Posted by
Barbara Foster on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Boosting-bright-field-resolution-with-dichroic-filters-tp7583983p7583987.html
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Hi, Ben
Whew! You've hit on lots of theory vs practice
here. Here are some short answers
1. Re: Your equation
First: congratulations in using the NA of the
condenser in your equation... Most people don't
know to do that (Dr. Robert Hoffman, inventor of
Hoffman Modulation Contrast, fought this battle
for years and would be so proud of you!)
Secondly, if you are using 0.6 for the Bessel
function (related to the round aperture), you
don't need to divide by 2. The equation is just
1.2 x wavelength / (NAo+NAc) where NAo = NA of
objective, NAc = NA condenrser.
2. Re: higher NA condensers
They require oil immersion. (Don't do this with
a condenser that is marked 0.9NA because the
lenses are not sealed against oil and the oil
will migrate down between elements and ruin the
optics).. To work properly, you need to put a
drop of oil on top of the condenser, bring the
down into optical contact, put a drop of oil
between slide + objective and bring objective
into optical cotact then set up Koehler (which
involves focusing both the objective and focusing
and centering the condenser).
Oil makes a huge difference in NA. It's all derived from Snell's Law:
NA = n sine a where n = refractive index of the
immersing fluid (air = 1.00, water = 1.33, imm
oil - which is highly engineered = 1.5 or often
1.512) and sine a = the sine of 1/2 the collecting angle of the optics.
Ex: for an objective
if the 1/2 angle is about 10 degrees, as measured
from the optic axis, sine a = 0.1; if nearly 90 degress, sin a = close to 1.0
An interesting trick which you can use with your
0.9 NA condenser is to insert a diffusing
filter into its front focal plane (about where
the condenser aperture iris is located. Use a
beaded filter (beads toward sample) if
possible. It acts like a micro array of lenses,
throwing light off at all angles, making the
condenser behave more like the 1.4NA you are
striving for.. You still have to fight Snell's
Law at the glass/air interface, but you have lots
more angle to work with. FEI has used this
approach to wonderful advantage in their iMic.
3. Re: Seeing better definition with blue, it all
comes down to diffraction theory.
Have you tried looking for the diffraction
pattern from your diatoms? I don't know your
full set up but you may be able to catch it with
the diatoms that have larger spacing. It's been a
while since I've done this, but here is what I remember.
For a sample, use the diatome (simple gratings also work well).
Set the microscope up for Koehler illumiation.
Cut a piece of black paper to fit over the light
port. Put a pinhole in it (poke a hole with the
end of an opened paperclip... regular sized paper
clips typically work well). Center the pinhole
on the light port. Make sure that both the field
iris and condeneser aperture iris are full open.
Remove the eyepice and look down into the
tube. You may need to adjust the pinhole a bit,
but you should see a bright white spot and an
array of rainbow dots around it. That's the
diffraction pattern which carries the imaging
information from your object to form the image.
The bright white central spot (the "Zerio order")
is the undiffracted light responsible for all
background illumination in the image. The shape
and spacing of the rainbox spots about
orientation, resolution, and edges from the specimen.
Orientation: always at right angles to the
structure in the specimen (ex: if you had
grating with ruling that went N-S; according to
Diffraction Theory, the spots would be arrayed E-W)
Spacing: Related to the Fourier Transform of
spacing in the specimen; Broad spacing in the
specimen = narrow spacing in the diffraction pattern)
Resolution: Capturing any two spots will carry spacing/resolution
Edge information: Capturing any three or more
spots (except the zero order and spots on either
side) contributes to edge information. Ideally,
a sharp edge in the specimen would create a sharp
"top hat" type of wave form, but diffraction
effects cause all sorts of distortion. The more
spots captured, the closer the image will represent the edge of the sample.
So what does all of this have to do with blue
light? Blue resists diffraction. If you look at
the diffraction pattern with white light, you'll
see all the dispersion and "cross talk" from the
full spectrum. If you keep watching and insert
your blue dichroic, you'll see the diffraction
pattern clean up instantly... and you'll notice
two other things: (1) Because you have affected
the zero order (made it blue), the background in
your image has become blue and (2) Because blue
resists diffraction, you can capture more blue
dots or portions of blue dots hence higher
resolution and better quality edges.
4. Re; Ringing
The condenser aperture controls coherence. Using
anything that narrows the beam emerging from the
condenser automatically selects a narrow
population of waves which are traveling through
space. Since those waves come from a similar
spot on the source and are traveling essentially
the same optical path, they will be in step or
"in phase" with each other. As a result, any
small local change in optical path (ex:
refractive index) in the sample, can put them out
of step just enough to either constructively (in
step or in step by whole wavelengths) or
destructively (out of step by 1/2 wave or odd 1/2
multiples) with each other. Constructive
interference results in bright rings; destructive
interference results in dark rings and the small
difference in refractive index at the boundary
between the diatom (which has n~1.5) and its
mounting (which will also be close to ~1.5) are
enough to enable those events to happen.
