darkfield references

[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

[John Oreopoulos]

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Jeff,

See this paper:

Vainrub, A., O. Pustovyy, and V. Vodyanoy, Resolution of 90 nm (lambda/5) in an optical transmission microscope with an annular condenser. Optics Letters, 2006. 31(19): p. 2855-2857.

John Oreopoulos
Staff Scientist
Spectral Applied Research Inc.
A Division of Andor Technology
Richmond Hill, Ontario
Canada
www.spectral.ca



On 2015-01-30, at 5:44 PM, Jeff Spector wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> 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

[Zdenek Svindrych-2]

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Hi Jeff,
I bet you can see 50 nm particles, especially when 'dry mounted'. The best
way is to try it yourself. Most today's (fluorescent) microscopes do not
have immersion condensers, but you may get a pretty good idea if you use e.
g. Olympus IX70 / IX80 microscope 'PH3' aperture for illumination and 10x-20
x objective (NA < 0.4).

To detect weakly scattering/refracting objects you need 1. clean sample, 2.
clean condenser. Quantitative treatment is always challenging. You may try
to read on Mie theory, Raighley scattering and the like...

Good luck!

zdenek

--
Zdenek Svindrych, Ph.D.
W.M. Keck Center for Cellular Imaging (PLSB 003)
University of Virginia, Charlottesville, USA
http://www.kcci.virginia.edu/workshop/index.php



---------- Původní zpráva ----------
Od: Jeff Spector <[hidden email]>
Komu: [hidden email]
Datum: 30. 1. 2015 18:06:36
Předmět: darkfield references

"*****
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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"

[George McNamara]

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Hi Jeff,

See       http://en.wikipedia.org/wiki/Dark_field_microscopy

John O cited:

Vainrub, A., O. Pustovyy, and V. Vodyanoy, Resolution of 90 nm (lambda/5) in an optical transmission microscope with an annular condenser. Optics Letters, 2006. 31(19): p. 2855-2857.

This is the CytoViva adapter - mnore on that at
http://en.wikipedia.org/wiki/CytoViva,_Inc
http://www.cytoviva.com/products/microscopy-2/cytoviva-optical-performance/
http://www.cytoviva.com/wp-content/documents/CytoViva-Use-Manual-5-7-09.pdf

Vainrub and CytoViva confuse "resolution' with "detection".

See also http://www.ncbi.nlm.nih.gov/pubmed/23520500

PubMed search for:     "darkfield microscopy"    (with quotes) turned up
194 hits, going back to 1926 - many of the early papers are on venereal
diseases, before microtubules start appearing in 1979,
http://www.ncbi.nlm.nih.gov/pubmed/511939 (wow - a biochem dept with a
light microscope).

Many microscopes phase contrast turrets include a darkfield position ...
also deliberately mismatching the objective lens and phase ring can get
pretty good darkfield (sometimes helps to tweak condenser focus). 40x or
higher objective lens with PhL condenser phase ring as one potential
combination.

Biggest problem: any dust anywhere in the entire optical path also
scatters into the detection path. some of the dust is not (readily)
accessible. Speaking of which - on inverted scopes I tape either a 50x50
mm neutral density filter(s) or a glass slide (or large glass coverslip)
over the opening at the top of the condenser - decreases dust
accumulation inside the condenser. Also lets me operate the tungsten
bulb at a higher voltage of more stable output (I am looking forward to
going to LEDs in the future).

Enjoy,
George


On 1/30/2015 4:44 PM, Jeff Spector wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> 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
>
>    

--



George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
Tattletales http://works.bepress.com/gmcnamara/42

[Barbara Foster]

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*****

Hi, Jeff

The concept is really quite simple. Darkfield doesn't "block" the
light.  Rather, a darkfield system "conditions" the light. This is a
bit difficult to do without a diagram, but let's try.  First:  There
are two lens systems involved:  The condenser and the objective.

