fluo questions

[johan]

Hello,

Can I ask, is it a requirement to have high NA objectives for epi fluorescence? I currently have DIC objectives; the implication is I need a different set for phase/BF/DF/oblique and yet another different set for fluo. Is that correct?

Thank you

[75RR]

Can't find any reference to what equipment you have. Do tell us. It would also be nice to see images in the My microscope section.

Phase needs a dedicated objective but then it can also be used for brightfield, darkfield, oblique and a basic Pol.
My DIC (Zeiss finite) works with Planachromats which can of course also be used for brightfield, darkfield, oblique, a basic Pol and also COL.

Can't speak for Epifluorescence, though a higher NA would of course increase resolution.

[apochronaut]

You would need to check with the maker of the objectives. The parameters regarding your question have changed over time and are somewhat different, depending on the mfg.

Fluorescence often works at quite dim light levels, so higher N.A. objectives, having greater light transmission, are useful where weak fluorescence might be encountered. The glass and cement in some objectives can auto fluoresce too, causing background "noise", so objectives used for fluorescence applications have special glasses, designated as such by the mfg. High uv transmission is needed, so special glass formulations are required. Also iris diaphragms are needed in high N.A. objectives, to reduce scattering. These objectives can also be used for BF and DF. With certain older fluorescence objectives uv absorptive coverslips are required.

D.I.C. and polarization objectives need to be "strain free", with the cement and glass free from internal stresses that could cause spurious image artifacts. These can also be used for BF and DF.

Phase contrast objectives as 75RR pointed out , need a wave retardation annulus installed, in order to effect phase. They can also be used for Bright Field, which demands nothing special by way of construction, aside from being a microscope objective, however the image obtained , using a phase objective for BF, is less contrasty than if a standard BF objective is employed.

Dark Field objectives only need to have the N.A. lower than the N.A. of the condenser, otherwise they have the same material specification as standard Bright Field objectives. High N.A. would be a bonus, due to the higher light transmission.

In the past, many mfg. purpose built objectives and would designate their application either in their literature or on the barrel. Those made for fluorescence applications for instance, would be considerably more expensive than a similar magnification and N.A. standard objective. Over time, it became more economically viable to streamline production and increasingly objectives are being made , which cover many bases. Reichert, as early as the mid-70's, when they converted to infinity correction, made all the objectives available for the Univar research microscope compatible with all contrast methods, then available ; BF, Phase, Anoptral Phase, I Fluor., T fluor., Pol., D.I.C., DF, or combinations thereof, they claimed 17 in all.. Any objective above .75 N.A. contained an iris, oddly, with the exception of the 50x .85 oil immersion planachro..

[johan]

Can't find any reference to what equipment you have. Do tell us. It would also be nice to see images in the My microscope section.

Phase needs a dedicated objective but then it can also be used for brightfield, darkfield, oblique and a basic Pol.
My DIC (Zeiss finite) works with Planachromats which can of course also be used for brightfield, darkfield, oblique, a basic Pol and also COL.

Can't speak for Epifluorescence, though a higher NA would of course increase resolution.

Thank you. No microscope images to show - I'm taking delivery of my first microscope this weekend. It has 3 DIC objectives, M plans. I gather these are only for use without a cover slip, ie metals. The scope has epi and regular bottom lighting.

So I'm trying to research what objectives I need to buy on a separate nosepiece to be able to do regular brightfield, darkfield, oblique and a basic Pol, ie the standard microscopy stuff rather than this fancy DIC. One day I might want to do epi fluoresence though. So what I was trying to understand was do I need different objectives for this, or will high quality plans do fluorescence and brightfield, darkfield, oblique and a basic Pol

[johan]

You would need to check ... etc

Thank you for taking the time, that helps a lot

[hkv]

M Plan and DIC? Then I assume you mean reflected DIC? Reflected DIC has very limited applications such as circuit board/surface analysis.

I use regular UplanFL and UplanSAPO for FLuo and it works perfectly. I cannot see why Mplans would not work.

You have not said what scope you have bought, but I assume it is a newer model. Olympus list clearly in their documentation and brochures what illumination techniques that can be used by which objective series. Perhaps if you share more info on what you have bought we could possible give you more precise advice.

[johan]

M Plan and DIC? Then I assume you mean reflected DIC? Reflected DIC has very limited applications such as circuit board/surface analysis. I use regular UplanFL and UplanSAPO for FLuo and it works perfectly. I cannot see why Mplans would not work.

You have not said what scope you have bought, but I assume it is a newer model. Olympus list clearly in their documentation and brochures what illumination techniques that can be used by which objective series. Perhaps if you share more info on what you have bought we could possible give you more precise advice.

Hi,

My bad - a Labophot 2, allegedly used for fibre optic inspection in its former life. Yes, apparently it is reflected DIC - are you saying that Mplans might work, that I don't need to spend a fortune on new objectives?

Thank you

[zzffnn]

MPlans won't work, at higher than NA 0.4, for biological fluoresce under 0.17 mm cover slip.

[hkv]

Well, MPLAN will most likely work OK if you don't use a cover glass. It is the cover glass as such that the MPLANs will have problems with. Especially for high N.A objectives. If you use a cover glass, it is like zzffnn says, anything above n.a. 0.4 will be a challenge.

[johan]

Right, as I thought. Ah well, I have a lot to learn.