David Lenzi |
*****
To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi all, I am trying to solve a field flatness issue, and was wondering if anyone else had come across something similar. If focussed at the center of the field, the edges are out of focus, and vice versa, with the difference in z about 0.5 to 1.0 um, the center lower than the edges. The microscope is a Leica DMI6000B (inverted), and the objective is a 63x/1.4NA plan apo oil immersion. The camera we're using has a large chip (Fairchild CMOS, 2560 x 2160 pixels), which captures a field of view of 21.7 mm (on the diagonal) at the objective's back aperture. I'm still waiting to get an answer from Leica on what the flatness specification is, but it seems to me it should be better than this. Here is what else I know: - i don't think it is the stage because the effect is radially symmetrical, and object go in and out of focus as they are moved from one corner, through the center, and to the opposite corner of the field. - The effect is independent of wavelength or filter cube. - The effect is also visible at 40x/1.3 NA (oil) and 20x (air). - The other key test is whether the effect is visible at the camera as well as through the eye pieces. This is harder to tell, but I think it is visible through the eye pieces. Any suggestions appreciated! -David Lenzi |
JOEL B. SHEFFIELD |
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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 David, I can't tell from your note whether your camera has a relay lens of some sort. Frequently, these lenses are a source of this kind of curvature of the field. Have you checked the camera itself? Joel On Thu, May 31, 2012 at 3:11 PM, David Lenzi <[hidden email]> wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hi all, > I am trying to solve a field flatness issue, and was wondering if anyone > else > had come across something similar. If focussed at the center of the > field, the > edges are out of focus, and vice versa, with the difference in z about 0.5 > to > 1.0 um, the center lower than the edges. The microscope is a Leica > DMI6000B > (inverted), and the objective is a 63x/1.4NA plan apo oil immersion. The > camera we're using has a large chip (Fairchild CMOS, 2560 x 2160 pixels), > which > captures a field of view of 21.7 mm (on the diagonal) at the objective's > back > aperture. I'm still waiting to get an answer from Leica on what the > flatness > specification is, but it seems to me it should be better than this. > > Here is what else I know: > - i don't think it is the stage because the effect is radially > symmetrical, and > object go in and out of focus as they are moved from one corner, through > the > center, and to the opposite corner of the field. > - The effect is independent of wavelength or filter cube. > - The effect is also visible at 40x/1.3 NA (oil) and 20x (air). > > - The other key test is whether the effect is visible at the camera as > well as > through the eye pieces. This is harder to tell, but I think it is visible > through > the eye pieces. > > Any suggestions appreciated! -David Lenzi > -- Joel B. Sheffield, Ph.D Department of Biology Temple University Philadelphia, PA 19122 Voice: 215 204 8839 e-mail: [hidden email] URL: http://astro.temple.edu/~jbs |
Gregg Jarvis |
In reply to this post by David Lenzi
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** David, Do you have any other microscopes available to see if it is in fact the microscope? It seems it could be the slide depth/poor slide? or the microscope objective is out of alignment or not seated all the way into the microscope housing,"spherical aberration" did someone bump it or change out the objectives recently? On Thu, May 31, 2012 at 3:11 PM, David Lenzi <[hidden email]> wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hi all, > I am trying to solve a field flatness issue, and was wondering if anyone > else > had come across something similar. If focussed at the center of the > field, the > edges are out of focus, and vice versa, with the difference in z about 0.5 > to > 1.0 um, the center lower than the edges. The microscope is a Leica > DMI6000B > (inverted), and the objective is a 63x/1.4NA plan apo oil immersion. The > camera we're using has a large chip (Fairchild CMOS, 2560 x 2160 pixels), > which > captures a field of view of 21.7 mm (on the diagonal) at the objective's > back > aperture. I'm still waiting to get an answer from Leica on what the > flatness > specification is, but it seems to me it should be better than this. > > Here is what else I know: > - i don't think it is the stage because the effect is radially > symmetrical, and > object go in and out of focus as they are moved from one corner, through > the > center, and to the opposite corner of the field. > - The effect is independent of wavelength or filter cube. > - The effect is also visible at 40x/1.3 NA (oil) and 20x (air). > > - The other key test is whether the effect is visible at the camera as > well as > through the eye pieces. This is harder to tell, but I think it is visible > through > the eye pieces. > > Any suggestions appreciated! -David Lenzi > -- Gregg Jarvis Advanced Products Group Omega Optical Inc. 24 Omega Drive Brattleboro, VT 05301 [hidden email] 1-802-251-7316 |
John Oreopoulos |
In reply to this post by David Lenzi
