Boosting bright field resolution with dichroic filters

[Smith, Benjamin E.]

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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  [phone]
http://www.microscopy.ou.edu/

[Sergey Tauger]

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Dear Mr. Smith,

It is a common practice to insert a light filter into optical path, but more
common way is to place 50 mm wide filter (a cheap one, like stained glass)
between lamp and condenser. A common choice is blue-green to green light
(500-550 nm). The reasons are:
1. Condensers are not usually compensated for blue or red. Surprisingly,
Nikon manufactures good objectives, but their condensers really suffer from
chromatic aberrations.
2. When speaking about brightfield we should not forget about light spread
and absorbance. Both are in reverse proportion to wavelength
3. When limited to a narrow light spectrum, we should increase lamp voltage
that can shorten its life significantly.

Best,
Sergey Tauger

[Smith, Benjamin E.]

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Hey James,
   This site has a nice intro to image FFTs:

https://www.cs.unm.edu/~brayer/vision/fourier.html

   I also recommend this site to my students, because it allows you to modify an FFT and immediately see the impact on the corresponding image:

http://www.brainflux.org/java/classes/FFT2DApplet.html

   In short, as long as the pixel dimensions are scaled correctly in ImageJ, then when you take the FFT, it will give you the spatial frequencies in actual units (i.e. um/cycle).  To read an FFT, the center pixel is the DC component, which you can think of like the offset on a PMT, where it represents the average intensity of the whole image.  As you move the cursor away from the center, you will notice imageJ will give you an angle, amplitude, and frequency.  The angle is the direction the spatial frequency is propagating.  The amplitude is the amplitude (peak to trough) height of that frequency.  Another way to think of this, the greater the amplitude, the greater the contrast of the corresponding feature in the image.  Finally, frequency is the physical peak to peak distance (hence um per cycle, etc.).

   To determine maximum optical resolution, you need to acquire an image where the pixel resolution is much higher than the optical resolution (in this case the pixels were 64nm x 64nm).  If the optical resolution is higher than the pixel resolution, then the FFT cannot show spatial frequencies at the limit of the optical resolution, as they are not contained within the image in the first place.  In other words, in a digital image the highest spatial frequency is 2 pixels, one high, next low, next high, etc.  So, if your pixels are 400nm across and you expect your resolution to be 200nm, then the highest spatial frequency contained within your image is 800nm/cycle so you will never be to resolve down to 200nm.  This is similar to if in an image a person's face is a single pixel, you can't reconstruct the face from that one pixel, no matter what CSI says.

   So, once you have an image where the pixel dimensions are much smaller than your optical resolution, then you take the FFT, and you are looking for the highest spatial frequencies (i.e. the points farthest from the center) where the amplitude of those frequencies is significantly greater than the background amplitude (i.e. look for the pixels that are significantly brighter than the background and farthest from the center).  There is no discrete cut-off, and the amplitude (i.e. contrast) decreases as you approach the resolution limit (this is explained in the link in my original post, in term of the modulation transfer function).  Therefore, I simply looked for a block of high amplitude pixels that were farthest from the center, and measured the spatial frequency at that cutoff.

   You can do the same exercise, with the images posted.  Load the images into ImageJ or FIJI, and make sure the image properties are set to 0.0645um x 0.0645um (ctrl+shift+P).  Then crop the image to remove the scale bar, or the scale bar will show up in the FFT (draw a rectangular selection and press ctrl+shift+X).  Finally, take the FFT of the image (Process->FFT->FFT).  Autocontrast the FFT to make the amplitudes more clear against the background noise (ctrl+shift+C then "Auto").  Then put the cursor over the outer most frequencies that clearly contrast against the background noise.  The bottom of the title bar (the bar with all the menu options) will then automatically give you all the info about the frequency at your cursor position. It should read something like:

r=0.30 micron/c (434), theta= 8.88°, value=121

Where "r" is your resolution, which in this case would be 300nm.  You can also have some fun with the FFT, where you can use the draw tool to black out some of the frequencies and then take the inverse FFT (Process->FFT->Inverse FFT) to see how this modifies the image.  I have found there is no better way to get and intuitive sense of FFTs then to play with them, and see how it effects the image.  FFTs can also be extremely powerful tool in isolating specific features in an image.

