Protistiary - Photographing living protists

Photographing living protists
It is hard to preserve many protists that lack walls, scales and other resilient components. As a result these organisms, many protozoa and some motile algae, are best documented when living. The micro*scope environment is dominated by images of living microbes, and is a good guide to the standards that one should aspire to. Use a good microscope. Understand how to set it up for Köhler illumination, and whatever contrast enhancement techniques you intend to use - most like Phase Contrast or Nomarski (Differential Interference Contrast). Use new slides and coverslips (no. 1 thickness, 22 mm X 22mm). Clean both slide and coverslip (yes, assume that you always need to have a coverslip) Use a dissecting scope to pick out cells with a fine pipette and transfer them to a clean slide.
The best images will be obtained when the organism is in contact or very close to the under surface of the coverslip. Make the preparation with the least fluid that you can manage. You may need to draw off excess fluid with a piece of tissue at the edge of the coverslip. If you withdraw fluid while watching at a low magnification, you will be able to control the amount of fluid so that the coverslip presses gently on the cell to stop it moving. Too much fluid, and the cells will move freely and images will be of a poor quality.
If the organism is large enough to be damaged by an overlying coverslip, you can create chambers. Fragments (shards) of coverslips or full coverslips can be laid onto a slide and a full coverslip placed over the top. A similar arrangement can be made with bits of slide. The trick is to keep the glass surfaces parallel. It is usually not possible to take high magnification images of organisms in chambers.
Photography The image cast on the camera’s chip needs to fill as much of the chip as possible, and this favoir higher magnifications or intermediate magnifiers or zoom systems Living organisms need to be imaged within 10 msec, and ideally within 5 msec. Many cameras have a slow capture speed, and scan each color separately. If this is the kind of camera you have got, give up now. Allow for an hour to get a nice picture or set of pictures of an organism Take the picture a little dull and with no colors saturated It at all possible orient the frame to centre the organism and align it along the axis of the frame, it will cut down the amount of post capture image processing * Pictures look best when symmetrical, and it is simplest to compare pictures if all cells are oriented in the same way – so take this into account as you frame the images. Post photographic image manipulation There is debate as to whether any or how much manipulation of an image is appropriate after it is capture. All imaging is a kind of artefact, and this justifies any manipulation of the image (that does not interefere with the factual content of the image) that serves to communicate the content of the image more effectively. Orient the specimen with its anterior to top where possible Frame the specimen, cropping surplus content Adjust levels Edit and add scale bars (pure yellow often works well) If material is heading for micro*scope , then an image family will be required, for detailed advice
Protistiary home page Top of microscopy

Photographing living protists

It is hard to preserve many protists that lack walls, scales and other resilient components. As a result these organisms, many protozoa and some motile algae, are best documented when living. The micro*scope environment is dominated by images of living microbes, and is a good guide to the standards that one should aspire to.

Use a good microscope. Understand how to set it up for Köhler illumination, and whatever contrast enhancement techniques you intend to use - most like Phase Contrast or Nomarski (Differential Interference Contrast).

Use new slides and coverslips (no. 1 thickness, 22 mm X 22mm). Clean both slide and coverslip (yes, assume that you always need to have a coverslip)

Use a dissecting scope to pick out cells with a fine pipette and transfer them to a clean slide.

The best images will be obtained when the organism is in contact or very close to the under surface of the coverslip. Make the preparation with the least fluid that you can manage. You may need to draw off excess fluid with a piece of tissue at the edge of the coverslip. If you withdraw fluid while watching at a low magnification, you will be able to control the amount of fluid so that the coverslip presses gently on the cell to stop it moving. Too much fluid, and the cells will move freely and images will be of a poor quality.

If the organism is large enough to be damaged by an overlying coverslip, you can create chambers. Fragments (shards) of coverslips or full coverslips can be laid onto a slide and a full coverslip placed over the top. A similar arrangement can be made with bits of slide. The trick is to keep the glass surfaces parallel. It is usually not possible to take high magnification images of organisms in chambers.

Photography

The image cast on the camera’s chip needs to fill as much of the chip as possible, and this favoir higher magnifications or intermediate magnifiers or zoom systems

Living organisms need to be imaged within 10 msec, and ideally within 5 msec. Many cameras have a slow capture speed, and scan each color separately. If this is the kind of camera you have got, give up now.

Allow for an hour to get a nice picture or set of pictures of an organism

Take the picture a little dull and with no colors saturated

It at all possible orient the frame to centre the organism and align it along the axis of the frame, it will cut down the amount of post capture image processing
*

Pictures look best when symmetrical, and it is simplest to compare pictures if all cells are oriented in the same way – so take this into account as you frame the images.

Post photographic image manipulation

There is debate as to whether any or how much manipulation of an image is appropriate after it is capture. All imaging is a kind of artefact, and this justifies any manipulation of the image (that does not interefere with the factual content of the image) that serves to communicate the content of the image more effectively.

Orient the specimen with its anterior to top where possible

Frame the specimen, cropping surplus content

Adjust levels

Edit and add scale bars (pure yellow often works well)

If material is heading for micro*scope , then an image family will be required, for detailed advice

Protistiary home page Top of microscopy