Re: Objective lens chromatic aberration
Locked
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** I originally posted the message below to the microscopy listserv but reviewing the discussion here on chromatic aberration, I thought it would be apropos here too even though it is not really regarding confocal. A few years ago we were having problems with the first commercial Olympus TIRF system because we could not get consistent evanescent waves with the one angle adjustment with the laser lines we had from 405 to 568 nm that were delivered via a single fiber (it was worse when we later added a 633 nm laser). I suggested we pump each laser in through a separate path that could be angled independently. We didn't build it, but I think Olympus now sells a TIRF system that does this. Another issue is that when I first heard about TIRF maybe 15 years ago, it was introduced as a ring illumination at the outer edge of the back aperture, not as a single point or crescent at the periphery on only one side. A ring, or at least a series of points around the periphery, seems like a better way to provide a uniform field due to aberrations from coherent light in the imperfect optics. Any thought on this? Sincerely, Michael -----------------------ORIGINAL MESSAGE------------------------------- We have the Nikon TIRF system and have three laser lines going into the TIRF arm via a single fiber. When we project through the 100X objective through the sample onto the wall we see that the lines go through the sample at different angles. (You can see a picture of the projection at approx 45 degrees at http://www.flickr.com/photos/mcammer/5359189090/ .) It is also noticeable in the TIRF images that the field depth is different for each wavelength. Is this unavoidable due to the different wavelengths or is it possible to align the optics better so these spots would be more coincident? _________________________________________ Michael Cammer, Assistant Research Scientist Skirball Institute of Biomolecular Medicine Lab: (212) 263-3208 Cell: (914) 309-3270 ------------------------------------------------------------ This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email. ================================= |
 
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Gary G. Li-2 |
Re: Objective lens chromatic aberration
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Michael, There are a few factors that need to be compromised in TIRF. 1) The TIRF penetration depth is dependent upon the wavelength of the incident illumination, the angle of incidence, and the refractive indices of the media at the interface. 2) An apochromatic lens brings lasers of three wavelengths on to the same focal plane, but may not to the same incident angle. Therefore, the incident angles for the three lasers should be able to be adjusted independently to get optimal TIRF images. You may want to take your sample off first, to see how different the angles of the three lasers projecting to the wall, to get an idea of the incident angles of your lasers coming out of the objective. Regards Gary G Li, PhD On Sun, Jan 16, 2011 at 1:02 PM, Cammer, Michael <[hidden email] > wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > I originally posted the message below to the microscopy listserv but > reviewing the discussion here on chromatic aberration, I thought it would be > apropos here too even though it is not really regarding confocal. > > A few years ago we were having problems with the first commercial Olympus > TIRF system because we could not get consistent evanescent waves with the > one angle adjustment with the laser lines we had from 405 to 568 nm that > were delivered via a single fiber (it was worse when we later added a 633 nm > laser). I suggested we pump each laser in through a separate path that > could be angled independently. We didn't build it, but I think Olympus now > sells a TIRF system that does this. > > Another issue is that when I first heard about TIRF maybe 15 years ago, it > was introduced as a ring illumination at the outer edge of the back > aperture, not as a single point or crescent at the periphery on only one > side. A ring, or at least a series of points around the periphery, seems > like a better way to provide a uniform field due to aberrations from > coherent light in the imperfect optics. Any thought on this? > > Sincerely, > > Michael > > -----------------------ORIGINAL MESSAGE------------------------------- > We have the Nikon TIRF system and have three laser lines > going into the TIRF arm via a single fiber. When we project through > the 100X objective through the sample onto the wall we see that the > lines go through the sample at different angles. (You can see a > picture of the projection at approx 45 degrees at > http://www.flickr.com/photos/mcammer/5359189090/ .) It is also > noticeable in the TIRF images that the field depth is different for > each wavelength. Is this unavoidable due to the different > wavelengths or is it possible to align the optics better so these > spots would be more coincident? > > > > _________________________________________ > Michael Cammer, Assistant Research Scientist > Skirball Institute of Biomolecular Medicine > Lab: (212) 263-3208 Cell: (914) 309-3270 > > > ------------------------------------------------------------ > This email message, including any attachments, is for the sole use of the > intended recipient(s) and may contain information that is proprietary, > confidential, and exempt from disclosure under applicable law. Any > unauthorized review, use, disclosure, or distribution is prohibited. If you > have received this email in error please notify the sender by return email > and delete the original message. Please note, the recipient should check > this email and any attachments for the presence of viruses. The organization > accepts no liability for any damage caused by any virus transmitted by this > email. > ================================= > |
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Mark Cannell |
Re: Objective lens chromatic aberration
