different fields of view with different objectives
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Jeff Spector |
different fields of view with different objectives
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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. ***** Greetings, I am trying to take two images of teh same field of view using two different objectives. One of them is a 1.4 NA 100X Nikon lens, and the other is a 0.5-1.3 Adjustable iris 100x lens. When I switch from one to the other and re focus. I find that I am imaging a different part of the sample, sometime by as much as 10 microns or more! I suspect this means that the nosepiece turret holding the objectives doesn't center properly for some reason, but I thought I'd ask the experts on this list if anyone has ever seen anything like this? thanks. -Jeff |
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Craig Brideau |
Re: different fields of view with different objectives
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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. ***** The nosepiece usually can't center all the lenses exactly and there will always be some slack. If you switch back and forth, or even come from different objectives, you will find a different center point. A 10 um differential is actually not that bad considering how little it would take for the lenses to be a fraction of a degree off from each other. To achieve the kind of repeatability you want you would need a precision rail or slider system, and even then there's no guarantee that the lenses have similar XY centers compared to their bodies. Craig On Fri, Mar 27, 2015 at 4:38 PM, Jeff Spector <[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. > ***** > > Greetings, > I am trying to take two images of teh same field of view using two > different objectives. > One of them is a 1.4 NA 100X Nikon lens, and the other is a 0.5-1.3 > Adjustable iris 100x lens. > When I switch from one to the other and re focus. I find that I am > imaging a different part of the sample, sometime by as much as 10 microns > or more! I suspect this means that the nosepiece turret holding the > objectives doesn't center properly for some reason, but I thought I'd ask > the experts on this list if anyone has ever seen anything like this? > thanks. > -Jeff > |
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Barbara Foster |
Re: different fields of view with different objectives
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In reply to this post by Jeff Spector
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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, Jeff All of the manufacturers have specs for both parcentration (imaging exactly the same field) and parfocalization (same plane of focus). I would recommend that you investigate the type of nosepiece used for polarized light. Those nosepieces often have 1 fixed position (usually for the 10x objective) which you can use as reference then have centerable positions for the other objectives centration screws for each objective). The procedure is fairly simple: You can either use a target slide with a cross hair and center the cross hair to the 10x then center all of the other objectives to the same field of view or, you can center a piece of dirt/debris in what you think is the center of the 10x field of view, then center the other objectives (less precise but equally valid). Hope this was helpful. Good hunting, Barbara Foster, President & Chief Consultant Microscopy/Microscopy Education* 7101 Royal Glen Trail, Suite A - McKinney, TX 75070 - P: 972-924-5310 www.MicroscopyEducation.com "Education, not Training" MME is currently scheduling courses for now and through the end of 2015. We can customize a course on nearly any topic, from fluorescence to confocal to image analysis to SEM/TEM. Call us today for a free training evaluation. *A subsidiary of The Microscopy & Imaging Place, Inc. At 09:04 PM 3/27/2015, 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. >***** > >Greetings, > I am trying to take two images of teh same field of view using two >different objectives. > One of them is a 1.4 NA 100X Nikon lens, and the other is a 0.5-1.3 >Adjustable iris 100x lens. > When I switch from one to the other and re focus. I find that I am >imaging a different part of the sample, sometime by as much as 10 microns >or more! I suspect this means that the nosepiece turret holding the >objectives doesn't center properly for some reason, but I thought I'd ask >the experts on this list if anyone has ever seen anything like this? > thanks. >-Jeff |
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*****
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. ***** Barbara's suggestion is excellent but I do suggest before you go out spending money you do some more tests. You are using oil immersion lenses - the drag of the oil might be moving your sample by the small amount you see. In this case you will see the movement in the direction you moved the lens (taking the inverted image into account) and it will reverse if you use the lenses in the reverse order. You don't mention what type of microscope you are using, but for inverted microscopes a high-viscosity oil is normally used, and that may be what you have even if this is an upright microscope. So if this looks like it's the problem get the lowest-viscosity oil you can and investigate locking up the stage, by fair means or foul. Is your turret central? You can check this with dry lenses since you'll see a consistent inward and outward movement as you rotate it. On some microscopes this can be adjusted. If neither of these tips helps then play 'musical lenses' - swap positions until