Darkfield questions

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Walter Piorkowski
Posts: 693
Joined: Mon Aug 14, 2006 6:42 pm
Location: South Beloit, Ill

Darkfield questions

Post by Walter Piorkowski »

I would like to open a discussion on darkfield imaging. I have recently posted my best efforts to date but there is always room for improvement.

I always have micro air bubbles not visible to the unaided eye in my immersion oil application above and below the slide. These as is well known destroy contrast due to flair and unfocused rings.

Question #1 – What is the proper technique and applicator for bubble free immersion oil application for darkfield imaging? I have used ball ended glass rods, cone tapered glass rods to remove my immersion oil from its storage container and will soon try direct application from a squeeze tube.

Question #2 -What issues affect the immersion medium? Does oil temperature play a factor? Should the storage container be placed in a vacuum to drive out the air bubbles?

My dry objectives consistently out perform oiled objectives for contrast and blackness of field. Even objectives of equal numerical aperture do not perform the same. I chalk this up to the fact that the oiled objectives are working through a second zone of contrast destroying immersion medium.

Question #3 – Are oiled objectives known to produce a less dark and contrast image?

The numerical aperture of high magnification oiled objectives plays a great part in their interaction with the light cone of the condenser. Early books (pre 1950) on microscopy clearly state that the numerical aperture of a darkfield condenser could not exceed 0.66 NA. This would be achieved with funnels inserted into achromatic objective of the time. Some objectives of 63x -100x were supplied with an iris to allow the high NA to be reduced. Darkfield condensers of the time, I assume, did not exceed 1.20NA

Question #4- If you put a funnel into an objective what NA are you ending up with? Are they even necessary these days if you have a 1.40 NA condenser.

Things have changed with darkfield condenser designs. Per the Molecular Expressions web sight the aperture blocked Abby and paraboloid have been supplanted with cardioid, bicentric, bispheric and cassegrain designs. NA’s are now up to 1.50 NA!

Question #5 – With all the different darkfield condenser designs out there and the manufactures pension for not labeling them, how are you to know what design you have? I have 5 condensers from various manufacturers collected over the years and only one is marked as to its’ design.

Question #6 – How do I know how high in NA I can go with any specific condenser that I may have. Most early condensers are not marked except with slide thickness data. The Molecular Expressions web sight chart is interesting in the relationships between design, condenser NA and maximum objective NA.

Walt

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