For example, take this image of a dandelion: https://drive.google.com/file/d/0B7pDqE0lTjQXbVZjTFlwVWhDT1U/view?usp=sharing

If I asked you to crop the image so that it shows only the dandelion head, it would take years to precisely crop out ever background pixel around every fluff strand.  Conversely, you may notice that the background is blurry (low spatial frequencies), while the fluff is sharp and fine (high spatial frequencies).  Therefore, if rather than cropping out the background positionally (i.e. in an image), you can crop it out via frequency (i.e. in an FFT).  Therefore, in 10 seconds (rather than years) you can use an FFT to separate the fluff from the background based on frequency rather than position and get the following result:

https://drive.google.com/file/d/0B7pDqE0lTjQXU2U0cGZ4OHJlQnM/view?usp=sharing

To recreate this trick exactly.  Open the original image in ImageJ.  Think of this image as a combination of both the high frequency information you want to keep and the low frequency information you want to remove.  To make a purely low frequency version of the image, apply a 20 sigma Gaussian blur to the image (Process->Filters->Gaussian Blur).  Why a Gaussian blur specifically makes a image low frequency only is explained nicely here:

http://homepages.inf.ed.ac.uk/rbf/HIPR2/gsmooth.htm

Then use the image calculator to subtract the blurred image from the original image (Process->Image Calculator).  The math you are doing is basically:
      Original Image    -    Blur Image = Fluff only
                                 OR
(High Freq + Low Freq) - (Low Freq) = High Freq

This is one of the more simple tricks with spatial frequencies, but it drives the point home as to how some problems that are near impossible to solve in positional space (an image) are trivial in frequency space (an FFT).

Hope this helps,
  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  [phone]
http://www.microscopy.ou.edu/
 





________________________________________
From: WAINWRIGHT James [[hidden email]]
Sent: Friday, July 10, 2015 9:24 AM
To: Smith, Benjamin E.
Subject: RE: Boosting bright field resolution with dichroic filters

Hi Benjamin,

Cool post!

Hope you don't mind me asking how you worked out the resolution from the FFT?

Bear in mind that I'm not a mathematician nor computer scientist.

I can kind of see that the FFT of the blue image has a larger "bright" area / distance from centre, but I'm not sure what that means and how you then show the resolution of the original?

Thanks for any explanation you can provide or handy website for explaining FFTs...

Best regards,

James

James Wainwright
Product Support Engineer (Microscopy Systems)
Europe, Middle East & Africa

Tel:   [phone]
Mob: [phone]
Fax: [phone]
Web:  www.andor.com/ContactSupport.aspx?type=s



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Smith, Benjamin E.
Sent: 10 July 2015 14:59
To: [hidden email]
Subject: Boosting bright field resolution with dichroic filters

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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  [phone]
http://www.microscopy.ou.edu/


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[Barbara Foster]

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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


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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  [phone]
>http://www.microscopy.ou.edu/

[Michael Model]

*****
To join, leave or search the confocal microscopy listserv, go to:
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Post images on http://www.imgur.com and include the link in your posting.
*****

Hi Ben,

The more accurate (so they claim) calculation of BF resolution was
published by Hopkins and Barham in 1950

"The Influence of the Condenser on Microscopic Resolution"

and they show that better resolution is achieved at NA(cond) < NA(obj)
(Fig. 2). You can find their paper on
http://iopscience.iop.org/0370-1301/63/10/301/pdf/0370-1301_63_10_301.pdf


Mike Model




On Fri, Jul 10, 2015 at 9:59 AM, Smith, Benjamin E. <[hidden email]>
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  [phone]
> http://www.microscopy.ou.edu/
>

[George McNamara]

*****
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 Ben,
thanks for initiating a great thread (ex. Barbara Foster's reply).