you get the least movement. Guy -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Barbara Foster Sent: Saturday, 28 March 2015 2:19 PM To: [hidden email] Subject: Re: different fields of view with different objectives ***** 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, Jeff All of the manufacturers have specs for both parcentration (imaging exactly the same field) and parfocalization (same plane of focus). I would recommend that you investigate the type of nosepiece used for polarized light. Those nosepieces often have 1 fixed position (usually for the 10x objective) which you can use as reference then have centerable positions for the other objectives centration screws for each objective). The procedure is fairly simple: You can either use a target slide with a cross hair and center the cross hair to the 10x then center all of the other objectives to the same field of view or, you can center a piece of dirt/debris in what you think is the center of the 10x field of view, then center the other objectives (less precise but equally valid). Hope this was helpful. Good hunting, Barbara Foster, President & Chief Consultant Microscopy/Microscopy Education* 7101 Royal Glen Trail, Suite A - McKinney, TX 75070 - P: 972-924-5310 www.MicroscopyEducation.com "Education, not Training" MME is currently scheduling courses for now and through the end of 2015. We can customize a course on nearly any topic, from fluorescence to confocal to image analysis to SEM/TEM. Call us today for a free training evaluation. *A subsidiary of The Microscopy & Imaging Place, Inc. At 09:04 PM 3/27/2015, 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. >***** > >Greetings, > I am trying to take two images of teh same field of view using two >different objectives. > One of them is a 1.4 NA 100X Nikon lens, and the other is a 0.5-1.3 >Adjustable iris 100x lens. > When I switch from one to the other and re focus. I find that I am >imaging a different part of the sample, sometime by as much as 10 >microns or more! I suspect this means that the nosepiece turret holding >the objectives doesn't center properly for some reason, but I thought >I'd ask the experts on this list if anyone has ever seen anything like this? > thanks. >-Jeff |
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Michael Giacomelli |
Re: different fields of view with different objectives
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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. ***** Would just imaging with both objectives and then cross-correlating to align them in post processing be an option? Mike On Sat, Mar 28, 2015 at 3:04 AM, Guy Cox <[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. > ***** > > Barbara's suggestion is excellent but I do suggest before you go out spending money you do some more tests. > > You are using oil immersion lenses - the drag of the oil might be moving your sample by the small amount you see. In this case you will see the movement in the direction you moved the lens (taking the inverted image into account) and it will reverse if you use the lenses in the reverse order. You don't mention what type of microscope you are using, but for inverted microscopes a high-viscosity oil is normally used, and that may be what you have even if this is an upright microscope. So if this looks like it's the problem get the lowest-viscosity oil you can and investigate locking up the stage, by fair means or foul. > > Is your turret central? You can check this with dry lenses since you'll see a consistent inward and outward movement as you rotate it. On some microscopes this can be adjusted. > > If neither of these tips helps then play 'musical lenses' - swap positions until you get the least movement. > > Guy > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Barbara Foster > Sent: Saturday, 28 March 2015 2:19 PM > To: [hidden email] > Subject: Re: different fields of view with different objectives > > ***** > 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, Jeff > > All of the manufacturers have specs for both parcentration (imaging exactly the same field) and parfocalization (same plane of focus). > > I would recommend that you investigate the type of nosepiece used for polarized light. Those nosepieces often have 1 fixed position (usually for the 10x objective) which you can use as reference then have centerable positions for the other objectives centration screws for each objective). The procedure is fairly simple: You can either use a target slide with a cross hair and center the cross hair to the 10x then center all of the other objectives to the same field of view or, you can center a piece of dirt/debris in what you think is the center of the 10x field of view, then center the other objectives (less precise but equally valid). > > Hope this was helpful. > > Good hunting, > Barbara Foster, President & Chief Consultant Microscopy/Microscopy Education* > 7101 Royal Glen Trail, Suite A - McKinney, TX 75070 - P: 972-924-5310 www.MicroscopyEducation.com > > "Education, not Training" > > > MME is currently scheduling courses for now and through the end of 2015. We can customize a course on nearly any topic, from fluorescence to confocal to image analysis to SEM/TEM. Call us today for a free training evaluation. > > *A subsidiary of The Microscopy & Imaging Place, Inc. > > At 09:04 PM 3/27/2015, 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. >>***** >> >>Greetings, >> I am trying to take two images of teh same field of view using two >>different objectives. >> One of them is a 1.4 NA 100X Nikon lens, and the other is a 0.5-1.3 >>Adjustable iris 100x lens. >> When I switch from one to the other and re focus. I find that I am >>imaging a different part of the sample, sometime by as much as 10 >>microns or more! I suspect this means that the nosepiece turret holding >>the objectives doesn't center properly for some reason, but I thought >>I'd ask the experts on this list if anyone has ever seen anything like this? >> thanks. >>-Jeff |