Step 1: The optics
Imagine light approaching the CONDENSER in a bundle of parallel
rays.  The curve on the incoming side of the condenser causes the
light to emerge at  wide variety of angles (You can prove this by
moving objectives out of the way and placing a business card on its
edge over the condenser.  P. S. - open the condenser aperture fully
for this experiment).  Light coming through the center "sees" the
flat tangent of that curve so continues straight on.  Light coming
through the edges sees maximum angle of that curve so is bent at a
high angle.  The higher the NA of the condenser, the broader the
range of angles emitted from the condenser.  To simplifiy, use the
optic axis as 0o (zero degrees) deviation.  Hypothetically, the
maximum angle emitted would be +/- 90o.  (Interesting experiment:
test the effect of opening and closing the condenser aperture on the
angle emerging from the condenser.  Second experiment: if you have a
turret condenser, close the aperture and very slightly rotate the
condenser so that the light approaches from other angles. Use the
card to watch what happens to the angle).

On the receiving side, the OBJECTIVE will collect a range of angles
set by its numerical aperture (NA = n sine a, where n = refractive
index of the medium between the top of the sample prep and sine a =
sine of half the collecting angle).

Step 2: The sample
Light emitted from the condenser interacts with the specimen in a
variety of ways (diffraction, refraction, reflection, fluorescence,
etc.).  The objective collects that light to form the image ("light
is the messenger").  In the simplest terms, the image is formed by
the interference between the undiffracted background light and the
diffracted light from the specimen.  The undiffracted light is
responsible for the background of the image;  the diffracted light
contributes to resolution, edge fidelity, and intensity of the sample
detail.  Part of the image is also be formed by interference between
specific of components of the diffracted light, but will not
contribute to the background (a further discussion is beyond the
scope of this posting).

Step 3: Enter Darkfield
The goal of a darkfield system is to select, at the condenser, only
those peripheral rays which will emerge at a very high angle.  We
want to select those rays of light which will have such a high angle
that they will miss being collected by the objective.  Since this is
undiffracted light, it contributes to the background information in
the image.  If we don't collect it (zero light), the background will
be black (hence, the term "darkfield").
As in all imaging, some of this highly angled light WILL interact
with the specimen.  It will be scattered at the appropriate angles to
be collected by the objective and go on to form an image.

There are two general approaches for engendering darkfield
microscopy:  A central patch stop to block all rays except those
highly angled peripheral rays or a highly curved, hemispherical
mirror mounted in the condenser, which will reflect light at very
high angles as it emerges from the condenser.  The first approach
creates angles effective to generate darkfield with lower NA
objectives (about 0.15, associated with magnifications up to about
10x).  The more elaborate mirror systems use oil immersion both
between the condenser and the back of the slide and the top of the
prep and  the objective) and are effective for higher NAs (~1.4,
associated with 60x or 100x oil immersion objectives).

Regarding the smallest object you can "see":
Darkfield is limited by DETECTION (how many photons of light can be
scattered to form the image) not RESOLUTION (based on diffraction and
the interaction with the undiffracted + diffracted light).  The
detection is limited by the light (quantum) efficiency of your optics
and camera and, for direct viewing, your eye.  Since your eye can
detect just a few photons, using darkfield (especially the oil
immersion variety) you will be able to "DETECT" objects as small as
about 50-60nm.  You won't be able to define their size or tell much
about their shape or edges (parameters inherent in resolution), but
you will be able to tell that something is there.

ARTIFACTS:
1. Because the photons are coming to you from throughout the entire
depth of the sample ("infinitely great" depth of field), unless the
sample is thin, darkfield images will often be "busy" with images
from one plane overlaying images from those above and below.
2. Because you are using a very narrow range of angles to illuminate
the sample, the light is highly coherent, so you will see a lot of
internal diffraction effects ("ringing" around the edges)
3. Because scatter is the main source of the imaging information, you
will also see lot of local chromatic aberration (rainbows or colors
at the edges).  For that reason, we always recommend that you view
the sample in brightfield first to asses if color is real or just an
artifact of darkfield.

