Imaging Tip of the Month: What to put in a Materials and Methods

In order to critically read and understand your microscopy data, there are a
few basics that should be included in a Materials and Methods section
describing how you acquired your images. This information should always be
provided as it is necessary for estimating the resolution and sensitivity of
your microscope, and therefore deciding whether you chose to use the
appropriate equipment.

1. Make and model of microscope (and confocal, if used)
2. Type, magnification, and numerical aperture of the objective lenses
3. Imaging environmental conditions: chamber, media, temperature, buffer,
etc.
4. Fluorophores
5. Filter sets (with manufacturer part number, so I can look up spectra)
and/or laser used
6. Camera make and model
7. Other motorized components used
8. Acquisition software
9. 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.)

Here is an example of a complete Materials and Methods (based on Station #2
in the NIC@HMS):

Cells were grown on No. 1.5 coverslips and mounted in a 20/20 Technologies
Bionomic microscope stage heated chamber warmed to 37C. 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 spinning disk
confocal on a Nikon TE2000U inverted microscope equipped with 100x Plan Apo
NA 1.4 objective lens. Histone-EGFP fluorescence was excited with the 488nm
line (selected with a 488/10 filter, Chroma # 53044) from a 100mW Melles
Griot argon krypton laser and collected with a triple band pass dichroic
mirror (Chroma # 53055) and a 525/50 emission filter (Chroma # 53051).
Images were acquired with a Hamamatsu ORCA ER cooled CCD camera controlled
with MetaMorph 7 software. For timelapse experiments, images were collected
every 1 min, using an exposure time of 500 ms and 2x2 binning, with
illumination light shuttered between acquisitions. Focus was maintained by
running a MetaMorph autofocus algorithm on the corresponding transmitted
light image every 10th timepoint. At each time point, 6 z-series optical
sections were collected with a step size of 0.5 microns, using a Prior
Proscan focus motor. Z-series were deconvolved using AutoQuant blind
deconvolution software, and are displayed as maximum z-projections. Gamma,
brightness, and contrast were adjusted on displayed images (identically for
compared image sets) using MetaMorph 7 software.