Unmasking spherical aberration
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Hello All,
I have what may be an amateurish question, but I have noticed something very strange today with my Olympus IX81 DSU spinning disk confocal. While acquiring a 3D stack with a PlanApo60X1.42NA objective with disk in place, intra-nuclear structures (foci of gamma-H2AX, 0.4-1.5um in diameter) appeared to become blurred and no longer in focus at a certain point in the acquisition. I did not notice anything odd while looking through the eyepiece. The same field of view, when imaged in widefield appeared normal. Maximum intensity projection of the confocal stack resulted in blurred foci, whereas the widefield MIP resulted in punctate, bright foci. X-Z orthogonal planes from the confocal stack showed a positive spherical aberration, but the widefield did not show anything marked. Can confocal imaging un-mask spherical aberration that may otherwise be obscured by widefield illumination? I am at a loss and would appreciate any feedback. I have a powerpoint that I prepared for the folks at Olympus if my question/concern is not very clear. Thanks alot to the group for considering my question. Cheers Farid -- Farid Jalali MSc Senior Research Technician/ Lab Manager Dr. Robert Bristow Lab Applied Molecular Oncology Princess Margaret Hospital Toronto, Canada 416-946-4501 X4351 (Princess Margaret Hospital) 416-581-7754 STTARR at MaRS Building 416-581-7791 STTARR Micrroscopy Suite |
 
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Julio Vazquez |
Re: Unmasking spherical aberration
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Hi Farid, I am not familiar with that particular instrument, or spinning disk units in general, but I would be surprised that the disk by itself would generate spherical aberration, especially suddenly out of the blue. How positive are you that what you are seeing is spherical aberration? What is the overall image quality (e.g. signal to noise, sharpness in the x,y plane, etc..) when you slide the DSU in? Does the effect appear only when you are focusing at a certain depth or is it always there? Do you see the effect with other lenses? Is it sample dependent? Is this a new problem that just appeared or did you see it in the past (and if so, under what conditions)? If the stacks you collect without the DSU look good, my inclination would be to think there is a mechanical or electronic problem with the DSU... I would shut down and reinitialize the instrument. Then, I would collect a couple of sample stacks with a nice test slide, with and without DSU. If the problem persists, I would contact the vendor and show them the test images. This might require a service visit. -- Julio Vazquez Fred Hutchinson Cancer Research Center Seattle, WA 98109-1024 Tel: Office: 206-667-1215/ Lab: 206-667-4205 FAX: 206-667-6845 -------------------------------------------------- This message is confidential, intended only for the named recipient(s) and may contain information that is privileged or exempt from disclosure under applicable law. If you are not the intended recipient(s), you are notified that the dissemination, distribution or copying of this information is strictly prohibited. If you received this message in error, please notify the sender then delete this message. On Dec 4, 2007, at 5:42 PM, Farid Jalali wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hello All, |
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James Pawley |
Re: Unmasking spherical aberration
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Hi Farid,
You speak of making a z-stack but don't mention how far it goes into the water. You are using an oil lens and presumably looking into an aqueous specimen. If the illumination fills the BFP of the 1.42 objective, you should expect to get severe SA only few microns into the water. Again, if the BFP is filled, all the light entering it at positions and angles that correspond to great than about NA 1.3 will be TIR and will make the signal from fluorescent markers located near the coverslip very much brighter. As you focus into the water therefore, you will not only have no TIRF light to enhance the brightness of the signal from fluorophors located near the interface, you will lose signal rapidly from SA. I am guessing that you don't notice this SA when viewing in widefield because the image you see is dominated by this near interface signal that doesn't have much SA (because is it near the interface). I suggest that you either use a water objective or look at cells made out of oil. Cheers, Jim P. Prof. James B. Pawley, Room 223, Zoology Research Building, FAX 608-265-5315 1117 Johnson Ave., Madison, WI, 53706 3D Microscopy of Living Cells Course, June 14-26, 2008, UBC, Vancouver Canada Info: http://www.3dcourse.ubc.ca/ "If it ain't diffraction, it must be statistics." Anon. |
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Farid Jalali |
Re: Unmasking spherical aberration
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Hi Jim,
Thanks to you and the rest of the responders. All very good suggestions and comments. I am looking at fixed cells grown on No 1.5 coverslips, mounted in Vectashield. Granted there is an RI mismatch between the Vectashield (RI 1.47 or so) and the immersion oil I am using (1.5156), it is not as severe as if I was looking into an aqueous media. Now if I did that after the Pawley course, well that would be scandalous (smile!). The cells are grown on the coverslip, and the cells are maybe 5-10um thick depending on cell cycle phase. I typically image at 0.25um steps, taking 80 images to generate the stack. I have imaged 200nm PSF beads and seen them to be generally symmetrical through Z, using this same objective, same Z step interval and number of slices. This bead is not the same as a cell obviously, but above all, it is something that I have not come across since we got this instrument 18 months ago. I have spent countless hours imaging with this objective and have all of a sudden encountered this problem. The point re. why I don't see the blurring of the max intensity projection makes sense. Olympus is going to follow-up on this matter and has pointed to making sure that the disk is on the correct focal plane. Thanks again to all for your helpful and insightful replies. Cheers Farid On Dec 5, 2007 5:58 PM, Jim Pawley <[hidden email]> wrote:
