Microphotography

Micrograph of a teratoma. H&E stain.

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.
  • 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:

• Köhler illumination.
• Ensure diaphragm is open.

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:

• http://www.gimp.org/release-notes/gimp-2.4.html.
• http://www.gimp.org/release-notes/gimp-2.4-videos.html.

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:

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:

• Hugin (sourceforge.net).

Images

• 

  Example of stitching. (WC)

• 

  Another example of stitching. (WC)

Focus stacking

• A technique to increase the depth of field.

Software:

• Hugin (sourceforge.net).

Image:

• Example of focus stacking (WC).

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)

• GIMP has a nice tutorial (gimp.org) on how it can be done.
• Animations are particularly useful for cytopathology.

See also

• Microscope.
• Digital ISO.
• Micrographs.
• Image annotation.
• Micrograph wishlist.

External links

• Martin Microscope company (martinmicroscope.com).