Fluor objective vs Apochromat objective with same NA

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Robert Berdan
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Fluor objective vs Apochromat objective with same NA

Post by Robert Berdan »

I was wondering if anyone has tested a Fluor objectives vs an Apochromat objective of the same numerical aperture. I have a Nikon Flour NA 0.75 and a 20X Apochromat NA 0.65 which is not a fair comparison as the NA is more important and the Flour objective is brighter in this case. I understand that brightness is proportional to the square of the NA. I am wondering if high NA Apochromat objectives would be equally bright for Fluorescence microscopy?

Has anyone tested this?

RB

viktor j nilsson
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Post by viktor j nilsson »

I have a Nikon CF Fluor 20x 0.75 and a Nikon CFN Plan Apo 20x 0.75 and I've compared them many times. In brightfield for visual use I'd say they are very very close in brightness and resolution. For fluorescence, however, transmission in the UV spectrum can matter a lot. The Nikon CF catalogue on Charles Krebs' site explicitly states that the Fluor series features "extra-high transmission extending into the ultraviolet excitation range". This could be important to you.

Pau
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Post by Pau »

Take a look at the Objective Brightness Values at https://www.olympus-lifescience.com/en/ ... nceerrors/

Brightness in Epi-Fluorescence Microscopy is determined by
Brightness Index = (NA)4 / (Magnification)2 × Transmission Ratio, so you want high NA (forth power! because the objective doubles as condenser and as objective)

Classic Apos have much more elements than fluorites and the transmission is inferior and this is more important in the UV range being good for blue or green excitation while many modern Plan Apos and Apos are excellent, at least from 405nm upwards.
In my limited experience old Plan Apos are very good excepted some ones in the UV range.
Last edited by Pau on Fri Apr 24, 2020 6:49 am, edited 1 time in total.
Pau

Ichthyophthirius
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Post by Ichthyophthirius »

Hi Robert,

Full agreement with what was said above. Just to add that older objectives not only had low UV transmission but also high autofluorescence (mainly from lens cement).

Fluorites that were optimised for epi-fluorescence from that time also had lens cements with lower autofluorescence. That was offset by having fewer lenses (lower colour correction and higher field curvature compared to plan achromats).

On the other hand high intensity UV is not really compatible with live biological samples. I would only recommend it at low intensity for background structures like a nuclear stain and use blue excitation instead whenever possible.

Robert Berdan
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Thank you your commments are much appreciated

Post by Robert Berdan »

Thank you for the information and it sounds reasonable. I tested an 20X APO objective NA 0.65 against a 20X Flour objective NA 0.75 and of course the Flour was much brighter because of the higher NA but I don't have two objectives a Flour and Apo with the same NA to test and was wondering if anyone else has.

Most of the Apochromats have smaller front objective openings as well. just wondered if 2 objectives had the same NA would they be equally bright and from the sounds of it - the Apochromat would not be as brights as the Fluor.

The Nikon Flour objectives I have so far - Flour 10, 20X are the brightest. I am waiting on a used 40X Flour to arrive to test with NA 0,85. I am using these objectives for wide field flouorescence microscopy and a small increase in NA seems to make a big difference - I have read about the mathematical relationship with brightness vs NA.

Unfortunately flouorescence with all 4X objectives I have tested so far are very poor for flouorescence- presumbably because of the low NA, but would be nice for some larger subjects. At the moment I am creating pans.

Thank you for taking the time to reply its nice to get the opinions of other microscopists.
RB

viktor j nilsson
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Location: Lund, Sweden

Post by viktor j nilsson »

I can highly recommend the Nikon Fluor 40x 1.30. It is incredible in brightfield, must be ideal for fluorescence.

Also, you said:
Robert Berdan wrote:" just wondered if 2 objectives had the same NA would they be equally bright and from the sounds of it - the Apochromat would not be as brights as the Fluor.
I just want to re-iterate what Pau said. From violet and up (405nm+), any difference in transmission between a modern PlanApo and a Fluor is going to be very small. So within the visual spectrum, you would hardly see any difference in brightness between a PlanApo and Fluor as long as they have the same NA (this is consistent with my experience). So if you use blue fluorescence, a Plan Apo will surely work fine.

But for UV fluorescence, Fluor objective will likely produce noticably stronger fluorescence as it was designed to have higher UV transmission.

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