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** I suggest it is much more likely that the fiber/rear aperture coupling lens was at fault. I must admit I don't see how with rear aperture illumination the same and same focal length for the 3 lasers how the incident rays could have different angles ... Cheers > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hi Michael, > > There are a few factors that need to be compromised in TIRF. 1) > The TIRF > penetration depth is dependent upon the wavelength of the incident > illumination, the angle of incidence, and the refractive indices of > the > media at the interface. 2) An apochromatic lens brings lasers of three > wavelengths on to the same focal plane, but may not to the same > incident > angle. Therefore, the incident angles for the three lasers should > be able > to be adjusted independently to get optimal TIRF images. > > You may want to take your sample off first, to see how different the > angles > of the three lasers projecting to the wall, to get an idea of the > incident > angles of your lasers coming out of the objective. > > Regards > > Gary G Li, PhD > > > On Sun, Jan 16, 2011 at 1:02 PM, Cammer, Michael <[hidden email] >> wrote: > >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> ***** >> >> I originally posted the message below to the microscopy listserv but >> reviewing the discussion here on chromatic aberration, I thought it >> would be >> apropos here too even though it is not really regarding confocal. >> >> A few years ago we were having problems with the first commercial >> Olympus >> TIRF system because we could not get consistent evanescent waves >> with the >> one angle adjustment with the laser lines we had from 405 to 568 nm >> that >> were delivered via a single fiber (it was worse when we later added >> a 633 nm >> laser). I suggested we pump each laser in through a separate path >> that >> could be angled independently. We didn't build it, but I think >> Olympus now >> sells a TIRF system that does this. >> >> Another issue is that when I first heard about TIRF maybe 15 years >> ago, it >> was introduced as a ring illumination at the outer edge of the back >> aperture, not as a single point or crescent at the periphery on >> only one >> side. A ring, or at least a series of points around the periphery, >> seems >> like a better way to provide a uniform field due to aberrations from >> coherent light in the imperfect optics. Any thought on this? >> >> Sincerely, >> >> Michael >> >> -----------------------ORIGINAL >> MESSAGE------------------------------- >> We have the Nikon TIRF system and have three laser lines >> going into the TIRF arm via a single fiber. When we project through >> the 100X objective through the sample onto the wall we see that the >> lines go through the sample at different angles. (You can see a >> picture of the projection at approx 45 degrees at >> http://www.flickr.com/photos/mcammer/5359189090/ .) It is also >> noticeable in the TIRF images that the field depth is different for >> each wavelength. Is this unavoidable due to the different >> wavelengths or is it possible to align the optics better so these >> spots would be more coincident? >> >> >> >> _________________________________________ >> Michael Cammer, Assistant Research Scientist >> Skirball Institute of Biomolecular Medicine >> Lab: (212) 263-3208 Cell: (914) 309-3270 >> >> >> ------------------------------------------------------------ >> This email message, including any attachments, is for the sole use >> of the >> intended recipient(s) and may contain information that is >> proprietary, >> confidential, and exempt from disclosure under applicable law. Any >> unauthorized review, use, disclosure, or distribution is >> prohibited. If you >> have received this email in error please notify the sender by >> return email >> and delete the original message. Please note, the recipient should >> check >> this email and any attachments for the presence of viruses. The >> organization >> accepts no liability for any damage caused by any virus transmitted >> by this >> email. >> ================================= >> |
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Andreas Bruckbauer |
TIRF and chromatic aberration
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In reply to this post by mcammer
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** For the point illumination it is just much easier to adjust the angle, i think the Till Photonics system can rotate the point very fast to give the same effect as the ring. An advantage of a ring would be that one does not destroy the coating on the objective when using high laser power. Olympus had a three laser TIRF illuminator for several years, at least in Europe. This allows to adjust the angle, position and focus for each wavelength independetly and it works very well for simultaenous multi-color TIRF with an image splitter (I have no commercial interest). They now have a four color version which is motorized and also marketed in the US. The Olympus 150x TIRF objective which we use for single molecule work is an Apochromat but not a PLAN Apo and there is some measurable difference is focus over the field of view. In the center i measured an axial chromatic aberration of only 190 nm for emision at 520 nm and 650 nm . best wishes Andreas -----Original Message----- From: Cammer, Michael <[hidden email]> To: [hidden email] Sent: Sun, 16 Jan 2011 18:02 Subject: Re: Objective lens chromatic aberration ***** o join, leave or search the confocal microscopy listserv, go to: ttp://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy **** I originally posted the message below to the microscopy listserv but reviewing he discussion here on chromatic aberration, I thought it would be apropos here oo even though it is not really regarding confocal. A few years ago we were having problems with the first commercial Olympus TIRF ystem because we could not get consistent evanescent waves with the one angle djustment with the laser lines we had from 405 to 568 nm that were delivered ia a single fiber (it was worse when we later added a 633 nm laser). I uggested we pump each laser in through a separate path that could be angled ndependently. We didn't build it, but I think Olympus now sells a TIRF system hat does this. Another issue is that when I first heard about TIRF maybe 15 years ago, it was ntroduced as a ring illumination at the outer edge of the