Posted by
Steffen Dietzel on
URL: http://confocal-microscopy-list.275.s1.nabble.com/darkfield-references-tp7583371p7583377.html
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Jeff,
concerning resolution, normal dark field is not any different from
bright field, except maybe that Rayleigh may make more sense than Abbe,
since you have a kind of self-luminous objects. In practical terms,
resolution will be less since you have to cut down NA either from the
objective or from the condenser. Concerning the smallest observable
object, dark field is somewhat like fluorescence: Not the size is
important, but the amount of light you can get out of it. If you want to
dig deeper, the search term is ultramicroscopy, named so because it can
visualize particles below the resolution limit. I estimate the term was
en vogue from around 1900 to maybe the 1950ies.
A 1902 experiment studied particles down to 4 nm, with sun light as
light source, probably still the record for size: H. Siedentopf, R.
Zsigmondy: Über Sichtbarmachung und Größenbestimmung
ultramikroskopischer Teilchen, mit besonderer Anwendung auf
Goldrubingläser. In: Annalen der Physik. 315, 1902, S. 1–39,
doi:10.1002/andp.19023150102
Henry Siedentopf (Zeiss company) and Richard Zsigmondy also developed
the initial version of the Slit-Ultramicroscope (Spaltultramikroskop),
which uses an illumination principle much like todays light sheet
fluorescence microscopes, to study colloids.
There are two Nobels associated with the technique.
Ultramicroscopy was used in the Millikan-Experiment
(
https://en.wikipedia.org/wiki/Oil_drop_experiment) which was rewarded
with the physics Nobel in 1923.
And in 1925 Zsigmondy got the Chemistry Nobel for his colloid studies.
His Nobel Lecture is online
(
http://nobelprize.org/nobel_prizes/chemistry/laureates/1925/zsigmondy-lecture.pdf)
Dark field seems to be particularly useful in visualizing living
Spirochaete bacteria, thus the technique boomed from 1906 when the
Syphilis bacteria where discovered.
Concerning Rheinberg Illumination that Barbara mentioned, that was
developed actually a little later than she thought: Julius Rheinberg of
London first described it in 1896 (according to one source). In
professional microscopy it seems to have been replaced mostly by phase
contrast. But Hobbyists still use it to make beautiful images.
If you should learn German anyway to read the Siedentopf & Zsigmondy
paper (don't know if there is a translated version somewhere), you also
can have a look at the German Wikipedia article on
Dunkelfeldmikroskopie, which I think is quite good. But then, I may not
be entirely impartial on that particular subject :-)
Cheers
Steffen
Am 30.01.2015 um 23:44 schrieb Jeff Spector:
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>
> Greetings,
> Can someone please point me to some references involving the theory
> behind darkfield microscopy? I understand the basic idea, but all I can
> find are different iterations of the basic idea that you block most of the
> light and only image scattered light. I'd like to learn a bit more about
> technical aspects of darkfield, i.e. what is the smallest object you can
> observe? What role do illumination power and camera exposure play in the
> quality of the final image. What role does specimen thickness/size play in
> the final image and can you discern objects of different size
> etc...
> Any help would be greatly appreciated. Perhaps I simply need to read up on
> scattering theory?
> thanks..
> -jeff
>
>
> --
> ------------------------------------------------------------
> 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