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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 This is probably a very foolish and basic question, but I am going to ask it any way and just apologise for my ignorance as a biological specialist using a confocal microscope. In microscopy I would generally multiply the objective lens magnification by the ocular lens (often 10x) to get the total magnification of an image. I want to know if this also applies for the confocal microscope or is the light path such that the objective magnification is the only one relevant? If I do need to include additional magnification what would these magnifications be, are they dependent upon the system being used (LSM 510 META) or is there a standard magnification? Thanks for your help in clarifying this for me. Regards Nicola |
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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, magnification can be very dangerous parameter. You obtain a magnification of 1 per definition only if you look at an object at the conventional viewing distance of 25 cm. Thus either changing the viewing distance or changing the size of an electronic image will change the magnification of the image. Therefore it is much better to include a scale bar in the image. This automatically also changes if you change the viewing distance and or the size of the image. In order to create a scale for an image of an wide-field microscope you need to know the total magnification of you microscope (=magnification of the objective fold intermediate magnification; latter on can also be 1) and the pixel size of your camera (if you use binning on the camera you also have to account for this). If you are unsure of the total magnification and/or the pixel size you can also use a calibration slide to measure it (see following link: http://www.swarthmore.edu/NatSci/nkaplin1/scalebar.htm). For confocal microscopy it is a little more complicated as you have various other parameters which influence the pixel size in an image (e.g. zoom factor, image size). Therefore all of the images of commercially available confocal microscopes save this information with the image (sometimes maybe not for all of the image formats). So you do not have to care about it. If you open/imort the images with ImageJ (with the LOCI Bioformats plugin you can read even the proprietary company formats http://loci.wisc.edu/software/bio-formats) you can directly add a scale bar via: Analyze-->Tools-->Scale Bar... Best regards Arne > -----Original Message----- > From: Confocal Microscopy List > [mailto:[hidden email]] On Behalf Of Nicola > Green > Sent: vendredi 16 août 2013 12:57 > To: [hidden email] > Subject: magnification > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hi > This is probably a very foolish and basic question, but I am going to ask it any > way and just apologise for my ignorance as a biological specialist using a > confocal microscope. > > In microscopy I would generally multiply the objective lens magnification by > the ocular lens (often 10x) to get the total magnification of an image. > I want to know if this also applies for the confocal microscope or is the light > path such that the objective magnification is the only one relevant? > If I do need to include additional magnification what would these > magnifications be, are they dependent upon the system being used (LSM > 510 > META) or is there a standard magnification? > > Thanks for your help in clarifying this for me. > > Regards > Nicola |
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In reply to this post by Nicola Green
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** In a confocal image the magnification depends on the objective magnification and the area scanned on the sample. There is of course an 'ocular' or transfer lens but that is not variable and thus is included in the calibration of the system. So when you 'zoom' in a confocal image you are just scanning a smaller part of the field of view. The system should still always give you the correct magnification, but since this is a digital image the appropriate units are pixels per micrometre (or the converse, the size of a pixel). Every confocal microscope will give you these figures. You should check once in a while, of course, using a standard specimen such as a stage micrometer. Guy -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Nicola Green Sent: Friday, 16 August 2013 8:57 PM To: [hidden email] Subject: magnification ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi This is probably a very foolish and basic question, but I am going to ask it any way and just apologise for my ignorance as a biological specialist using a confocal microscope. In microscopy I would generally multiply the objective lens magnification by the ocular lens (often 10x) to get the total magnification of an image. I want to know if this also applies for the confocal microscope or is the light path such that the objective magnification is the only one relevant? If I do need to include additional magnification what would these magnifications be, are they dependent upon the system being used (LSM 510 META) or is there a standard magnification? Thanks for your help in clarifying this for me. Regards Nicola |
