Insider photographic tricks for protozoa

[pippo1234]

I am trying to improve my photographic technique and have kind of hit a wall.

There are two main difficulties when taking pictures of protozoa:

1. They move
2. Their thickness often exceeds the available depth of field.

One can deal with 1. using a combination of high shutter speed, which requires high wattage lighting or a flash, and some viscosity enhancing medium (e.g. protoslo, methylcellulose) or reducing the amount of water under the coverslip.

2. is a problem in conjunction with 1. as focus stacking of a non-static subject is unfeasible. The attached picture is an example of my quandary. High shutter speed 1/4000s, paper towel to draw water from under the coverslip, oblique illumination. Still not enough depth of field with a 20x plan objective. I see beautiful images of protozoa (e.g. Robert Berdan). Most of them use DIC. Does DIC increase depth of field (I guess not)? If it's just an illusion, it's amazing.
Are there are any tricks I am missing?

I have a beam splitter on order in order to be able to use a flash, but that does not address 2.

TIA

[MichaelG.]

.

To the best of my knowledge ... the only currently practical way to address [2] is to reduce the objective lens aperture and thereby increase depth of field.

Unfortunately this also reduces optical resolution; so it’s a ‘trade-off’

MichaelG.
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Edit: __ for a glimpse of the future ... have a look at this https://youtu.be/0Xxfm2pSPBA
[ not sure it will ever translate to what we want to do; but it’s certainly impressive ]

[RobBerdan]

I watched the video - Keyence is a company that is offering zoom video microscopes that I am looking into, but costs are high, software is proprietary. i hope to test a system in the near future. The ability to focus stack on the fly is amazing. The top of the line microscope offers 6,000X but I saw price tag around $75,000. I would love to own it, but I think it is out of my financial reach.
RB