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Craig Brideau |
Re: different fields of view with different objectives
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*****
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. ***** Post processing should be viable assuming you didn't have to sacrifice too much of your field for the correction. The catch would be that the offset between lenses would have to be consistent as you switch back and forth. If the final point is not repeatable then you would need some way of identifying the same features at different magnifications and correlating them. A mathematical transform and correlation function *might* be able to do this. Craig On Sat, Mar 28, 2015 at 2:02 PM, Michael Giacomelli <[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. > ***** > > Would just imaging with both objectives and then cross-correlating to > align them in post processing be an option? > > Mike > > On Sat, Mar 28, 2015 at 3:04 AM, Guy Cox <[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. > > ***** > > > > Barbara's suggestion is excellent but I do suggest before you go out > spending money you do some more tests. > > > > You are using oil immersion lenses - the drag of the oil might be moving > your sample by the small amount you see. In this case you will see the > movement in the direction you moved the lens (taking the inverted image > into account) and it will reverse if you use the lenses in the reverse > order. You don't mention what type of microscope you are using, but for > inverted microscopes a high-viscosity oil is normally used, and that may be > what you have even if this is an upright microscope. So if this looks like > it's the problem get the lowest-viscosity oil you can and investigate > locking up the stage, by fair means or foul. > > > > Is your turret central? You can check this with dry lenses since you'll > see a consistent inward and outward movement as you rotate it. On some > microscopes this can be adjusted. > > > > If neither of these tips helps then play 'musical lenses' - swap > positions until you get the least movement. > > > > Guy > > > > -----Original Message----- > > From: Confocal Microscopy List [mailto:[hidden email]] > On Behalf Of Barbara Foster > > Sent: Saturday, 28 March 2015 2:19 PM > > To: [hidden email] > > Subject: Re: different fields of view with different objectives > > > > ***** > > 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, Jeff > > > > All of the manufacturers have specs for both parcentration (imaging > exactly the same field) and parfocalization (same plane of focus). > > > > I would recommend that you investigate the type of nosepiece used for > polarized light. Those nosepieces often have 1 fixed position (usually for > the 10x objective) which you can use as reference then have centerable > positions for the other objectives centration screws for each objective). > The procedure is fairly simple: You can either use a target slide with a > cross hair and center the cross hair to the 10x then center all of the > other objectives to the same field of view or, you can center a piece of > dirt/debris in what you think is the center of the 10x field of view, then > center the other objectives (less precise but equally valid). > > > > Hope this was helpful. > > > > Good hunting, > > Barbara Foster, President & Chief Consultant Microscopy/Microscopy > Education* > > 7101 Royal Glen Trail, Suite A - McKinney, TX 75070 - P: 972-924-5310 > www.MicroscopyEducation.com > > > > "Education, not Training" > > > > > > MME is currently scheduling courses for now and through the end of > 2015. We can customize a course on nearly any topic, from fluorescence to > confocal to image analysis to SEM/TEM. Call us today for a free training > evaluation. > > > > *A subsidiary of The Microscopy & Imaging Place, Inc. > > > > At 09:04 PM 3/27/2015, 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. > >>***** > >> > >>Greetings, > >> I am trying to take two images of teh same field of view using two > >>different objectives. > >> One of them is a 1.4 NA 100X Nikon lens, and the other is a 0.5-1.3 > >>Adjustable iris 100x lens. > >> When I switch from one to the other and re focus. I find that I am > >>imaging a different part of the sample, sometime by as much as 10 > >>microns or more! I suspect this means that the nosepiece turret holding > >>the objectives doesn't center properly for some reason, but I thought > >>I'd ask the experts on this list if anyone has ever seen anything like > this? > >> thanks. > >>-Jeff > |
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*****