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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 is actually an issue that befuddles many microscopists. Magnification, after all, really depends on the final image. That is, an object viewed with a 10X lens and viewed through the eyepiece could be said to have a certain magnification as compared to an object seen with the unaided eye at a given distance. However, when that image is captured with a digital, or photographic system, and displayed on a 10" monitor, or projected on a 30' screen, the magnification is clearly different. A printed image, might be yet another size. It should be standard procedure to produce images with an embedded size indication, rather than give the magnification. The important information is provided by specifying the objective lens (ideally), which makes the major contribution to resolution, So, to get back to the original question, and echo Guy's comment, , what matters are the dimensions of the area captured by the system. Most confocal microscopes, as Guy mentions, include this kind of information about each image, and can be extracted by software. Joel On Fri, Aug 16, 2013 at 7:59 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 > ***** > > In a confocal image the magnification depends on the objective > magnification and the area scanned on the sample. There is of course an > 'ocular' or transfer lens but that is not variable and thus is included in > the calibration of the system. So when you 'zoom' in a confocal image you > are just scanning a smaller part of the field of view. The system should > still always give you the correct magnification, but since this is a > digital image the appropriate units are pixels per micrometre (or the > converse, the size of a pixel). Every confocal microscope will give you > these figures. You should check once in a while, of course, using a > standard specimen such as a stage micrometer. > > Guy > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] > On Behalf Of Nicola Green > Sent: Friday, 16 August 2013 8:57 PM > To: [hidden email] > Subject: magnification > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hi > This is probably a very foolish and basic question, but I am going to ask > it any way and just apologise for my ignorance as a biological specialist > using a confocal microscope. > > In microscopy I would generally multiply the objective lens magnification > by the ocular lens (often 10x) to get the total magnification of an image. > I want to know if this also applies for the confocal microscope or is the > light path such that the objective magnification is the only one relevant? > If I do need to include additional magnification what would these > magnifications be, are they dependent upon the system being used (LSM 510 > META) or is there a standard magnification? > > Thanks for your help in clarifying this for me. > > Regards > Nicola > -- 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 |
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In reply to this post by Guy Cox-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 ***** So how does the manufacturer know the size of the pixel? I guess this will be calibrated at setup of the system with a target of known size for one objective and then stored somewhere in the parameters and hopfully regularly checked. Do they then just change it proportionally for the other objectives? Sometimes objective magnification can be a little bit different than what is written on the barrel, epecially when the lens has a correction collar, did anyone check this? We had the case that a journal insisted on us giving the magnification in the figure caption, so we gave the objective magnification and insisted on having the scale bar in addition to that. Andreas -----Original Message----- From: Guy Cox <[hidden email]> To: CONFOCALMICROSCOPY <[hidden email]> Sent: Fri, 16 Aug 2013 13:01 Subject: Re: magnification ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** In a confocal image the magnification depends on the objective magnification and the area scanned on the sample. There is of course an 'ocular' or transfer lens but that is not variable and thus is included in the calibration of the system. So when you 'zoom' in a confocal image you are just scanning a smaller part of the field of view. The system should still always give you the correct magnification, but since this is a digital image the appropriate units are pixels per micrometre (or the converse, the size of a pixel). Every confocal microscope will give you these figures. You should check once in a while, of course, using a standard specimen such as a stage micrometer. Guy -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Nicola Green Sent: Friday, 16 August 2013 8:57 PM To: [hidden email] Subject: magnification ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi This is probably a very foolish and basic question, but I am going to ask it any way and just apologise for my ignorance as a biological specialist using a confocal microscope. In microscopy I would generally multiply the objective lens magnification by the ocular lens (often 10x) to get the total magnification of an image. I want to know if this also applies for the confocal microscope or is the light path such that the objective magnification is the only one relevant? If I do need to include additional magnification what would these magnifications be, are they dependent upon the system being used (LSM 510 META) or is there a standard magnification? Thanks for your help in clarifying this for me. Regards Nicola |