SEVERAL OTHER THINGS TO CONSIDER:
Other imaging techniques remove the undiffracted, background-forming
light, but other imaging parameters are in operation which determine
whether they are diffraction or detection limited.
EX 1: Polarized light (which IS diffraction limited)
EX 2: DIC (which is based on polarized light)  (which is also
diffraction limited)
EX 3: Fluorescence (which is detection limited)
EX 4: CytoViva (cytoviva.com) use a darkfield condenser in a novel
way which (a) actually improves resolution and (b) improves optical
sectioning.  It does so by placing the light source essentially at
the front focal plane of the objective, changing part of the normal
darkfield optics.  As a result, it can actually RESOLVE fine detail
on the order of 90 nm (my old eyes resolved at the 90nm level; the
younger tech specialist which whom I had a chance to work early on,
could resolve about 82nm on the Richardson test slide).   And, unlike
darkfield, it CAN optically section.  Also, more recent work suggests
that it does not suffer from the chromatic effects of conventional
darkfield and, as a result, has been having considerable success in
hyperspectral imaging.  So, even though a darkfield condenser is
involved and even though the image has a dark background, its
behavior and capabilities put it into a category of its own.

  By the way, you can create your own darkfield patch stops using
India Ink on overhead transparency film.  To determine the size of the patch:
1.  Set up Koehler illumination then move to a clear part of your slide
2.  Remove the eyepiece and peer down the tube into the back focal
place of the objective (BFPo)
3. Close the CONDENSER aperture until you can just barely see the
edge of its leaves in the BFPo)
4, Gently remove the condenser from the microscope, flip it upside
down, and measure the size of the opening of the aperture. That is
the size you need for the patch stop.
(Luckily, the manufacturers make these available for very little money).
5. To operate: make sure that the  patch stop is in the Front Focal
Plane of the condesner (in the location of the aperture iris.

Also something fun:
Rheinberg illumination was an important contrast technique in the mid
1800's and is derived from the same principle but uses colored patch
stops instead of black.  With a black patch stop on a white
background, you get white images on a black field;  With a green
patch stop on a white background, you get white objects on a green
field.  One of my favorites is a blue patch stop on a yellow
background, which gives you yellow objects on a blue field (a highly
effective contrast combination for the human eye).   I once taught a
week long course for a company to whom  counting filamentous mold was
important.  One of the biggest successes came by accident when we
used Rheinberg with their samples.  This combination made mold
counting really easy!  One note: you can make Rheinberg filters from
any colored plastic film but may need to (a) use a double thickness
to get good rich color and (b) crank up your light source.

Hope this is all helpful.

Good hunting!
Barbara Foster, President & Chief Consultant
Microscopy/Microscopy Education*
www.MicroscopyEducation.com

*A subsidiary of The Microscopy & Imaging Place, Inc.
7101 Royal Glen Trail, Suite A
McKinney, TX 75070
P: 972-924-5310
F: 214-592-0277

MME is currently scheduling courses for now and through June 2015.
Call us today for a free training evaluation.






At 04:19 PM 1/30/2015, Jeff Spector wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>Post images on http://www.imgur.com and include the link in your posting.
>*****
>
>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]

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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:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> 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

[Barbara Foster]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hi, Steffen

Many thanks for the correction on the date for
Rheinberg!  Funny... I think that that came from
some ancient literature ... I will have to search
my files to see if there is still a copy.

One point about darkfield and NA: Actually, the
rays emerging from the condenser are at the
condenser's maximum NA.  The  cone in the middle
excludes the components of smaller NAs.  For this
reason, patch stops will only work with low NA
objectives.  However, because only a narrow band
of illumination is selected, the light is highly
coherent.  Again, scattering theory rather than
diffraction theory determines what can be detected.

Best regards
Barbara Foster, President & Chief Consultant
Microscopy/Microscopy Education*
www.MicroscopyEducation.com

"Education, not Training"


MME is currently scheduling courses for now and
through June 2015. Call us today for a free training evaluation.

*A subsidiary of The Microscopy & Imaging Place, Inc.
7101 Royal Glen Trail, Suite A  - McKinney, TX 75070 - P: 972-924-5310

At 04:31 AM 2/2/2015, you wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>Post images on http://www.imgur.com and include the link in your posting.
>*****
>
>Jeff,
>
>concerning resolution, normal dark field is not any differfent 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. 1­39,,
>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:
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com and include the link in your posting.
> > *****
> >
> > 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