How can you narrow the beam: (a) close down the
condenser aperture iris (the basis for axial
illumination and, if off-set, for oblique
illumination) or (2) open the iris completely and
insert a slit, either on axis or off-axis
(another way to create oblique illumination and
the part of the foundation for Hoffman Modulation
Contrast) or (3) introduce an annulus, which is
like having an infinite number of off-axis spots
arranged in a ring. If the IMAGE of the ring
placed in the condenser is smaller than the NA of
the objective, you have the first step toward
phase contrast (you need to have a matching phase
plate in the back focal plane of the
objective.... but that's another discussion). If
the image of the ring is larger than the NA of
the objective, that is the foundation for
Darkfield, Rheinberg (a trully delightful
experiment, especiallly with diatoms!),and Dr. McCrone's Dispersion Staining.
I think that covers it.
Good hunting!
Barbara Foster, President & Chief Consultant
Microscopy/Microscopy Education ... "Education, not Training"
7101 Royal Glen Trail, Suite A - McKinney, TX 75070 - P: 972-924-5310
www.MicroscopyEducation.com
NEW! Getting involved in Raman or FTIR?
MME is now offering courses in these areas specifically for microscopists!
Now scheduling courses through the end of
2015. We can customize a course on nearly any
topic, from fluorescence to confocal to image analysis to SEM/TEM.
Call today for a free training evaluation.
At 07:17 AM 7/10/2015, 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.
>*****
>
>Hey microscopists,
> I had a student ask if the department had a
> 1.4NA condenser for high resolution imaging of
> diatoms. This is a pretty specialized piece of
> equipment, and the highest NA condenser I could
> find on hand was 0.9NA, so I started thinking
> about how we could get a comparably high resolution with our setup.
>
> For a 1.4NA objective and a 1.4NA
> condenser, with white light BF illumination,
> one would calculate the lateral resolution to be approximately:
>
> (0.6 * 575nm) / ((1.4 + 1.4) / 2) = 246nm
>
> For a 1.4NA objective and a 0.9NA
> condenser, with white light BF illumination,
> one would calculate the lateral resolution to be approximately:
>
>
> (0.6 * 575nm) / ((1.4 + 0.9) / 2) = 300nm
>
> However, if you then simply put a blue
> emission filter (such as a DAPI filter cube)
> into the light path, then one would calculate the lateral resolution to be:
>
>
> (0.6 * 445nm) / ((1.4 + 0.9) / 2) = 232nm
>
> Which is now a slightly better lateral
> resolution then even the 1.4NA condenser setup.
>
> I tested this out on a diatom slide, and
> the results perfectly matched the theory, with
> the white BF image maxing out at 300nm
> resolution, and the blue BF image maxing out at
> 230nm resolution. You can also clearly see
> additional detail in the blue BF image:
>
>White BF Image -
>
https://drive.google.com/file/d/0B7pDqE0lTjQXT3VKc2Y0ckFEU2s/view>Blue BF Image -
>
https://drive.google.com/file/d/0B7pDqE0lTjQXVUhBODJ4NUZMS3c/view>FFT of White BF -
>
https://drive.google.com/file/d/0B7pDqE0lTjQXb2lBR2dwRXEzVVE/view>FFT of Blue BF -
>
https://drive.google.com/file/d/0B7pDqE0lTjQXZU5GQWNaTE5aUGM/view>
> Upon further investigation, I found this
> great write-up by René van Wezel discussing the
> same and other ideas for boosting resolution:
>
http://www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopy-uk.org.uk/mag/artapr09/rvw-contrast.html>
>
> However, in my hands, annular illumination
> generated a ringing artifact, although this is
> likely because the NA of the condenser is much
> lower than the NA of the objective. All in
> all, I'm sure for experienced microscopists
> this is likely an obvious solution, but for
> newer microscopists, it may be surprising just
> how much higher the resolution becomes simply
> by putting a short wavelength dichroic filter
> into the light path (especially when comparing
> the FFTs), and serves as a reminder that
> transmitted light resolution isn't primarily
> about NA alone. I know for myself, I
> qualitatively knew that blue light would boost
> resolution, but it wasn't until I did out the
> math, and verified it experimentally, that I
> realized that blue light with a conventional
> dry condenser can even out-perform white light
> with a 1.4NA oil immersion condenser.
>
>Have a great Friday,
> Ben Smith
>
>Benjamin E. Smith, Ph.D.
>Samuel Roberts Noble Microscopy Laboratory
>Research Scientist, Confocal Facility Manager
>University of Oklahoma
>Norman, OK 73019
>E-mail:
[hidden email]
>Voice 405-325-4391
>FAX 405-325-7619
>
http://www.microscopy.ou.edu/