View a different, even less permissive take on what can be excluded from the methods section.