back aperture, not as single point or crescent at the periphery on only one side. A ring, or at east a series of points around the periphery, seems like a better way to rovide a uniform field due to aberrations from coherent light in the imperfect ptics. Any thought on this? Sincerely, Michael -----------------------ORIGINAL MESSAGE------------------------------- e have the Nikon TIRF system and have three laser lines oing into the TIRF arm via a single fiber. When we project through he 100X objective through the sample onto the wall we see that the ines go through the sample at different angles. (You can see a icture of the projection at approx 45 degrees at ttp://www.flickr.com/photos/mcammer/5359189090/ .) It is also oticeable in the TIRF images that the field depth is different for ach wavelength. Is this unavoidable due to the different avelengths or is it possible to align the optics better so these pots would be more coincident? _________________________________________ ichael Cammer, Assistant Research Scientist kirball Institute of Biomolecular Medicine ab: (212) 263-3208 Cell: (914) 309-3270 ----------------------------------------------------------- his email message, including any attachments, is for the sole use of the ntended recipient(s) and may contain information that is proprietary, onfidential, and exempt from disclosure under applicable law. Any unauthorized eview, use, disclosure, or distribution is prohibited. If you have received his email in error please notify the sender by return email and delete the riginal message. Please note, the recipient should check this email and any ttachments for the presence of viruses. The organization accepts no liability or any damage caused by any virus transmitted by this email. ================================ |
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Craig Brideau |
Re: Objective lens chromatic aberration
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In reply to this post by Mark Cannell
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** This would also lead to chromatically dependent NA, which would be a big no-no for a corrected objective. Craig On Sun, Jan 16, 2011 at 2:16 PM, Mark Cannell <[hidden email]>wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > I suggest it is much more likely that the fiber/rear aperture coupling lens > was at fault. I must admit I don't see how with rear aperture illumination > the same and same focal length for the 3 lasers how the incident rays could > have different angles ... > > Cheers > > > > ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> ***** >> >> Hi Michael, >> >> There are a few factors that need to be compromised in TIRF. 1) The TIRF >> penetration depth is dependent upon the wavelength of the incident >> illumination, the angle of incidence, and the refractive indices of the >> media at the interface. 2) An apochromatic lens brings lasers of three >> wavelengths on to the same focal plane, but may not to the same incident >> angle. Therefore, the incident angles for the three lasers should be able >> to be adjusted independently to get optimal TIRF images. >> >> You may want to take your sample off first, to see how different the >> angles >> of the three lasers projecting to the wall, to get an idea of the incident >> angles of your lasers coming out of the objective. >> >> Regards >> >> Gary G Li, PhD >> >> >> On Sun, Jan 16, 2011 at 1:02 PM, Cammer, Michael < >> [hidden email] >> >>> wrote: >>> >> >> ***** >>> To join, leave or search the confocal microscopy listserv, go to: >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >>> ***** >>> >>> I originally posted the message below to the microscopy listserv but >>> reviewing the discussion here on chromatic aberration, I thought it would >>> be >>> apropos here too even though it is not really regarding confocal. >>> >>> A few years ago we were having problems with the first commercial Olympus >>> TIRF system because we could not get consistent evanescent waves with the >>> one angle adjustment with the laser lines we had from 405 to 568 nm that >>> were delivered via a single fiber (it was worse when we later added a 633 >>> nm >>> laser). I suggested we pump each laser in through a separate path that >>> could be angled independently. We didn't build it, but I think Olympus >>> now >>> sells a TIRF system that does this. >>> >>> Another issue is that when I first heard about TIRF maybe 15 years ago, >>> it >>> was introduced as a ring illumination at the outer edge of the back >>> aperture, not as a single point or crescent at the periphery on only one >>> side. A ring, or at least a series of points around the periphery, seems >>> like a better way to provide a uniform field due to aberrations from >>> coherent light in the imperfect optics. Any thought on this? >>> >>> Sincerely, >>> >>> Michael >>> >>> -----------------------ORIGINAL MESSAGE------------------------------- >>> We have the Nikon TIRF system and have three laser lines >>> going into the TIRF arm via a single fiber. When we project through >>> the 100X objective through the sample onto the wall we see that the >>> lines go through the sample at different angles. (You can see a >>> picture of the projection at approx 45 degrees at >>> http://www.flickr.com/photos/mcammer/5359189090/ .) It is also >>> noticeable in the TIRF images that the field depth is different for >>> each wavelength. Is this unavoidable due to the different >>> wavelengths or is it possible to align the optics better so these >>> spots would be more coincident? >>> >>> >>> >>> _________________________________________ >>> Michael Cammer, Assistant Research Scientist >>> Skirball Institute of Biomolecular Medicine >>> Lab: (212) 263-3208 Cell: (914) 309-3270 >>> >>> >>> ------------------------------------------------------------ >>> This email message, including any attachments, is for the sole use of the >>> intended recipient(s) and may contain information that is proprietary, >>> confidential, and exempt from disclosure under applicable law. Any >>> unauthorized review, use, disclosure, or distribution is prohibited. If >>> you >>> have received this email in error please notify the sender by return >>> and delete the original message. Please note, the recipient should check >>> this email and any attachments for the presence of viruses. The >>> organization >>> accepts no liability for any damage caused by any virus transmitted by >>> this >>> email. >>> ================================= >>> >>> |
