Posted by
James Pawley on
URL: http://confocal-microscopy-list.275.s1.nabble.com/PSF-measurement-using-Au-beads-tp7581962p7582015.html
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Hi Lu,
As a great fan of what I like to call
"backscattered light imaging" (BSL), and also of
using gold beads to determine the PSF, I am
intrigued by your problems.
You mention that the specimen has only glass or
oil between the objective and the focus plane.
That is good. However, if one of your paired
images relates to light light reflecting at the
glycerol/glass surface on the far side of the
particle, it would have considerable SA because
of the light having passed twice through some
microns of glycerol. Remember that in the
Handbook chapter that you mentioned, Juskaitis
found that he got SA even using oil lenses used
with the correct oil if the oil was the wrong
temperature or the coverslip was the wrong
thickness. It is all very sensitive.
In fact, I admit to being surprised that you can
use BSL to see a bead at a glass/glycerol
interface at all. You should have an immense
signal from the glass/glycerol interface that
would swamp anything you get from a submicron
gold bead. (This reflected light signal will have
more intensity at high-NA angles because the
reflectivity at an RI interface increases
strongly as the angle between the ray and the
surface becomes smaller. This may be related to
your observation that there is less problem when
you close down your objective.)
Embedding the beads in the proper immersion oil should reduce this problem.
In terms of the "double image," I think we need
to look at the effects of reflections,
particularly those from the flat or nearly flat
optic al surfaces such as the ends of the fibres
and the slide/coverslip. I didn't see any mention
of efforts to use the normal "antiflex"
techniques that use polarizers and 1/4 wave
plates to discriminate against signals caused by
reflections from the optical components such as
the scan lens and the objective.
I clearly remember the problems we had trying to
get rid of reflections that seemed to originate
from in the Nikon widefield eyepiece used for a
scan lens in the BioRad MRC confocals.
Reflections from curved surfaces have virtual
origins and if the optics between this origin and
the pinhole (sensor-fibre) plane have the effect
of focusing this virtual origin onto the sensor
plane, will create a "blobby" artifact.
Of course, our blobs didn't go in and out of
focus as you move the objective in z so it is
unlikely that this is the exact cause of the
artifact that you are seeing, but it is the only
example that I have.
As you seem to have a copy of The Handbook
available, you might look at the pages shown in
the Index under the headings "backscattered
light" and "antiflex".
And you might try looking at some larger beads...
Good luck,
Jim Pawley
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>
>Hi Zdenek,
>
>Thanks for your advices. We are using a multimode fiber acting as a
>detection pinhole. We use an 60X/1.35 objective (olympus), f= 300 mm lens
>to focus the light into detection fiber. The excitation laser line is 650
>nm, so one airy unit= 1.22*650nm/1.35 * 300mm/30mm = 58.7 um. I have three
>fibers with core diameters of 50 um, 100 um, 150 um. I am currently using
>50 um one. I tried 150 um, the image was actually similar in terms of
>overall shape, i.e. 4 lobes.
>
>For the achromat doublet. Our intention was to use the same lens to
>collimate several colors (from 450 nm~650 nm), but we found also that
>Thorlabs achromat shifted the focal points of 532 nm and 650 nm by 600 nm
>on the sample plane. We are thinking of changing to some other
>lenses/objective lens. Do you have any suggestions? I noticed that low mag.
>objective usually does not do a lot chromatic aberration corrections so
>it's kind of tricky to find one, with the right focal length.
>
>Thanks,
>Lu
>
>-----------------------------------------------------
>Lu Yan
>Nanostructured Fibers and Nonlinear Optics Laboratory
>Electrical and Computer Engineering
>Boston University
>8 St. Mary St., Boston, MA, 02215
>(617)353-0286
>
[hidden email]
>-----------------------------------------------------
>
>
>On Fri, May 2, 2014 at 6:15 AM, Zdenek Svindrych <
[hidden email]> wrote:
>
>> *****
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>>
>> Hi Lu,
>> 1. You can reduce the two reflective layers by closely matching the
>> refractive indices using proper oil instead of glycerol. The mismatch may
>> also influence the PSF itself, but not dramatically. Also by opening up the
>> detection pinhole you can scan the laser focus profile, this can tell you
>> whether the problem is due to excitation or detection.
>>
>> 2. 30 mm achromat doublet can introduce some aberrations. I often use low
>> power microscope objectives when it comes to short focal lengths. Also note
>> that to get the best diffraction-limited excitation you should
>> significantly
>> overfill the back aperture of the objective. So a 100 mm collimating lens
>> (achromat doublet) could solve both problems.
>>
>> Finally, try fluorescent beads. They should give you more accurate
>> representation of your PSF...
>>
>> Regards, zdenek svindrych
>>
>>
>>
>> ---------- PÛvodní zpráva ----------
>> Od: Lu <
[hidden email]>
>> Komu:
[hidden email]
>> Datum: 2. 5. 2014 0:04:14
>> PÞedmût: PSF measurement using Au beads
>>
>> "*****
>> To join, leave or search the confocal microscopy listserv, go to:
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>> *****
>>
>> Hello all,
>>
>> We have a home-build confocal microscopy setup in our lab, and we have been
>> trying to evaluate the PSF using gold beads (d=150 nm), but currently we
>> are
>> having some difficulty on interpreting some of our results. I am hopeful
>> that I could find some help here.
>>
>> Backgroud: Our excitation light is a 650 nm laser diode. Objective lens is
>> Olympus 60X 1.35/Oil, and the illumination light from a single mode fiber
>> is
>> collimated using a Thorlabs achromatic doublet with focal length of 30 mm,
>> and then sent into the objective by folding mirrors (the beam is slightly
>> underfilling the back aperture of the objective). Piezo scanning is used
>> instead of resonant mirrors scanning. We collect the reflected/scattered
>> light from the bead to form image. No filter was used in front of our
>> detector. For the beads sample, 99% Glycerol was used as mountant.
>>
>> Problems:
>> 1. Two reflective layers showed up as we do axially scanning, separated by
>> about 8 um, and the bead turned out to be attached to one of them. The
>> axial
>> PSF looks terribly distorted. It is very much like a four lobes pattern,
>> i.e. an intensity null surrounded by 4 lobes on top/bottom and right/left.
>> You could imagine that at some z positions, the lateral intensity pattern
>> has a donut-shape. We do have a good explanation why this happened. Does
>> any
>> one ever have similar problem? Is my sample preparation wrong?
>>
>> 2. If I put a iris before the back aperture of the objective, and closed it
>> a little bit to truncated my collimated beam to half of its original size,
>> then the axial PSF suddenly got cleaned up, i.e. a single nice vertical
>> lobe
>> appeared. But 2 reflective layers were still there observable. Any idea
>> why?
>> We thought the achromatic double for collimation might induce some higher
>> order free space mode other than pure Gaussian mode, such that when we
>> close
>> the iris we effectively cut off some high k vectors of those 'other modes',
>> leaving nicer Gaussian going into the objective to produce nicer axial PSF.
>> Does this make sense to you guys?
>>
>> 3. A question often confuses me, which exactly quantity, in my case, should
>> I correlate my measured FWHM of the bead image, in order to check if my
>> setup is of diffraction limited performance? I have not been able to find a
> > consistent criteria in literatures.
>>
>> Thanks in advance.
>> Lu"
>>
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