-- Farid Jalali MSc Senior Research Technician/ Lab Manager Dr. Robert Bristow Lab Applied Molecular Oncology Princess Margaret Hospital Toronto, Canada 416-946-4501 X4351 (Princess Margaret Hospital) 416-581-7754 STTARR at MaRS Building 416-581-7791 STTARR Micrroscopy Suite |
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Chris Tully |
Re: Unmasking spherical aberration
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Farid,
Vectashield sounds like an almost perfect RI match for glycerin (1.47399 according to Dow Chemical: http://www.dow.com/glycerine/resources/table12_81100.htm ). I would suggest that you contact your microscope sales person about getting a glycerin immersion lens to try out for a few days and see if that makes a difference. Chris Tully Applications Engineer Vashaw Scientific Inc. http://www.vashaw.com http://www.leica-microsystems.com [hidden email]
On Dec 5, 2007 8:40 PM, Farid Jalali <[hidden email]> wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Jim, |
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Stephen Cody |
Re: Unmasking spherical aberration
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In reply to this post by Farid Jalali
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G’day Farid,
I wouldn’t say that confocal ‘unmasks spherical abberation’, but in a fashion you are right. Confocal microscopy is highly susceptible to spherical aberration. Unless you are using reflection microscopy, the pinhole (slits in the case of the DSU) will reject the infocus signal, and out-of-focus blurr will come through. Because of this there will be a dramatic drop of intensity of the signal with increasing depth into the specimen.
The spherical aberration is no different in widefield microscopy, its just that there is no pinhole, so all the signal reaches your eye. You say the PSF with a bead looked OK, but confocal loses performance dramatically at depth with only a slight aberration. So I wouldn’t rule out this as being an effect of Refractive Index mis-match. If it is not possible to remove the RI-mismatch altogether (as other people have suggested), on a point scanner confocal I would suggest opening up the pinhole a little. On the DSU I presume you could use an alternative disk with wider slits as a compromise.
Cheers Stephen H. Cody Tip: Learn how to receive reminders about you microscope
booking: -----Original Message-----
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Jim, On Dec 5, 2007 5:58 PM, Jim Pawley <[hidden email]> wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Farid,
You speak of making a z-stack but don't mention how far it goes into the water. You are using an oil lens and presumably looking into an aqueous specimen. If the illumination fills the BFP of the 1.42 objective, you should expect to get severe SA only few microns into the water. Again, if the BFP is filled, all the light entering it at positions and angles that correspond to great than about NA 1.3 will be TIR and will make the signal from fluorescent markers located near the coverslip very much brighter. As you focus into the water therefore, you will not only have no TIRF light to enhance the brightness of the signal from fluorophors located near the interface, you will lose signal rapidly from SA.
I am guessing that you don't notice this SA when viewing in widefield because the image you see is dominated by this near interface signal that doesn't have much SA (because is it near the interface).
I suggest that you either use a water objective or look at cells made out of oil.
Cheers,
Jim P.
Prof. James B.
Pawley,
Ph. 608-263-3147 "If it ain't diffraction, it must be statistics." Anon.
This communication is intended only for the named recipient and may contain information that is confidential, legally privileged or subject to copyright; the Ludwig Institute for Cancer Research does not waiver any rights if you have received this communication in error. The views expressed in this communication are those of the sender and do not necessarily reflect the views of the Ludwig Institute for Cancer Research. |
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Farid Jalali |
Re: Unmasking spherical aberration
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In reply to this post by Farid Jalali
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Well the saga of my Olympus IX81 DSU going astray has come to an end. Seems the disk was out of alignment; not exactly sure if this speaks to the disk itself or the mechanism that holds and houses it. The Olympus guys were here yesterday and sorted it out. I suspect this has something to do with the fact that we are switching between disk 3 and 4, as we switch between 60X and 100X objectives.
Thanks to the group for thought provoking comments and helpful ideas.
Best to All.
Happy Holidays.
Farid -- Farid Jalali MSc Senior Research Technician/ Lab Manager Dr. Robert Bristow Lab Applied Molecular Oncology Princess Margaret Hospital Toronto, Canada 416-946-4501 X4351 (Princess Margaret Hospital) 416-581-7754 STTARR at MaRS Building 416-581-7791 STTARR Microscopy Suite |
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