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Andreas Bruckbauer |
Re: Objective lens chromatic aberration
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** I guess the initial question about chromatic aberration and the new one about TIRF got muddeled up. I guess chromatically different NA in TIRF could arise when part of the correction is build in the tube lens? The illumination light forTIRF "through the objective" does not pass the tube lens and is thus is not well corrected. Furthermore slight imperfections in the dichroic which relfects the laser light can lead to distortions and these can be wavelength dependent. best wishes Andreas -----Original Message----- From: Craig Brideau <[hidden email]> To: [hidden email] Sent: Mon, 17 Jan 2011 19:01 Subject: Re: Objective lens chromatic aberration ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** This would also lead to chromatically dependent NA, which would be a big no-no for a corrected objective. Craig On Sun, Jan 16, 2011 at 2:16 PM, Mark Cannell <[hidden email]>wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > I suggest it is much more likely that the fiber/rear aperture coupling lens > was at fault. I must admit I don't see how with rear aperture illumination > the same and same focal length for the 3 lasers how the incident rays could > have different angles ... > > Cheers > > > > ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> ***** >> >> Hi Michael, >> >> There are a few factors that need to be compromised in TIRF. 1) The TIRF >> penetration depth is dependent upon the wavelength of the incident >> illumination, the angle of incidence, and the refractive indices of the >> media at the interface. 2) An apochromatic lens brings lasers of three >> wavelengths on to the same focal plane, but may not to the same incident >> angle. Therefore, the incident angles for the three lasers should be able >> to be adjusted independently to get optimal TIRF images. >> >> You may want to take your sample off first, to see how different the >> angles >> of the three lasers projecting to the wall, to get an idea of the incident >> angles of your lasers coming out of the objective. >> >> Regards >> >> Gary G Li, PhD >> >> >> On Sun, Jan 16, 2011 at 1:02 PM, Cammer, Michael < >> [hidden email] >> >>> wrote: >>> >> >> ***** >>> To join, leave or search the confocal microscopy listserv, go to: >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >>> ***** >>> >>> I originally posted the message below to the microscopy listserv but >>> reviewing the discussion here on chromatic aberration, I thought it would >>> be >>> apropos here too even though it is not really regarding confocal. >>> >>> A few years ago we were having problems with the first commercial Olympus >>> TIRF system because we could not get consistent evanescent waves with the >>> one angle adjustment with the laser lines we had from 405 to 568 nm that >>> were delivered via a single fiber (it was worse when we later added a 633 >>> nm >>> laser). I suggested we pump each laser in through a separate path that >>> could be angled independently. We didn't build it, but I think Olympus >>> now >>> sells a TIRF system that does this. >>> >>> Another issue is that when I first heard about TIRF maybe 15 years ago, >>> it >>> was introduced as a ring illumination at the outer edge of the back >>> aperture, not as a single point or crescent at the periphery on only one >>> side. A ring, or at least a series of points around the periphery, seems >>> like a better way to provide a uniform field due to aberrations from >>> coherent light in the imperfect optics. Any thought on this? >>> >>> Sincerely, >>> >>> Michael >>> >>> -----------------------ORIGINAL MESSAGE------------------------------- >>> We have the Nikon TIRF system and have three laser lines >>> going into the TIRF arm via a single fiber. When we project through >>> the 100X objective through the sample onto the wall we see that the >>> lines go through the sample at different angles. (You can see a >>> picture of the projection at approx 45 degrees at >>> http://www.flickr.com/photos/mcammer/5359189090/ .) It is also >>> noticeable in the TIRF images that the field depth is different for >>> each wavelength. Is this unavoidable due to the different >>> wavelengths or is it possible to align the optics better so these >>> spots would be more coincident? >>> >>> >>> >>> _________________________________________ >>> Michael Cammer, Assistant Research Scientist >>> Skirball Institute of Biomolecular Medicine >>> Lab: (212) 263-3208 Cell: (914) 309-3270 >>> >>> >>> ------------------------------------------------------------ >>> This email message, including any attachments, is for the sole use of the >>> intended recipient(s) and may contain information that is proprietary, >>> confidential, and exempt from disclosure under applicable law. Any >>> unauthorized review, use, disclosure, or distribution is prohibited. If >>> you >>> have received this email in error please notify the sender by return >>> and delete the original message. Please note, the recipient should check >>> this email and any attachments for the presence of viruses. The >>> organization >>> accepts no liability for any damage caused by any virus transmitted by >>> this >>> email. >>> ================================= >>> >>> |
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Craig Brideau |
Re: Objective lens chromatic aberration
