Microphotography

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The article deals with microphotography, i.e. creating microphotos.

Taking the picture

The keys to good pictures

  • Spend time on set-up.
    • Clean the slide.
    • Look for the pristine areas without artifacts, e.g. folds.
  • Composition.
    • Put normal beside pathologic - so one has a reference point.
      • Transititions are usually interesting.
    • Where to put the centre of interest:
      • Truly beautiful: use the rule of thirds - see composition section.
      • Functional: centre of interest in the centre.
  • Focus.
    • Sharpness of nuclear membrane and detail in the cytoplasm.
  • White balance.
  • De-vignetting.
  • Pictures at different magnification.
    • Sets are usually better than one.
    • Many entities have high and low power features.
      • It is often impossible to capture them with one picture.

Camera settings

Exposure compensation:

  • None.

White balance:

  • Normal.

Focusing

Setting live view:

  • Rench (II).
    • Live view function settings.
      • Live View shoot.
  • Press "Set" button to open the shutter.

Use the zoom button (on the camera): press twice to digitally magnify 10x.

  • Focus microscope as usual (@ 10x digital magnification).

Note:

  • Post-processing will not fix a blurry image. The way to get sharp images is take sharp pictures!

Composition

A picture that uses the rule of thirds. (WC)

Functional pictures that tell a story and are easy to understand:

  • The centre of interest at the centre.

An artsy look can be achieved by making use of the rule of thirds.

  • Rule of thirds: centre of interest is at one of the four intersects of the imaginary lines that divide the image into thirds.

Post-processing

GIMP scripts:

  • White-balance.
  • Shadows & highlight.

White balance

  • May be done with:
    • White balance plugin.[1]
    • Curves function (in GIMP).

Curves function (in GIMP)

  • Adjust magnitude, then blue, then green -- red should be last.
    • Low magnification images tend toward "too pink" with the white-balance script.

White balance plugin (script)

Procedure:

  1. Select
    • Filters.
      • Colors.
        • White balance.
  2. Use eye dropper to select what should be white on the image.
  3. Set to "background color".

Vignetting

Defintion:

  • The edge of images are darker than the centre.

Microscope configuration:

Some random notes:

  • Software for microscope cameras usually have de-vignetting tools. The tools go by different names and usually entail taking a background image. Ideally, one should shoot the background image at the same magnification as the (primary) image.
  • Vignetting is usually worse at lower magnification.

Random links:

De-vignetting

Student method

  1. Take picture as one wants.
  2. Remove slide and shoot the background.

In GIMP:

  1. Load image.
  2. Load background as a layer ("Open as layer... ").
  3. Make background into an "overlay" in Layers dialog box.
  4. Invert background.
  5. Save as jpg (merge with background).

Adapted from codeforhire.com

Procedure:[2]

  1. Load image in GIMP.
  2. Duplicate layer (right click on layer and select Duplicate Layer).
  3. Apply Gaussian blur to top layer.
    • Filters -> Blur -> Gaussian Blur
      • Select a blur radius 1/5 of the smallest dimension of the image.
        • If the image is 2768x2110, the blur radius should be 2110/5=422.
  4. Invert top layer (Colors - Invert).
  5. Change top layer to Overlay mode.
  6. Normal top (masking) layer (Colors - Auto - Normalize).
  7. Adjust brightness of (top) layer (Colors - Brightness-Contrast).
  8. Merge layers.
    • Right click on top layer and select Merge Down.

Stitching

  • A technique to increase the field of view.

Software:

Images

Focus stacking

Software:

Image:

Dirt and defects

  • It is best to clean the slide.
    • Photons are free... time is not.
  • Dirt and defects can be removed with the clone tool in GIMP.

Sharpening

  • Images should be sharpened to enhance edges.
    • This is particularly important if the image is projected on a large screen and/or enlarged.
    • Over-sharpening makes images look like caricatures.
  • Sharpening should be the last step in post-processing.

Animation

An animated GIF showing squamous carcinoma of the lung. (WC)

See also

References

  1. URL: http://registry.gimp.org/node/72. Accessed on: 26 July 2010.
  2. URL: http://codeforhire.com/2013/06/29/simple-image-devignetting/. Accessed on: October 16, 2014.

External links