Re: Boosting bright field resolution with dichroic filters

Posted by Steffen Dietzel on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Boosting-bright-field-resolution-with-dichroic-filters-tp7583983p7584000.html

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Joachim,

thanks for the link. (Barbara, the mail server broke it in two lines,
you have to copy both parts, then it worked for me.)

I am far from understanding all the formulas there, but I seem to
understand that it boils down to a Rayleigh criterion for two point
light sources (on page 3).
The circular aperture in this example would be the magnifying lens (with
diameter d) with a distance s between to point light sources. Then it is
said that sin(alpha)= 1.22* lambda/d

Not sure how to transform this approach that seems to be developed for
Astronomy to the resolution in the microscope,  but assuming that works
(and I don't doubt it), I am still not convinced that this point light
source approach is the right one for brightfield = absorption
microscopy, where we do not have light sources in the focal plane. Text
books tend to suggest fluorescence -> Rayleigh and bright field -> Abbe.

Steffen

Am 13.07.2015 um 17:13 schrieb Hehl Joachim:

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> Hi Steffen,
> Good question! The factor 0.61 comes from the calculation of the intensity
> distribution of light after the diffraction at circular apertures.
> Here is a link in german
> http://www.chemgapedia.de/vsengine/vlu/vsc/de/ph/14/ep/einfuehrung/wellenop
> tik/interferenz2e.vlu.html
> Best
> Jo
>
> ETH Zürich
> Joachim Hehl
> Staff Scientist
> ScopeM - Scientific Center for Optical and Electron Microscopy
> HPM E 14
> Otto-Stern-Weg 3
> CH-8093, Zurich
> Switzerland
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> Web: www.lmc.ethz.ch
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>
>
>
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>
>
> On 7/13/15 11:26 AM, "Steffen Dietzel" <[hidden email]> wrote:
>
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>>
>> Dear all,
>>
>> two questions, first about the formula:
>>
>> I thought 0.61*lambda/(NA)  (Rayleigh) would be the appropriate formula
>> for self-luminous objects (dark field, fluorescence) while for bright
>> field the fitting variant of the Abbe formula should be applied:
>> lambda/NA-obj for central illumination, lambda/2NA for NA-obj = NA cond
>> and, in general, lambda/(NA-obj + NA cond), provided that NA-obj is
>> equal or bigger than NA-cond.
>>
>> It seems though, that others are happy with 1.2*lambda/(NA-obj + NA
>> cond). Where does the 1.2 (or 2*0.6)  in a bright field situation come
>> from? Would that be the paper by Hopkins and Barham (1950), that Mike
>> was referring to? Because I have no hope of understanding this paper by
>> reading through during this life (Lots of formulas my biologist's brain
>> is not flexible enough to adjust for).
>>
>> Second:
>> Is there a good source for diatoms that can be stained with fluorescent
>> dyes and self-mounted, as George suggested? I have a few diatom slides
>> for teaching (Pleurosigma), but they came already mounted.
>>
>> Steffen
>>
>>>
>>> --
>>> ------------------------------------------------------------
>>> Steffen Dietzel, PD Dr. rer. nat
>>> Ludwig-Maximilians-Universität München
>>> Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
>>> Head of light microscopy
>>>
>>> Marchioninistr. 27
>>> D-81377 München
>>> Germany

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
Head of light microscopy

Marchioninistr. 27
D-81377 München

Phone: [phone]
Fax-to-email: +49/89/2180-9976509
skype: steffendietzel
e-mail: [hidden email]


--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
Head of light microscopy

Marchioninistr. 27
D-81377 München
Germany