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Tube lens, or scan lens? Craig On Mon, Jan 17, 2011 at 3:06 PM, Andreas Bruckbauer <[hidden email]>wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > I guess the initial question about chromatic aberration and the new one > about TIRF got muddeled up. I guess chromatically different NA in TIRF could > arise when part of the correction is build in the tube lens? The > illumination light forTIRF "through the objective" does not pass the tube > lens and is thus is not well corrected. Furthermore slight imperfections in > the dichroic which relfects the laser light can lead to distortions and > these can be wavelength dependent. > > > best wishes > > Andreas > > > > -----Original Message----- > From: Craig Brideau <[hidden email]> > To: [hidden email] > Sent: Mon, 17 Jan 2011 19:01 > Subject: Re: Objective lens chromatic aberration > > > ***** > > To join, leave or search the confocal microscopy listserv, go to: > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > ***** > > > > This would also lead to chromatically dependent NA, which would be a big > > no-no for a corrected objective. > > > > Craig > > > > > > On Sun, Jan 16, 2011 at 2:16 PM, Mark Cannell <[hidden email] > >wrote: > > > > > ***** > > > To join, leave or search the confocal microscopy listserv, go to: > > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > > ***** > > > > > > I suggest it is much more likely that the fiber/rear aperture coupling > lens > > > was at fault. I must admit I don't see how with rear aperture > illumination > > > the same and same focal length for the 3 lasers how the incident rays > could > > > have different angles ... > > > > > > Cheers > > > > > > > > > > > > ***** > > >> To join, leave or search the confocal microscopy listserv, go to: > > >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > >> ***** > > >> > > >> Hi Michael, > > >> > > >> There are a few factors that need to be compromised in TIRF. 1) The > TIRF > > >> penetration depth is dependent upon the wavelength of the incident > > >> illumination, the angle of incidence, and the refractive indices of the > > >> media at the interface. 2) An apochromatic lens brings lasers of three > > >> wavelengths on to the same focal plane, but may not to the same incident > > >> angle. Therefore, the incident angles for the three lasers should be > able > > >> to be adjusted independently to get optimal TIRF images. > > >> > > >> You may want to take your sample off first, to see how different the > > >> angles > > >> of the three lasers projecting to the wall, to get an idea of the > incident > > >> angles of your lasers coming out of the objective. > > >> > > >> Regards > > >> > > >> Gary G Li, PhD > > >> > > >> > > >> On Sun, Jan 16, 2011 at 1:02 PM, Cammer, Michael < > > >> [hidden email] > > >> > > >>> wrote: > > >>> > > >> > > >> ***** > > >>> To join, leave or search the confocal microscopy listserv, go to: > > >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > >>> ***** > > >>> > > >>> I originally posted the message below to the microscopy listserv but > > >>> reviewing the discussion here on chromatic aberration, I thought it > would > > >>> be > > >>> apropos here too even though it is not really regarding confocal. > > >>> > > >>> A few years ago we were having problems with the first commercial > Olympus > > >>> TIRF system because we could not get consistent evanescent waves with > the > > >>> one angle adjustment with the laser lines we had from 405 to 568 nm > that > > >>> were delivered via a single fiber (it was worse when we later added a > 633 > > >>> nm > > >>> laser). I suggested we pump each laser in through a separate path that > > >>> could be angled independently. We didn't build it, but I think Olympus > > >>> now > > >>> sells a TIRF system that does this. > > >>> > > >>> Another issue is that when I first heard about TIRF maybe 15 years ago, > > >>> it > > >>> was introduced as a ring illumination at the outer edge of the back > > >>> aperture, not as a single point or crescent at the periphery on only > one > > >>> side. A ring, or at least a series of points around the periphery, > seems > > >>> like a better way to provide a uniform field due to aberrations from > > >>> coherent light in the imperfect optics. Any thought on this? > > >>> > > >>> Sincerely, > > >>> > > >>> Michael > > >>> > > >>> -----------------------ORIGINAL MESSAGE------------------------------- > > >>> We have the Nikon TIRF system and have three laser lines > > >>> going into the TIRF arm via a single fiber. When we project through > > >>> the 100X objective through the sample onto the wall we see that the > > >>> lines go through the sample at different angles. (You can see a > > >>> picture of the projection at approx 45 degrees at > > >>> http://www.flickr.com/photos/mcammer/5359189090/ .) It is also > > >>> noticeable in the TIRF images that the field depth is different for > > >>> each wavelength. Is this unavoidable due to the different > > >>> wavelengths or is it possible to align the optics better so these > > >>> spots would be more coincident? > > >>> > > >>> > > >>> > > >>> _________________________________________ > > >>> Michael Cammer, Assistant Research Scientist > > >>> Skirball Institute of Biomolecular Medicine > > >>> Lab: (212) 263-3208 Cell: (914) 309-3270 > > >>> > > >>> > > >>> ------------------------------------------------------------ > > >>> This email message, including any attachments, is for the sole use of > the > > >>> intended recipient(s) and may contain information that is proprietary, > > >>> confidential, and exempt from disclosure under applicable law. Any > > >>> unauthorized review, use, disclosure, or distribution is prohibited. If > > >>> you > > >>> have received this email in error please notify the sender by return > > > >>> and delete the original message. Please note, the recipient should > check > > >>> this email and any attachments for the presence of viruses. The > > >>> organization > > >>> accepts no liability for any damage caused by any virus transmitted by > > >>> this > > >>> email. > > >>> ================================= > > >>> > > >>> > > > > |
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In reply to this post by Andreas Bruckbauer