Be sure the microscope is optimally focused for each wavelength!

You wrote:

    (0.6 * 445nm) / ((1.4 + 0.9) / 2) = 232nm

but for $97 you could buy a 400 nm filter
https://marketplace.idexop.com/store/IdexCustom/PartDetails?pvId=35105
(or buy a filter from Semrock or Chroma).

    (0.6 * 400nm) / ((1.4 + 0.9) / 2) = 209nm

Could go shorter than this with appropriate light source, such as the
~370 nm peak of the XLED1, in transmitted mode
http://www.excelitas.com/Pages/Product/X-Cite-XLED1.aspx

    (0.6 * 370nm) / ((1.4 + 0.9) / 2) = 193nm

or in epi mode, 1.4 NA objective lens (and assuming clean light path):

    (0.6 * 370nm) / ((1.4 + 0.9) / 2) = 158nm


//

A bit more challenging: diameter of the pores of /Pleurosigma
angulatum/, instead of just the spacing of the pores - see bottom of

http://www.microscopy-uk.org.uk/mag/artjan13/fs-diatom-micro.html


enjoy,

George
p.s. reflection confocal microscopy of diatoms is also a lot of fun ...
especially <<1.0 Airy diameter pinhole. Hopefully someone will publish
results of this for the Zeiss AiryScan. STED with the right fluorophore
in the mounting medium (or thin coating the diatom) would also be
interesting.


On 7/10/2015 8:59 AM, Smith, Benjamin E. 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  [phone]
> http://www.microscopy.ou.edu/
>
>    

--



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]

*****
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, again

Just a comment on the George's discussion.  These
don't seem like big improvements but just
remember, everytime you are dropping resolution
you are also improving edge information but you
get a double bang for the buck.  It only takes a
difference of 10-20 nm for you to "see" a better quality image.

Barbara
[phone]

Microscopy/Microscopy Education
"Education, not just Training"

At 05:24 PM 7/11/2015, George McNamara 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.
>*****
>
>Hi Ben,
>thanks for initiating a great thread (ex. Barbara Foster's reply).
>
>Be sure the microscope is optimally focused for each wavelength!
>
>You wrote:
>
>    (0.6 * 445nm) / ((1.4 + 0.9) / 2) = 232nm
>
>but for $97 you could buy a 400 nm filter
>https://marketplace.idexop.com/store/IdexCustom/PartDetails?pvId=35105
>(or buy a filter from Semrock or Chroma).
>
>    (0.6 * 400nm) / ((1.4 + 0.9) / 2) = 209nm
>
>Could go shorter than this with appropriate
>light source, such as the ~370 nm peak of the XLED1, in transmitted mode
>http://www.excelitas.com/Pages/Product/X-Cite-XLED1.aspx
>
>    (0.6 * 370nm) / ((1.4 + 0.9) / 2) = 193nm
>
>or in epi mode, 1.4 NA objective lens (and assuming clean light path):
>
>    (0.6 * 370nm) / ((1.4 + 0.9) / 2) = 158nm
>
>
>//
>
>A bit more challenging: diameter of the pores of
>/Pleurosigma angulatum/, instead of just the
>spacing of the pores - see bottom of
>
>http://www.microscopy-uk.org.uk/mag/artjan13/fs-diatom-micro.html
>
>
>enjoy,
>
>George
>p.s. reflection confocal microscopy of diatoms
>is also a lot of fun ... especially <<1.0 Airy
>diameter pinhole. Hopefully someone will publish
>results of this for the Zeiss AiryScan. STED
>with the right fluorophore in the mounting
>medium (or thin coating the diatom) would also be interesting.
>
>
>On 7/10/2015 8:59 AM, Smith, Benjamin E. 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  [phone]
>>http://www.microscopy.ou.edu/
>>
>>
>
>
>--
>
>
>
>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

[Steffen Dietzel]

*****
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.
*****

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

[George McNamara]

*****
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.
*****

Diatoms in bulk ... maybe in your (or a colleague's) kitty litter.