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 Jeff, Two things has to be taken into consideration- - As suggested by Guy, the drag might be moving the slide itself when you switch the objectives. Just have to make sure that the slide is held in place when the objectives are switched. I have seen the slide move when the objectives are switched until unless you escape the objective and then refocus your sample again. - Are you using two different detectors or cameras? If that is the case, then there are chances of a slight off-set of the camera or detector and this might show as a shift. As Craig has suggested, post-processing can solve the issue too. Good luck. Sathya Srinivasan Manager RUN Advanced Optical Microscopy Facility (www.ucalgary.ca/runcore) University of Calgary Calgary, AB > Date: Sat, 28 Mar 2015 14:25:50 -0600 > From: [hidden email] > Subject: Re: different fields of view with different objectives > To: [hidden email] > > ***** > 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. > ***** > > Post processing should be viable assuming you didn't have to sacrifice too > much of your field for the correction. The catch would be that the offset > between lenses would have to be consistent as you switch back and forth. If > the final point is not repeatable then you would need some way of > identifying the same features at different magnifications and correlating > them. A mathematical transform and correlation function *might* be able to > do this. > > Craig > > On Sat, Mar 28, 2015 at 2:02 PM, Michael Giacomelli <[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. > > ***** > > > > Would just imaging with both objectives and then cross-correlating to > > align them in post processing be an option? > > > > Mike > > > > On Sat, Mar 28, 2015 at 3:04 AM, Guy Cox <[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. > > > ***** > > > > > > Barbara's suggestion is excellent but I do suggest before you go out > > spending money you do some more tests. > > > > > > You are using oil immersion lenses - the drag of the oil might be moving > > your sample by the small amount you see. In this case you will see the > > movement in the direction you moved the lens (taking the inverted image > > into account) and it will reverse if you use the lenses in the reverse > > order. You don't mention what type of microscope you are using, but for > > inverted microscopes a high-viscosity oil is normally used, and that may be > > what you have even if this is an upright microscope. So if this looks like > > it's the problem get the lowest-viscosity oil you can and investigate > > locking up the stage, by fair means or foul. > > > > > > Is your turret central? You can check this with dry lenses since you'll > > see a consistent inward and outward movement as you rotate it. On some > > microscopes this can be adjusted. > > > > > > If neither of these tips helps then play 'musical lenses' - swap > > positions until you get the least movement. > > > > > > Guy > > > > > > -----Original Message----- > > > From: Confocal Microscopy List [mailto:[hidden email]] > > On Behalf Of Barbara Foster > > > Sent: Saturday, 28 March 2015 2:19 PM > > > To: [hidden email] > > > Subject: Re: different fields of view with different objectives > > > > > > ***** > > > 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, Jeff > > > > > > All of the manufacturers have specs for both parcentration (imaging > > exactly the same field) and parfocalization (same plane of focus). > > > > > > I would recommend that you investigate the type of nosepiece used for > > polarized light. Those nosepieces often have 1 fixed position (usually for > > the 10x objective) which you can use as reference then have centerable > > positions for the other objectives centration screws for each objective). > > The procedure is fairly simple: You can either use a target slide with a > > cross hair and center the cross hair to the 10x then center all of the > > other objectives to the same field of view or, you can center a piece of > > dirt/debris in what you think is the center of the 10x field of view, then > > center the other objectives (less precise but equally valid). > > > > > > Hope this was helpful. > > > > > > Good hunting, > > > Barbara Foster, President & Chief Consultant Microscopy/Microscopy > > Education* > > > 7101 Royal Glen Trail, Suite A - McKinney, TX 75070 - P: 972-924-5310 > > www.MicroscopyEducation.com > > > > > > "Education, not Training" > > > > > > > > > MME is currently scheduling courses for now and through the end of > > 2015. We can customize a course on nearly any topic, from fluorescence to > > confocal to image analysis to SEM/TEM. Call us today for a free training > > evaluation. > > > > > > *A subsidiary of The Microscopy & Imaging Place, Inc. > > > > > > At 09:04 PM 3/27/2015, 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. > > >>***** > > >> > > >>Greetings, > > >> I am trying to take two images of teh same field of view using two > > >>different objectives. > > >> One of them is a 1.4 NA 100X Nikon lens, and the other is a 0.5-1.3 > > >>Adjustable iris 100x lens. > > >> When I switch from one to the other and re focus. I find that I am > > >>imaging a different part of the sample, sometime by as much as 10 > > >>microns or more! I suspect this means that the nosepiece turret holding > > >>the objectives doesn't center properly for some reason, but I thought > > >>I'd ask the experts on this list if anyone has ever seen anything like > > this? > > >> thanks. > > >>-Jeff > > |
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Jeff Spector |
Re: different fields of view with different objectives
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*****