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** What people seem to be missing in this thread is that glass and water are both dispersive media so the critical angle will be different for different wavelengths. You cannot expect to get optimal TIRF with two colours coming in at the same angle. Guy Optical Imaging Techniques in Cell Biology by Guy Cox CRC Press / Taylor & Francis http://www.guycox.com/optical.htm <http://www.guycox.com/optical.htm> ______________________________________________ Associate Professor Guy Cox, MA, DPhil(Oxon) Australian Centre for Microscopy & Microanalysis, Madsen Building F09, University of Sydney, NSW 2006 Phone +61 2 9351 3176 Fax +61 2 9351 7682 Mobile 0413 281 861 ______________________________________________ http://www.guycox.net <http://www.guycox.net> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Andreas Bruckbauer Sent: Tuesday, 18 January 2011 9:06 AM To: [hidden email] Subject: Re: Objective lens chromatic aberration ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** I guess the initial question about chromatic aberration and the new one about TIRF got muddeled up. I guess chromatically different NA in TIRF could arise when part of the correction is build in the tube lens? The illumination light forTIRF "through the objective" does not pass the tube lens and is thus is not well corrected. Furthermore slight imperfections in the dichroic which relfects the laser light can lead to distortions and these can be wavelength dependent. best wishes Andreas -----Original Message----- From: Craig Brideau <[hidden email]> To: [hidden email] Sent: Mon, 17 Jan 2011 19:01 Subject: Re: Objective lens chromatic aberration ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** This would also lead to chromatically dependent NA, which would be a big no-no for a corrected objective. Craig On Sun, Jan 16, 2011 at 2:16 PM, Mark Cannell <[hidden email]>wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > I suggest it is much more likely that the fiber/rear aperture coupling lens > was at fault. I must admit I don't see how with rear aperture illumination > the same and same focal length for the 3 lasers how the incident rays could > have different angles ... > > Cheers > > > > ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> ***** >> >> Hi Michael, >> >> There are a few factors that need to be compromised in TIRF. 1) The TIRF >> penetration depth is dependent upon the wavelength of the incident >> illumination, the angle of incidence, and the refractive indices of the >> media at the interface. 2) An apochromatic lens brings lasers of three >> wavelengths on to the same focal plane, but may not to the same incident >> angle. Therefore, the incident angles for the three lasers should be able >> to be adjusted independently to get optimal TIRF images. >> >> You may want to take your sample off first, to see how different the >> angles >> of the three lasers projecting to the wall, to get an idea of the incident >> angles of your lasers coming out of the objective. >> >> Regards >> >> Gary G Li, PhD >> >> >> On Sun, Jan 16, 2011 at 1:02 PM, Cammer, Michael < >> [hidden email] >> >>> wrote: >>> >> >> ***** >>> To join, leave or search the confocal microscopy listserv, go to: >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >>> ***** >>> >>> I originally posted the message below to the microscopy listserv but >>> reviewing the discussion here on chromatic aberration, I thought it would >>> be >>> apropos here too even though it is not really regarding confocal. >>> >>> A few years ago we were having problems with the first commercial Olympus >>> TIRF system because we could not get consistent evanescent waves with the >>> one angle adjustment with the laser lines we had from 405 to 568 nm that >>> were delivered via a single fiber (it was worse when we later added a 633 >>> nm >>> laser). I suggested we pump each laser in through a separate path that >>> could be angled independently. We didn't build it, but I think Olympus >>> now >>> sells a TIRF system that does this. >>> >>> Another issue is that when I first heard about TIRF maybe 15 years ago, >>> it >>> was introduced as a ring illumination at the outer edge of the back >>> aperture, not as a single point or crescent at the periphery on only one >>> side. A ring, or at least a series of points around the periphery, seems >>> like a better way to provide a uniform field due to aberrations from >>> coherent light in the imperfect optics. Any thought on this? >>> >>> Sincerely, >>> >>> Michael >>> >>> -----------------------ORIGINAL MESSAGE------------------------------- >>> We have the Nikon TIRF system and have three laser lines >>> going into the TIRF arm via a single fiber. When we project through >>> the 100X objective through the sample onto the wall we see that the >>> lines go through the sample at different angles. (You can see a >>> picture of the projection at approx 45 degrees at >>> http://www.flickr.com/photos/mcammer/5359189090/ .) It is also >>> noticeable in the TIRF images that the field depth is different for >>> each wavelength. Is this unavoidable due to the different >>> wavelengths or is it possible to align the optics better so these >>> spots would be more coincident? >>> >>> >>> >>> _________________________________________ >>> Michael Cammer, Assistant Research Scientist >>> Skirball Institute of Biomolecular Medicine >>> Lab: (212) 263-3208 Cell: (914) 309-3270 >>> >>> >>> ------------------------------------------------------------ >>> This email message, including any attachments, is for the sole use of the >>> intended recipient(s) and may contain information that is proprietary, >>> confidential, and exempt from disclosure under applicable law. Any >>> unauthorized review, use, disclosure, or distribution is prohibited. If >>> you >>> have received this email in error please notify the sender by return >>> and delete the original message. Please note, the recipient should check >>> this email and any attachments for the presence of viruses. The >>> organization >>> accepts no liability for any damage caused by any virus transmitted by >>> this >>> email. >>> ================================= >>> >>> ________________________________ No virus found in this message. Checked by AVG - www.avg.com Version: 10.0.1191 / Virus Database: 1435/3342 - Release Date: 12/27/10 Internal Virus Database is out of date. |
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In reply to this post by Gary G. Li-2