Diatomaceous earth
https://en.wikipedia.org/wiki/Diatomaceous_earth

Diatomaceous earth consists of fossilized remains of diatoms
<https://en.wikipedia.org/wiki/Diatom>, a type of hard-shelled algae
<https://en.wikipedia.org/wiki/Algae>. It is used as a filtration
<https://en.wikipedia.org/wiki/Filtration> aid, mild abrasive in
products including metal polishes and toothpaste
<https://en.wikipedia.org/wiki/Toothpaste>, mechanical insecticide
<https://en.wikipedia.org/wiki/Insecticide>, absorbent
<https://en.wikipedia.org/wiki/Absorption_%28chemistry%29> for liquids,
matting agent for coatings, reinforcing filler in plastics and rubber,
anti-block in plastic films, porous support for chemical catalysts, cat
litter <https://en.wikipedia.org/wiki/Cat_litter>, activator in blood
clotting <https://en.wikipedia.org/wiki/Blood_clotting> studies, a
stabilizing component of dynamite
<https://en.wikipedia.org/wiki/Dynamite>, and a thermal insulator
<https://en.wikipedia.org/wiki/Thermal_insulation>.

(I first learned of it from using kilograms in the filtration system of
our backyard pool).

Or
http://www.amazon.com/Diatomaceous-Earth-Food-Grade-10/dp/B00025H2PY


    Diatomaceous Earth Food Grade 10 Lb

<http://www.amazon.com/Diatomaceous-Earth-Food-Grade-10/dp/B00025H2PY/ref=sr_1_1?ie=UTF8&qid=1436789646&sr=8-1&keywords=Diatomaceous+earth>

by DiatomaceousEarth
Diatomaceous earth (de) is made of tiny, fossilized diatoms (aquatic
organisms) that accumulated over millennia in fresh water lakes. When
mined and left untreated, this amazing product can be used in hundreds
of different ways. However, not all diatomaceous earth is created equal.
While most other manufacturer's hand scoop de into zip lock bags,
diatomaceous earth brand de is professionally packaged using stainless
steel equipment. Because of this process, you can be confident there are
no contaminants in your food grade diatomaceous earth.


Maybe you could repackage this from "food grade" to "microscope grade".

Same order could include

http://www.amazon.com/100g-Fluorescein-Powder-Chemical-Reagent/dp/B00FJ64S3U


    100g of Fluorescein Powder, Chemical Reagent

<http://www.amazon.com/100g-Fluorescein-Powder-Chemical-Reagent/dp/B00FJ64S3U/ref=sr_1_1?ie=UTF8&qid=1436789750&sr=8-1&keywords=fluorescein>

by Aldon Corp
$44.95
<http://www.amazon.com/100g-Fluorescein-Powder-Chemical-Reagent/dp/B00FJ64S3U/ref=sr_1_1?ie=UTF8&qid=1436789750&sr=8-1&keywords=fluorescein>+
$6.74 shipping




Or make transgenic diatoms (http://www.ncbi.nlm.nih.gov/pubmed/25297896 
... centric diatoms sounds good for FFTs, and their abstract mentions
firefly luciferase and Azami-Green GP genes), fuse their silica binding
protein (Si-tag, see, for example,
http://www.ncbi.nlm.nih.gov/pubmed/21277372 ... Ecoli L2 ribosomal
protein and its 203-273 peptide apparently are SiBP's and has been fused
to EGFP, http://www.ncbi.nlm.nih.gov/pubmed/22926644) to GFP, RFP,
photoswitchable FP, iRFP, etcFP,
and sell that/those on amazon.com

enjoy,

George


On 7/13/2015 4:26 AM, Steffen Dietzel 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.
> *****
>
> 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
>

--



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

[Steffen Dietzel]

*****
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.
*****

Well, I once tried diatomaceous earth from the health food store, only
to find out that it came well ground, so it wasn't much use for
microscopy. Haven't tried again ever since. If somebody should know a
brand available in Germany that does work, I would be very interested,
would be a great resource for teaching.