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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 agree. With a confocal image the scale bar is really the only useful descriptor. 'Magnification' for confocals is more a function of the timing of the acquisition electronics, and talking about it in ocular terms is misleading. Craig On Fri, Aug 16, 2013 at 10:49 AM, 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 > ***** > > So how does the manufacturer know the size of the pixel? I guess this will > be calibrated at setup of the system with a target of known size for one > objective and then stored somewhere in the parameters and hopfully > regularly checked. Do they then just change it proportionally for the other > objectives? Sometimes objective magnification can be a little bit different > than what is written on the barrel, epecially when the lens has a > correction collar, did anyone check this? > > We had the case that a journal insisted on us giving the magnification in > the figure caption, so we gave the objective magnification and insisted on > having the scale bar in addition to that. > > Andreas > > > > > > > > -----Original Message----- > From: Guy Cox <[hidden email]> > To: CONFOCALMICROSCOPY <[hidden email]> > Sent: Fri, 16 Aug 2013 13:01 > Subject: Re: magnification > > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > In a confocal image the magnification depends on the objective > magnification and > the area scanned on the sample. There is of course an 'ocular' or > transfer lens > but that is not variable and thus is included in the calibration of the > system. > So when you 'zoom' in a confocal image you are just scanning a smaller > part of > the field of view. The system should still always give you the correct > magnification, but since this is a digital image the appropriate units are > pixels per micrometre (or the converse, the size of a pixel). Every > confocal > microscope will give you these figures. You should check once in a while, > of > course, using a standard specimen such as a stage micrometer. > > Guy > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] > On > Behalf Of Nicola Green > Sent: Friday, 16 August 2013 8:57 PM > To: [hidden email] > Subject: magnification > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hi > This is probably a very foolish and basic question, but I am going to ask > it any way and just apologise for my ignorance as a biological specialist > using a confocal microscope. > > In microscopy I would generally multiply the objective lens magnification > by the ocular lens (often 10x) to get the total magnification of an image. > I want to know if this also applies for the confocal microscope or is the > light path such that the objective magnification is the only one relevant? > If I do need to include additional magnification what would these > magnifications be, are they dependent upon the system being used (LSM 510 > META) or is there a standard magnification? > > Thanks for your help in clarifying this for me. > > Regards > Nicola > > > |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hello All, On wide field microscopes, the magnification (and calibration data ) can be calculated with remarkable accuracy! If any of you are familiar with Image-Pro Plus v5 or later, it includes a calibration estimation tool that I helped develop and tested extensively against every microscope I could get my hands on at the time. Rarely were my calculated calibrations off by more than +/-1%. However, as has been pointed out repeatedly the magnification of an image from a confocal is much more variable (I will skip the reasons why). With that said magnification is simply: Image Size Magnification = --------------------------------- Original object size So, with a scale bar that shows a known size the magnification can be calculated at any time with a ruler and calculator. In response to one posting about a journal editor insisting on listing the magnification in the image discussion I think that we should all push back pointing out that in today's world where it is easy, even trivial to capture an image at any resolution and print it out in sizes ranging from a few inches to many feet that a single number for magnification is irrelevant and even misleading. To interpret a statement of magnification I need to know many details of the way in which the image presented was captured and manipulated post acquisition (I do consider even cropping an image "manipulation"). But a scale bar applied by the acquisition software or post acquisition based on a spatial calibration gives me instant access to the scale of objects in the image and if I so desire to the real magnification (i.e the magnification after all manipulations that lead to it appearing on the page in front of me). Further since many of now read articles in PDF format, magnification becomes even squishier because I can used any PDF viewer to zoom in on a single pixel should I so desire! Chris Tully On 8/16/2013 3:32 PM, Craig Brideau wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > I agree. With a confocal image the scale bar is really the only useful > descriptor. 