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi all; Is anyone using a Tokai Hit incubation chamber for long-term live-cell imaging? May I contact you offline about humidity issues? Thanks. Kathy Kathy Spencer The Scripps Research Institute La Jolla, CA 92037 |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Kathy, Tokai Hit System I had been using was equipped with a doughnut-shaped water chamber and heated lid along with CO2 and heat supplier. I have done live imaging up to 5 hours with no humidity problem. What kind of humidity issues do you have? The water chamber was simple enough that nothing could go wrong, so it seemed. Ippei -- Ippei Kotera, Ph.D. Tanz CRND Tanz Neuroscience Building, Room 221 University of Toronto 6 Queen's Park Crescent West Toronto, Ontario M5S 3H2 Phone: (416)978-2503 [hidden email] |
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Boswell, Carl A - (cboswell) |
Re: tokai hit chamber
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In reply to this post by kspencer007
Hi Kathy,
If you're interested in offline conversations you might want to leave some offline contact info. I know of a unit being used here but I would need to defer to the person with experience with it. Cheers, C Carl A. Boswell, Ph.D. Molecular and Cellular Biology Univ. of Arizona 520-954-7053 FAX 520-621-3709 -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Kathryn Spencer Sent: Thursday, February 10, 2011 3:14 PM To: [hidden email] Subject: tokai hit chamber ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi all; Is anyone using a Tokai Hit incubation chamber for long-term live-cell imaging? May I contact you offline about humidity issues? Thanks. Kathy Kathy Spencer The Scripps Research Institute La Jolla, CA 92037 |
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In reply to this post by Ippei Kotera
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Thanks Ippei; We are imaging 2-3 days, and the reservoir is drying out. Short term, it does seem OK. I'm just unsure about why it is drying out, if we are just imaging much longer than normal. If we put in wet Kimwipes, everything seems fine. Somehow, we need to add water during our imaging. Kathy -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Ippei Kotera Sent: Thursday, February 10, 2011 3:56 PM To: [hidden email] Subject: Re: tokai hit chamber ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Kathy, Tokai Hit System I had been using was equipped with a doughnut-shaped water chamber and heated lid along with CO2 and heat supplier. I have done live imaging up to 5 hours with no humidity problem. What kind of humidity issues do you have? The water chamber was simple enough that nothing could go wrong, so it seemed. Ippei -- Ippei Kotera, Ph.D. Tanz CRND Tanz Neuroscience Building, Room 221 University of Toronto 6 Queen's Park Crescent West Toronto, Ontario M5S 3H2 Phone: (416)978-2503 [hidden email] |
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Romin, Yevgeniy/Sloan Kettering Institute |
Re: tokai hit chamber
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Kathy We have several Tokai Hit icubators in our facility, they all dry out within 24 hours, the company seems to be aware of it and they suggest to put in more water every 12 hours, so that is what we do. Wet Kimwipes also help. Or wet cotton balls. ________________________________________ From: Confocal Microscopy List [[hidden email]] On Behalf Of Kathryn Spencer [[hidden email]] Sent: Thursday, February 10, 2011 7:17 PM To: [hidden email] Subject: Re: tokai hit chamber ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Thanks Ippei; We are imaging 2-3 days, and the reservoir is drying out. Short term, it does seem OK. I'm just unsure about why it is drying out, if we are just imaging much longer than normal. If we put in wet Kimwipes, everything seems fine. Somehow, we need to add water during our imaging. Kathy -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Ippei Kotera Sent: Thursday, February 10, 2011 3:56 PM To: [hidden email] Subject: Re: tokai hit chamber ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Kathy, Tokai Hit System I had been using was equipped with a doughnut-shaped water chamber and heated lid along with CO2 and heat supplier. I have done live imaging up to 5 hours with no humidity problem. What kind of humidity issues do you have? The water chamber was simple enough that nothing could go wrong, so it seemed. Ippei -- Ippei Kotera, Ph.D. Tanz CRND Tanz Neuroscience Building, Room 221 University of Toronto 6 Queen's Park Crescent West Toronto, Ontario M5S 3H2 Phone: (416)978-2503 [hidden email] ===================================================================== Please note that this e-mail and any files transmitted with it may be privileged, confidential, and protected from disclosure under applicable law. If the reader of this message is not the intended recipient, or an employee or agent responsible for delivering this message to the intended recipient, you are hereby notified that any reading, dissemination, distribution, copying, or other use of this communication or any of its attachments is strictly prohibited. If you have received this communication in error, please notify the sender immediately by replying to this message and deleting this message, any attachments, and all copies and backups from your computer. |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Great advice. Thank you. Kathy -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Yevgeniy Romin Sent: Thursday, February 10, 2011 5:54 PM To: [hidden email] Subject: Re: tokai hit chamber ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Kathy We have several Tokai Hit icubators in our facility, they all dry out within 24 hours, the company seems to be aware of it and they suggest to put in more water every 12 hours, so that is what we do. Wet Kimwipes also help. Or wet cotton balls. ________________________________________ From: Confocal Microscopy List [[hidden email]] On Behalf Of Kathryn Spencer [[hidden email]] Sent: Thursday, February 10, 2011 7:17 PM To: [hidden email] Subject: Re: tokai hit chamber ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Thanks Ippei; We are imaging 2-3 days, and the reservoir is drying out. Short term, it does seem OK. I'm just unsure about why it is drying out, if we are just imaging much longer than normal. If we put in wet Kimwipes, everything seems fine. Somehow, we need to add water during our imaging. Kathy -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Ippei Kotera Sent: Thursday, February 10, 2011 3:56 PM To: [hidden email] Subject: Re: tokai hit chamber ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Kathy, Tokai Hit System I had been using was equipped with a doughnut-shaped water chamber and heated lid along with CO2 and heat supplier. I have done live imaging up to 5 hours with no humidity problem. What kind of humidity issues do you have? The water chamber was simple enough that nothing could go wrong, so it seemed. Ippei -- Ippei Kotera, Ph.D. Tanz CRND Tanz Neuroscience Building, Room 221 University of Toronto 6 Queen's Park Crescent West Toronto, Ontario M5S 3H2 Phone: (416)978-2503 [hidden email] ===================================================================== Please note that this e-mail and any files transmitted with it may be privileged, confidential, and protected from disclosure under applicable law. If the reader of this message is not the intended recipient, or an employee or agent responsible for delivering this message to the intended recipient, you are hereby notified that any reading, dissemination, distribution, copying, or other use of this communication or any of its attachments is strictly prohibited. If you have received this communication in error, please notify the sender immediately by replying to this message and deleting this message, any attachments, and all copies and backups from your computer. |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** FYI- Our Pathology Devices stage incubator does not have this problem and works for days with little media loss. Dave On Feb 10, 2011, at 10:06 PM, Kathryn Spencer wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Great advice. Thank you. > Kathy > > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Yevgeniy Romin > Sent: Thursday, February 10, 2011 5:54 PM > To: [hidden email] > Subject: Re: tokai hit chamber > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hi Kathy > > We have several Tokai Hit icubators in our facility, they all dry out within 24 hours, the company seems to be aware of it and they suggest to put in more water every 12 hours, so that is what we do. Wet Kimwipes also help. Or wet cotton balls. > > ________________________________________ > From: Confocal Microscopy List [[hidden email]] On Behalf Of Kathryn Spencer [[hidden email]] > Sent: Thursday, February 10, 2011 7:17 PM > To: [hidden email] > Subject: Re: tokai hit chamber > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Thanks Ippei; > We are imaging 2-3 days, and the reservoir is drying out. Short term, it does seem OK. I'm just unsure about why it is drying out, if we are just imaging much longer than normal. If we put in wet Kimwipes, everything seems fine. Somehow, we need to add water during our imaging. > Kathy > > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Ippei Kotera > Sent: Thursday, February 10, 2011 3:56 PM > To: [hidden email] > Subject: Re: tokai hit chamber > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hi Kathy, > > Tokai Hit System I had been using was equipped with a doughnut-shaped > water chamber and heated lid along with CO2 and heat supplier. I have > done live imaging up to 5 hours with no humidity problem. What kind of > humidity issues do you have? The water chamber was simple enough that > nothing could go wrong, so it seemed. > > Ippei > > -- > Ippei Kotera, Ph.D. > > Tanz CRND > Tanz Neuroscience Building, Room 221 > University of Toronto > 6 Queen's Park Crescent West > Toronto, Ontario M5S 3H2 > Phone: (416)978-2503 > [hidden email] > > > > ===================================================================== > > Please note that this e-mail and any files transmitted with it may be > privileged, confidential, and protected from disclosure under > applicable law. If the reader of this message is not the intended > recipient, or an employee or agent responsible for delivering this > message to the intended recipient, you are hereby notified that any > reading, dissemination, distribution, copying, or other use of this > communication or any of its attachments is strictly prohibited. If > you have received this communication in error, please notify the > sender immediately by replying to this message and deleting this > message, any attachments, and all copies and backups from your > computer. Dr. David Knecht Department of Molecular and Cell Biology Co-head Flow Cytometry and Confocal Microscopy Facility U-3125 91 N. Eagleville Rd. University of Connecticut Storrs, CT 06269 860-486-2200 860-486-4331 (fax) |
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In reply to this post by kspencer007
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Kathy, Hi Kathy, If you have a 5% CO2 gas flow into the chamber, have you tried bubbling it through a small reservoir of distilled water? Tank gas is quite dry. A while back I used that trick in a jerryrigged unit for 24-h. imaging. We have a Tokai-Hit now but have not yet needed it for very long-term imaging. cheers, Tim Feinstein On Feb 10, 2011, at 7:17 PM, Kathryn Spencer wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Thanks Ippei; > We are imaging 2-3 days, and the reservoir is drying out. Short term, it does seem OK. I'm just unsure about why it is drying out, if we are just imaging much longer than normal. If we put in wet Kimwipes, everything seems fine. Somehow, we need to add water during our imaging. > Kathy > > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Ippei Kotera > Sent: Thursday, February 10, 2011 3:56 PM > To: [hidden email] > Subject: Re: tokai hit chamber > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hi Kathy, > > Tokai Hit System I had been using was equipped with a doughnut-shaped > water chamber and heated lid along with CO2 and heat supplier. I have > done live imaging up to 5 hours with no humidity problem. What kind of > humidity issues do you have? The water chamber was simple enough that > nothing could go wrong, so it seemed. > > Ippei > > -- > Ippei Kotera, Ph.D. > > Tanz CRND > Tanz Neuroscience Building, Room 221 > University of Toronto > 6 Queen's Park Crescent West > Toronto, Ontario M5S 3H2 > Phone: (416)978-2503 > [hidden email] |
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