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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 David, My guess is the following: Since you're now using a CMOS camera with a much larger chip area, you're probably seeing the field curvature that was always there associated with the objective and tube lens of your microscope system. You probably didn't see it before simply because the camera you were using previously could only view a much smaller portion of the entire field of view presented by the objective and tube lens combination. One way to test that theory would be to switch back to your older camera and physically translate it across the field of view and see if you observe the same effect as before with the other camera. Instead of using a camera hard mount, put the camera on a lab jack and place a dark cloak over the camera the microscope side port when translating. John Oreopoulos On 2012-05-31, at 3:11 PM, David Lenzi wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hi all, > I am trying to solve a field flatness issue, and was wondering if anyone else > had come across something similar. If focussed at the center of the field, the > edges are out of focus, and vice versa, with the difference in z about 0.5 to > 1.0 um, the center lower than the edges. The microscope is a Leica DMI6000B > (inverted), and the objective is a 63x/1.4NA plan apo oil immersion. The > camera we're using has a large chip (Fairchild CMOS, 2560 x 2160 pixels), which > captures a field of view of 21.7 mm (on the diagonal) at the objective's back > aperture. I'm still waiting to get an answer from Leica on what the flatness > specification is, but it seems to me it should be better than this. > > Here is what else I know: > - i don't think it is the stage because the effect is radially symmetrical, and > object go in and out of focus as they are moved from one corner, through the > center, and to the opposite corner of the field. > - The effect is independent of wavelength or filter cube. > - The effect is also visible at 40x/1.3 NA (oil) and 20x (air). > > - The other key test is whether the effect is visible at the camera as well as > through the eye pieces. This is harder to tell, but I think it is visible through > the eye pieces. > > Any suggestions appreciated! -David Lenzi |
George McNamara |
In reply to this post by David Lenzi
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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 David, I was working with a Leica plan apo 63x/1.4 NA oil immersion objective lens on our SP5 confocal microscope (DMI6000). The user's specimens were flat cells on coverglasses - each coverglass had little specks of green thingies strewn over it (probably residue of cells that had mostly been blown off during sample prep). All the coverglasses had tiny specks that were in focus at the coverglass over the entire field of view of the eyepiece. Yes, my eyes are not calibrated in Z. Some suggestions: is there some optical component in the light path that is not needed - a 0.5x or 1.6x relay lens (in camera mount) or optovar (in the stand) for example? ... if yes, is it designed for this microscope? ... sometimes microscope components stop working - is it possible a motorized turret that is supposed to remove a relay lens is stuck in the wrong place? when you look at the same specimens on a different microscope with comparable lens - plan apo 63x or 60x 1.4 NA - is it flat or pincushioned? (especailly same lens on another DMI6000). are you looking at features directly at the coverglass ... is the coverglass 170 um thick (or at least out of a box that says #1.5)? ... do multiple specimens behave the same way? ... if you are looking at something far away, say cells on a Lab-Tek chamber slide with 50 um gap to the coverglass, this lens is designed to image at the [170 um thick] coverglass, not far (10+ um) away. are you using the right immersion oil? (though you mentioned your 20x dry lens has the same problems) is your lens "messed up"? (i.e. if you look at the front element on a stereomicroscope, does it look ok or "messed up") George On 5/31/2012 3:11 PM, David Lenzi wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hi all, > I am trying to solve a field flatness issue, and was wondering if anyone else > had come across something similar. If focussed at the center of the field, the > edges are out of focus, and vice versa, with the difference in z about 0.5 to > 1.0 um, the center lower than the edges. The microscope is a Leica DMI6000B > (inverted), and the objective is a 63x/1.4NA plan apo oil immersion. The > camera we're using has a large chip (Fairchild CMOS, 2560 x 2160 pixels), which > captures a field of view of 21.7 mm (on the diagonal) at the objective's back > aperture. I'm still waiting to get an answer from Leica on what the flatness > specification is, but it seems to me it should be better than this. > > Here is what else I know: > - i don't think it is the stage because the effect is radially symmetrical, and > object go in and out of focus as they are moved from one corner, through the > center, and to the opposite corner of the field. > - The effect is independent of wavelength or filter cube. > - The effect is also visible at 40x/1.3 NA (oil) and 20x (air). > > - The other key test is whether the effect is visible at the camera as well as > through the eye pieces. This is harder to tell, but I think it is visible through > the eye pieces. > > Any suggestions appreciated! -David Lenzi > > |
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