43x very dim

Here you can discuss different microscopic techniques and illumination methods, such as Brightfield, Darkfield, Phase Contrast, DIC, Oblique illumination, etc.
Message
Author
apochronaut
Posts: 3560
Joined: Fri May 15, 2015 12:15 am

Re: 43x very dim

#31 Post by apochronaut » Wed Feb 28, 2018 4:53 pm

I've done a lot of DF.
The question always comes up in using lower illumination for high resolution DF, whether the background need be black or is grey or a dark grey, good enough? I use the term grey field as opposed to dark field. Typically, one begins to achieve DF at an N.A. of about 1.0 but true DF does not occur until an N.A. of somewhere around .8 is achieved, somewhat limiting the brilliance of the image, while increasing the contrast and lowering the halo, or flare.

Many companies offer funnel stops for objectives with N.A.s over .85 or alternate objectives with iris diaphragms, due to grayfield occurrence, and therefore lowered contrast with those objectives performing at N.A.'s on the cusp.

To stay with one company as an example, Reichert : the 63X 1.0 oil immersion objective for the 160mm system, came with a funnel stop. Without it, the background is darker grey. In the literature covering the objectives for the Univar, their high end foray into infinity corrected optics, Reichert stipulates that all objectives with an N.A. over 0.75 are fitted with iris diaphragms, in the section covering dark field.

In many cases the proximity of the light emitter to the condenser is the critical factor. I have tried DF on an AO series 10 at 20 watts halogen and at a fairly close proximity to the specimen plane. The original bulb is located at around 20 cm. distance; 7 to the mirror and 13 from the mirror to the specimen plane. Closing up the iris on a 100x oil 1.25 advanced planachro, to achieve true DF, gives a usable but less than acceptable DF image. Very transparent structures and suspended particles in an aqueous substrate are difficult to discern. Opening the iris to achieve a brighter image , lowers the contrast sufficiently, that there is further loss of details. Using the same optics in a series 20 , with 100 watt halogen illumination, allows for a greater closure of the iris, with a stark black background , very high contrast and lots of detail, down to around the 1/2 μm level or even 1/4, with a planapo objective. One of the most evident characteristics of DF with the lowest possible aperture, is a relative freedom from flare, which can cause a severe loss of contrast and detail in imaging with an inadequately closed iris.

Hobbyst46
Posts: 2943
Joined: Mon Aug 21, 2017 9:02 pm

Re: 43x very dim

#32 Post by Hobbyst46 » Wed Feb 28, 2018 5:56 pm

Apochronaut:
Thanks a lot for the great info. I understand from it that the DF issue arises from the distance. It agrees with the decay of intensity of radiation with the squared distance to the condenser. So if I could place the source nearer to the condenser the problem will be diminished ? The collector optics, however, were designed and located by the manufacturer to provide optimum illumination. If I cannot change the location and size of these optics and the distance between the source and these optics, are the only means to achieve good high NA DF (1) iris-containing high NA objectives and (2) a much more powerful light source than originally provided ?
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

MichaelG.
Posts: 2414
Joined: Mon Apr 10, 2017 8:24 am
Location: NorthWest England

Re: 43x very dim

#33 Post by MichaelG. » Wed Feb 28, 2018 6:06 pm

Hobbyst46 wrote:Is German eBay a separate site from "English/American" eBay?
Yes ... but you can log-in with the same username & password.
https://www.ebay.de

MichaelG.
Too many 'projects'

Hobbyst46
Posts: 2943
Joined: Mon Aug 21, 2017 9:02 pm

Re: 43x very dim

#34 Post by Hobbyst46 » Wed Feb 28, 2018 6:11 pm

Thanks MichaelG.
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

Hobbyst46
Posts: 2943
Joined: Mon Aug 21, 2017 9:02 pm

Re: 43x very dim

#35 Post by Hobbyst46 » Wed Feb 28, 2018 7:24 pm

When nothing works read the manual !!!

Following the previous discussions I opened the original instruction manual of my scope, in a page that I have never noticed before, and so it says:

"For dark-field investigations with immersion objectives we produce an ultra-condenser... in conjunction with an immersion objective having an iris diaphragm."
A special lamp is not mentioned.

Anyway, my only iris objectives are 25x and 40x, so high mag DF, attractive as it is, will have to wait...
And I am afraid this discussion has shifted away from the original subject of the post...
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

apochronaut
Posts: 3560
Joined: Fri May 15, 2015 12:15 am

Re: 43x very dim

#36 Post by apochronaut » Wed Feb 28, 2018 10:07 pm

your GFL has an illuminator very close under the condenser, does it not? might be able to get away with 10 watts. the larger stands had facility for high wattage ilumination in the back of the base and yes, the thread is going off on a tangent.

for the AO series 4, there is a bit of distance., around 26 cm. from the filament to the specimen plane, so it will need some extra oomph in the light to get high magnification DF going.

Hobbyst46
Posts: 2943
Joined: Mon Aug 21, 2017 9:02 pm

Re: 43x very dim

#37 Post by Hobbyst46 » Wed Feb 28, 2018 10:33 pm

Apochronaut:

Just to answer the previous question - this GFL has the light bulb inside the round base plate, under the arm, not below the condenser.
the distance (and base of course). The distance is roughtly 11+5=16 cm. I would like high NA darkfield, though.

Also, below are some photos of 100x oil phase contrast under the unfiltered LED illumination. In each of them, the size of the dominant figure is about 50 micrometers. No processing except crop and resize. The diatom slides are crowded but demonstrate some resolution. Oiled condenser-to-slide as well.
Attachments
Oxalis pollen grain stack.jpg
Oxalis pollen grain stack.jpg (34.44 KiB) Viewed 1868 times
Single frame strew diatoms 1.jpg
Single frame strew diatoms 1.jpg (93.12 KiB) Viewed 1868 times
Single frame strew diatoms 2.jpg
Single frame strew diatoms 2.jpg (65.3 KiB) Viewed 1868 times
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

Micro-Bob
Posts: 30
Joined: Mon Dec 05, 2016 12:18 pm

Re: 43x very dim

#38 Post by Micro-Bob » Thu Mar 01, 2018 10:35 am

To stop hijacking this beginners thread I opened a new one exclusively for dark field illumination:
posting.php?mode=post&f=28

Bob

Timemaster1212
Posts: 119
Joined: Sun Dec 03, 2017 12:21 am

Re: 43x very dim

#39 Post by Timemaster1212 » Fri Mar 02, 2018 5:17 am

Haha thank you all for your help, should be done with robotics after my state competition Saturday. Then i can focus and post pictures with my scope :D

Post Reply