Slide thickness for Darkfield

[ajmckay]

Hi all, I'm looking to buy some slides to go with the used Fisher Micromaster I just purchased. I might also pick up some prepared slides for that "instant gratification" and to see how a prepared slide is supposed to look - but my real interest is in preparing my own.

In my researching I seem to recall that slide thickness matters for use with darkfield illumination. For some reason a slide + cover slip thickness of < 1.0mm seems to stick in my head.

I have an older but very nice micrometer so I could find the thinnest of the batch - do you think this set from Amscope would be a decent place to start? Certainly comes with a lot of them so I can do quite a bit of experimenting.

https://www.amazon.com/dp/B014UUNHVE/_e ... BIOF&psc=1

Thanks!

[einman]

Slides are supposed to be about 1.00 mm thick plus or minus 5%. Although you can experiment with slide thickness to match your specific equipment.

Most objectives are optimized for a coverslip thickness of 0.17 mm or 170 microns.

[charlie g]

I find that the dark field 'sweet spot' is quite adjustable by the positioning of your 'darkfield substage condenser' by use of this condensers focus control knob. I find folks 'go to great lengths' in selecting specific thickness of coverslips they use...for crucial cutting edge resolution with objectives of high NA specs, objectives over 60X magnification.

With your 4X, 10X, 20X, 40X objectives...simply adjust your substage condenser for the dark field 'sweet spot', aj. congrats on your shared microscopy. Charlie guevara

[einman]

I find that the dark field 'sweet spot' is quite adjustable by the positioning of your 'darkfield substage condenser' by use of this condensers focus control knob. I find folks 'go to great lengths' in selecting specific thickness of coverslips they use...for crucial cutting edge resolution with objectives of high NA specs, objectives over 60X magnification.

With your 4X, 10X, 20X, 40X objectives...simply adjust your substage condenser for the dark field 'sweet spot', aj. congrats on your shared microscopy. Charlie guevara

This is true. The method can also be used in lieu of a collar on a higher mag objective.

[apochronaut]

Slides for oil immersion DF tend to be thicker than slides for BF or phase. Some manufacturers , Spencer/AO and B & L, for sure, stamped the required slide thickness on the nose of the condenser and for glass slides, it is optimally, 1.2mm, and less for quartz slides. the tolerance is usually about .10mm, with the thinnest acceptable 1.15 and the thickest 1.25, for American Optical for instance.

[charlie g]

Hi, apo, thanks for info for 'aj'. Please specify what objective: NA, and objective magnification range you anticipate 'DF dedicated thicker slides are best to use for DF?

Are you suggesting standard quality slides hamper high NA/ high magnification DF method? Thanks for your input on this post, and all our microscopy discussions, Phil/ apochronaught.

Charlie guevara

[ajmckay]

Thanks everyone for the responses!

I'm also curious what's meant by DF slides actually being thicker than BF/phase... But that's not surprising - given I just started in this hobby lol.

It does make sense, however, that the "sweet spot" could be adjusted by moving the condenser up and down.

I checked my scope for any markings that would indicate slide thickness but did not find any. Sooo... I'm taking that to mean that, at least for now, it's probably not super important which slides/cover slips I start out with. This is based on the fact that I can't find where I thought I read that the slides need to be thinner than 1mm, as well as the comments of you all indicating that there's no such rule, the condenser is adjustable, and some manufacturers may even spec thicker slide glass than "standard".

Thanks! I've ordered the ones I linked to above - seems to be a good deal to get started.

** In related news I've started doing some research on simple hand held microtomes... Not sure if DIY or commerical product is the right call yet. I do have experience in lapping metals (CPU heatsinks).

[einman]

Slides for oil immersion DF tend to be thicker than slides for BF or phase. Some manufacturers , Spencer/AO and B & L, for sure, stamped the required slide thickness on the nose of the condenser and for glass slides, it is optimally, 1.2mm, and less for quartz slides. the tolerance is usually about .10mm, with the thinnest acceptable 1.15 and the thickest 1.25, for American Optical for instance.

Yes I had read that. But for Leitz, for example, there is no marking referring to slide thickness on my condensers, nor on either of my AO darkfield condensers 1096 or 1096B. Could it be that only applied to older condensers?

[einman]

As Phil indicated; "Make certain your glass slides are not too thick or too thin, especially if you are
using a high NA dark field condenser. High NA dark field condensers have a fixed focal
length at which they optimally work. The manufacturer may recommend a slide thickness
or this value may be printed on the condenser. " - https://www.med.unc.edu/microscopy/file ... dark-field


However-
Per Olympus "Since darkfield microscopy eliminates the bright undeviated light, this form of illumination is very wasteful of light and thus demands a high intensity illumination source. Microscope slides must be of the appropriate thickness, approximately one millimeter +/- 0.1 mm. " https://www.olympus-lifescience.com/en/ ... darkfield/

So I am assuming if there is no markings on the nose then a standard slide 1.0 mm is sufficient.

[apochronaut]

Probably but it would be well to err on the thicker side, since it is generally held that slides thinner than 1.0 mm are a problem with most condensers and average slides, untested for thickness, can often be below 1.0mm. Thinner slides require the condenser to be farther from the slide and maintaining oil contact can be an issue, with the standard immersion oils. The optimal thickness is condenser specific but any I have seen, have a .1mm latitude. 1.0 to 1.1, 1.05 to 1.15 etc. If you only have a box or boxes of 1 mm slides handy, and that is by far the most common, choose the thickest, as a starting point. Slides over about 1.3 are not good.

Regarding the AO/Reichert condensers of later production. I use 1.2mm slides with those. I have used slides as thin as 1.05 and there was a bit of a problem, with very high N.A. results. Can't recall what it was but I continued on with thicker slides.

Olympus, probably produced a shorter focal length condenser, reflecting the reality , that many people have only one size of slides and don't really want to have to buy another, for a special technique.

Fisher has gone through a few manufacturers over the years, even at one time AO or B & L. Your condenser would be the same as the original mfg.'s, so data might be available. Modern instruments have been Chinese for a while but earlier, I think they were Meiji?