Anyway, I was thinking more in the direction of a source of cultured,
well defined (dead) diatoms, that will avoid the trouble of finding a
specialist to identify the species. Like "Pleurosigma, 10 g, 50 Euro" or
the like.

I am afraid that transgenic diatoms available on Amazon won't be a
reality any time soon in Germany... But whatever they use for
photosynthesis is probably fluorescent anyway, as long as they are alive.

Steffen

Am 13.07.2015 um 14:26 schrieb George McNamara:

> Diatoms in bulk ... maybe in your (or a colleague's) kitty litter.
>
> Diatomaceous earth
> https://en.wikipedia.org/wiki/Diatomaceous_earth
>
> Diatomaceous earth consists of fossilized remains ofdiatoms
> <https://en.wikipedia.org/wiki/Diatom>, a type of hard-shelledalgae
> <https://en.wikipedia.org/wiki/Algae>. It is used as afiltration
> <https://en.wikipedia.org/wiki/Filtration>aid, mild abrasive in products
> including metal polishes andtoothpaste
> <https://en.wikipedia.org/wiki/Toothpaste>, mechanicalinsecticide
> <https://en.wikipedia.org/wiki/Insecticide>,absorbent
> <https://en.wikipedia.org/wiki/Absorption_%28chemistry%29>for liquids,
> matting agent for coatings, reinforcing filler in plastics and rubber,
> anti-block in plastic films, porous support for chemical catalysts,cat
> litter <https://en.wikipedia.org/wiki/Cat_litter>, activator inblood
> clotting <https://en.wikipedia.org/wiki/Blood_clotting>studies, a
> stabilizing component ofdynamite
> <https://en.wikipedia.org/wiki/Dynamite>, and athermal insulator
> <https://en.wikipedia.org/wiki/Thermal_insulation>.
>
> (I first learned of it from using kilograms in the filtration system of
> our backyard pool).
>
> Or
> http://www.amazon.com/Diatomaceous-Earth-Food-Grade-10/dp/B00025H2PY
>
>
>     Diatomaceous Earth Food Grade 10 Lb
>
> <http://www.amazon.com/Diatomaceous-Earth-Food-Grade-10/dp/B00025H2PY/ref=sr_1_1?ie=UTF8&qid=1436789646&sr=8-1&keywords=Diatomaceous+earth>
>
> byDiatomaceousEarth
> Diatomaceous earth (de) is made of tiny, fossilized diatoms (aquatic
> organisms) that accumulated over millennia in fresh water lakes. When
> mined and left untreated, this amazing product can be used in hundreds
> of different ways. However, not all diatomaceous earth is created equal.
> While most other manufacturer's hand scoop de into zip lock bags,
> diatomaceous earth brand de is professionally packaged using stainless
> steel equipment. Because of this process, you can be confident there are
> no contaminants in your food grade diatomaceous earth.
>
>
> Maybe you could repackage this from "food grade" to "microscope grade".
>
> Same order could include
>
> http://www.amazon.com/100g-Fluorescein-Powder-Chemical-Reagent/dp/B00FJ64S3U
>
>
>     100g of Fluorescein Powder, Chemical Reagent
>
> <http://www.amazon.com/100g-Fluorescein-Powder-Chemical-Reagent/dp/B00FJ64S3U/ref=sr_1_1?ie=UTF8&qid=1436789750&sr=8-1&keywords=fluorescein>
>
> byAldon Corp
> $44.95
> <http://www.amazon.com/100g-Fluorescein-Powder-Chemical-Reagent/dp/B00FJ64S3U/ref=sr_1_1?ie=UTF8&qid=1436789750&sr=8-1&keywords=fluorescein>+
> $6.74 shipping
>
>
>
>
> Or make transgenic diatoms (http://www.ncbi.nlm.nih.gov/pubmed/25297896
> ... centric diatoms sounds good for FFTs, and their abstract mentions
> firefly luciferase and Azami-Green GP genes), fuse their silica binding
> protein (Si-tag, see, for example,
> http://www.ncbi.nlm.nih.gov/pubmed/21277372 ... Ecoli L2 ribosomal
> protein and its 203-273 peptide apparently are SiBP's and has been fused
> to EGFP, http://www.ncbi.nlm.nih.gov/pubmed/22926644) to GFP, RFP,
> photoswitchable FP, iRFP, etcFP,
> and sell that/those on amazon.com
>
> enjoy,
>
> George
>
>
> On 7/13/2015 4:26 AM, Steffen Dietzel 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.
>> *****
>>
>> 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
>>
>
>
> --
>
>
>
> George McNamara, Ph.D.
> Single Cells Analyst
> L.J.N. Cooper Lab
> University of Texas M.D. Anderson Cancer Center
> Houston, TX 77054
> Tattletaleshttp://works.bepress.com/gmcnamara/42
>

