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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 <[log in to unmask]>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 <[log in to unmask]>
>> To: CONFOCALMICROSCOPY <[log in to unmask]>
>> 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:[log in to unmask]]
>> On
>> Behalf Of Nicola Green
>> Sent: Friday, 16 August 2013 8:57 PM
>> To: [log in to unmask]
>> 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

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