Editing Method

Usage Description

Suitable file types for raw images

- Always use the original file type from the software used for imaging

- Suitable alternative would be TIF but you might loose metadata and convert intensities to colors

- DO NOT use .jpg, .jpeg and similar file formats since they destroy all your data

Saving images of 8- to 32-bit type

- All intensity based images, such as fluorescence, Western Blots, EM, AFM, medical imaging

Saving images as RGB type

- Only image from photo-cameras, non-fluorescent immuno-histochemistry.

- DO NEVER convert or save intensity-based images as RGB type

Bit-depth conversion

- Avoid if possible and if necessary check that the image does not optically change.
- NEVER do before intensity measurements.

LUT changes

No problem (as long as the image is not converted into a color image)

Multi-color LUTs

- Choose wisely to not optically eliminate information and use a calibration bar to allow color interpretation

Channel Color Choices

- generally avoid blue and specifically combinations of red and green

- 2 channels: green + magenta

- 3 channels: cyan + magenta + yellow (careful with over-saturation)

- DO NOT merge more than 3 channels, rather make separate merges with 2 or max. 3 channels

Uneven Lighting - Flat-Field Correction

- Optimal with a real background image, taken during the imaging session.

- For fluorescent images use a autofluorescent plastic slide (e.g. from Chroma)

- Usable and necessary for intensity measurements.

Unspecific Signal Reduction

- The sliding paraboloid-based method described here (a simplistic deconvolution) can optically improve images in presence of diffuse unspecific signal. It partially reduces unspecific staining, uneven lighting and auto-fluorescence. Careful, it is based on an estimation and therefore not quantitative.
- DO NOT USE before intensity measurements!

- ALL images that need to be compared to each other need to be changed exactly the same

Contrast Adjustment

- Brightness adjustment is seldomly helpful

- For figures ONLY use linear-methods. AVOID gamma adjustment, histogram equalization

- Auto contrast CANNOT be used on multiple images which need to be compared to each other

- ALL images that need to be compared to each other need to be changed exactly the same (Use the Optimizer tools in Fiji)

- In color images, contrast should only be applied to the intensity information, not to color-tone (hue) or saturation.

- DO NEVER use before pixel intensity measurements

Image Scaling

- Check metadata for correct information about scaling

- Scaling is necessary before adding scale bars in Fiji, spatial measurements and 3D reconstructions

- DO NOT manually measure an existing scale bar to scale other images (this is only the last resort, if there is not information available)

Image Transformation

- DO NOT rotate images in pixel-based software (such as Fiji or Photoshop)

- DO NOT reduce image size in pixel-based software

- DO NOT use interpolation for transformations whenever possible

- DO transformations mainly in vector graphic-based software without interpolation

- Upscaling (making images bigger) MUST always be done by integer factors in pixel-based software

Image file types for figures

- Whenever possible use vector graphic supporting formats such as SVG, PDF, EPS