--
------------------------------------------------------------
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

[Hehl Joachim]

*****
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,
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


Web: www.lmc.ethz.ch
Phone:     [phone]
Natel:     [phone]
Fax:       [phone]
e-mail: Joachim.Hehl <mailto:[hidden email]>@scopem.ethz.ch








On 7/13/15 11:26 AM, "Steffen Dietzel" <[hidden email]> 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.
>*****
>
>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

[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.
*****

Unfortunately, the link didn't work.

Shouldn't it be 1.22?

Normally, you see it written as  0.6 only if it has been divided by 2NA.

Barbara Foster,  President and  Chief Strategic Consultant

The Microscopy & Imaging Place, Inc.
7101 Royal Glen Trail, Suite A   McKinney TX 75070
P: [phone]     W: www.MicroscopyMarket.com

The MIP:  Microscopy & Spectroscopy Business Specialists
Bending the curve toward faster sales and business development
At 08:22 AM 7/13/2015, 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.
>*****
>
>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
>
>
>Web: www.lmc.ethz.ch
>Phone:     [phone]
>Natel:     [phone]
>Fax:       [phone]
>e-mail: Joachim.Hehl <mailto:[hidden email]>@scopem.ethz.ch
>
>
>
>
>
>
>
>
>On 7/13/15 11:26 AM, "Steffen Dietzel" <[hidden email]> 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.
> >*****
> >
> >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]

*****
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.
*****

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:

> *****
> 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,
> 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
>
>
> Web: www.lmc.ethz.ch
> Phone:     [phone]
> Natel:     [phone]
> Fax:       [phone]
> e-mail: Joachim.Hehl <mailto:[hidden email]>@scopem.ethz.ch
>
>
>
>
>
>
>
>
> On 7/13/15 11:26 AM, "Steffen Dietzel" <[hidden email]> 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.
>> *****
>>
>> 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

[Michael Model]

*****
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.
*****

It must be a little different here because in bright field the sources are
partially coherent - the smaller the condenser aperture the more coherent.
Fluorescent sources are incoherent and astronomical objects are coherent, I
believe. - Mike

On Tue, Jul 14, 2015 at 8:55 AM, Steffen Dietzel <[hidden email]>
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.
> *****
>
> 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:
>
>> *****
>> 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,
>> 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
>>
>>
>> Web: www.lmc.ethz.ch
>> Phone:     [phone]
>> Natel:     [phone]
>> Fax:       [phone]
>> e-mail: Joachim.Hehl <mailto:[hidden email]>@
>> scopem.ethz.ch
>>
>>
>>
>>
>>
>>
>>
>>
>> On 7/13/15 11:26 AM, "Steffen Dietzel" <[hidden email]> 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.
>>> *****
>>>
>>> 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
>