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. ***** thanks for all the helpful suggestions. I am using an oil immersion inverted scope. I do escape the objective before switching them. I think it is just in the manufacturing tolerance. I can get around it for now by putting fiduciary markers in the sample and finding the offest between objectives and then always translating the stage the correct amount when changing lenses. This seems to be working so far. thanks for all the helpful discussion. -jeff On Mon, Mar 30, 2015 at 1:19 PM, Sathya Srinivasan <[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. > ***** > > Hi Jeff, > Two things has to be taken into consideration- > - As suggested by Guy, the drag might be moving the slide itself when you > switch the objectives. Just have to make sure that the slide is held in > place when the objectives are switched. I have seen the slide move when the > objectives are switched until unless you escape the objective and then > refocus your sample again. > - Are you using two different detectors or cameras? If that is the case, > then there are chances of a slight off-set of the camera or detector and > this might show as a shift. > > As Craig has suggested, post-processing can solve the issue too. > > Good luck. > > Sathya Srinivasan > Manager > RUN Advanced Optical Microscopy Facility > (www.ucalgary.ca/runcore) > University of Calgary > Calgary, AB > > > > Date: Sat, 28 Mar 2015 14:25:50 -0600 > > From: [hidden email] > > Subject: Re: different fields of view with different objectives > > To: [hidden email] > > > > ***** > > 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. > > ***** > > > > Post processing should be viable assuming you didn't have to sacrifice > too > > much of your field for the correction. The catch would be that the offset > > between lenses would have to be consistent as you switch back and forth. > If > > the final point is not repeatable then you would need some way of > > identifying the same features at different magnifications and correlating > > them. A mathematical transform and correlation function *might* be able > to > > do this. > > > > Craig > > > > On Sat, Mar 28, 2015 at 2:02 PM, Michael Giacomelli <[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. > > > ***** > > > > > > Would just imaging with both objectives and then cross-correlating to > > > align them in post processing be an option? > > > > > > Mike > > > > > > On Sat, Mar 28, 2015 at 3:04 AM, Guy Cox <[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. > > > > ***** > > > > > > > > Barbara's suggestion is excellent but I do suggest before you go out > > > spending money you do some more tests. > > > > > > > > You are using oil immersion lenses - the drag of the oil might be > moving > > > your sample by the small amount you see. In this case you will see the > > > movement in the direction you moved the lens (taking the inverted image > > > into account) and it will reverse if you use the lenses in the reverse > > > order. You don't mention what type of microscope you are using, but > for > > > inverted microscopes a high-viscosity oil is normally used, and that > may be > > > what you have even if this is an upright microscope. So if this looks > like > > > it's the problem get the lowest-viscosity oil you can and investigate > > > locking up the stage, by fair means or foul. > > > > > > > > Is your turret central? You can check this with dry lenses since > you'll > > > see a consistent inward and outward movement as you rotate it. On some > > > microscopes this can be adjusted. > > > > > > > > If neither of these tips helps then play 'musical lenses' - swap > > > positions until you get the least movement. > > > > > > > > Guy > > > > > > > > -----Original Message----- > > > > From: Confocal Microscopy List [mailto: > [hidden email]] > > > On Behalf Of Barbara Foster > > > > Sent: Saturday, 28 March 2015 2:19 PM > > > > To: [hidden email] > > > > Subject: Re: different fields of view with different objectives > > > > > > > > ***** > > > > 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, Jeff > > > > > > > > All of the manufacturers have specs for both parcentration (imaging > > > exactly the same field) and parfocalization (same plane of focus). > > > > > > > > I would recommend that you investigate the type of nosepiece used for > > > polarized light. Those nosepieces often have 1 fixed position > (usually for > > > the 10x objective) which you can use as reference then have centerable > > > positions for the other objectives centration screws for each > objective). > > > The procedure is fairly simple: You can either use a target slide > with a > > > cross hair and center the cross hair to the 10x then center all of the > > > other objectives to the same field of view or, you can center a piece > of > > > dirt/debris in what you think is the center of the 10x field of view, > then > > > center the other objectives (less precise but equally valid). > > > > > > > > Hope this was helpful. > > > > > > > > Good hunting, > > > > Barbara Foster, President & Chief Consultant Microscopy/Microscopy > > > Education* > > > > 7101 Royal Glen Trail, Suite A - McKinney, TX 75070 - P: > 972-924-5310 > > > www.MicroscopyEducation.com > > > > > > > > "Education, not Training" > > > > > > > > > > > > MME is currently scheduling courses for now and through the end of > > > 2015. We can customize a course on nearly any topic, from > fluorescence to > > > confocal to image analysis to SEM/TEM. Call us today for a free > training > > > evaluation. > > > > > > > > *A subsidiary of The Microscopy & Imaging Place, Inc. > > > > > > > > At 09:04 PM 3/27/2015, 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. > > > >>***** > > > >> > > > >>Greetings, > > > >> I am trying to take two images of teh same field of view using two > > > >>different objectives. > > > >> One of them is a 1.4 NA 100X Nikon lens, and the other is a > 0.5-1.3 > > > >>Adjustable iris 100x lens. > > > >> When I switch from one to the other and re focus. I find that I am > > > >>imaging a different part of the sample, sometime by as much as 10 > > > >>microns or more! I suspect this means that the nosepiece turret > holding > > > >>the objectives doesn't center properly for some reason, but I thought > > > >>I'd ask the experts on this list if anyone has ever seen anything > like > > > this? > > > >> thanks. > > > >>-Jeff > > > > > |
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