'Magnification' for confocals is more a function of the timing > of the acquisition electronics, and talking about it in ocular terms is > misleading. > > Craig > > > On Fri, Aug 16, 2013 at 10:49 AM, 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 >> ***** >> >> So how does the manufacturer know the size of the pixel? I guess this will >> be calibrated at setup of the system with a target of known size for one >> objective and then stored somewhere in the parameters and hopfully >> regularly checked. Do they then just change it proportionally for the other >> objectives? Sometimes objective magnification can be a little bit different >> than what is written on the barrel, epecially when the lens has a >> correction collar, did anyone check this? >> >> We had the case that a journal insisted on us giving the magnification in >> the figure caption, so we gave the objective magnification and insisted on >> having the scale bar in addition to that. >> >> Andreas >> >> >> >> >> >> >> >> -----Original Message----- >> From: Guy Cox <[hidden email]> >> To: CONFOCALMICROSCOPY <[hidden email]> >> Sent: Fri, 16 Aug 2013 13:01 >> Subject: Re: magnification >> >> >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> ***** >> >> In a confocal image the magnification depends on the objective >> magnification and >> the area scanned on the sample. There is of course an 'ocular' or >> transfer lens >> but that is not variable and thus is included in the calibration of the >> system. >> So when you 'zoom' in a confocal image you are just scanning a smaller >> part of >> the field of view. The system should still always give you the correct >> magnification, but since this is a digital image the appropriate units are >> pixels per micrometre (or the converse, the size of a pixel). Every >> confocal >> microscope will give you these figures. You should check once in a while, >> of >> course, using a standard specimen such as a stage micrometer. >> >> Guy >> >> -----Original Message----- >> From: Confocal Microscopy List [mailto:[hidden email]] >> On >> Behalf Of Nicola Green >> Sent: Friday, 16 August 2013 8:57 PM >> To: [hidden email] >> Subject: magnification >> >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> ***** >> >> Hi >> This is probably a very foolish and basic question, but I am going to ask >> it any way and just apologise for my ignorance as a biological specialist >> using a confocal microscope. >> >> In microscopy I would generally multiply the objective lens magnification >> by the ocular lens (often 10x) to get the total magnification of an image. >> I want to know if this also applies for the confocal microscope or is the >> light path such that the objective magnification is the only one relevant? >> If I do need to include additional magnification what would these >> magnifications be, are they dependent upon the system being used (LSM 510 >> META) or is there a standard magnification? >> >> Thanks for your help in clarifying this for me. >> >> Regards >> Nicola >> >> >> -- *Chris Tully* Principal Consultant 240-475-9753 Image Incyte, LLC <http:%5C%5Cwww.ImageIncyte.com> [hidden email] <mailto:[hidden email]> |
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In reply to this post by Nicola Green
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Nicola, When you export your image from the 510 as a TIFF file, the software should give you the option to add a scale bar to the image. Ask a Zeiss rep if you don't find it. Alternately, your lab may own or borrow a graticule (also referred to as reticule or stage micrometer), i.e. a glass slide engraved with a 1 or 2mm scale, with marks for 10 and 100 microns intervals. Take a transmitted light image of the scale using each of the lenses present on the confocal and with a set zoom factor (important). You can then store these images and go back to the appropriate one to create a scale bar for any image, past or future. Just don't forget to take the zoom factor into account. Evelyn Ralston, Ph.D. Light Imaging Section NIAMS, National Institutes of Health, Bethesda, MD 20892 On Aug 16, 2013, at 6:56 AM, Nicola Green wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hi > This is probably a very foolish and basic question, but I am going to ask > it any way and just apologise for my ignorance as a biological specialist > using a confocal microscope. > > In microscopy I would generally multiply the objective lens magnification > by the ocular lens (often 10x) to get the total magnification of an image. > I want to know if this also applies for the confocal microscope or is the > light path such that the objective magnification is the only one relevant? > If I do need to include additional magnification what would these > magnifications be, are they dependent upon the system being used (LSM 510 > META) or is there a standard magnification? > > Thanks for your help in clarifying this for me. > > Regards > Nicola |
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