RE: [Histonet] quick and dirty scale bar

From:Philip Oshel


They give a needed sense of scale to the subject. This may seem 
superfluous, but it's valuable. I rather suspect you have a set of 
neurons tucked away in your cortex that know how big a macrophage 
fixed with Bouin's is, or a methanol-fixed RBC, etc., so you know 
what you're looking at and if it's the right size.
How about a confocal image of the mitochondrial network of a 
crustacean larva? Or the size range of pollen grains? Or are the 
pores in that polymer about 1 micron or 10?
Obviously you're right that the sizes are "ishy" -- they're ishy even 
on non-processed samples like a chunk of semiconductor or a ceramic 
like bone (unless much time and money has been spent properly 
calibrating the microscopes). I've had more than one dispute about 
this, where some investigator wants to measure things to 0.01 
micrometer or better, and the for-real precision is more like 10%.
Still, it's useful to know if the micrograph was taken with a 10X 
lens a 40X lens or a 100X lens, and if the relay lens was 0.33X or 
0.45X or ... get me in the ballpark (sorry, cricket grounds) and I'll 
have a better shot and figuring out if that little blob is a tiny 
little one micron green algal cell or a 50 micro green algae -- in, 
most likey, a completely different kingdom.
The meaning really is in your second sentence. You've got lots of 
known sizes to reference against. Most of us don't. Even in animal 
tissues, the same kind of cell in a bird isn't going to be as big as 
in a human or a rat or a lizard or ... .
Scale bars become the reference to recognize orders of size and 
relative sizes. There are few internal standards or references.


>And what pray, is their meaning?
>One can tell the size of things by reference to known sizes, and
>recognize relative sizes. Is this not good enough, even though sizes are
>only "ishy"?
>If you want to measure things exactly, that's a different matter, but
>even then, you only get "ishy" results because of the unknown factor of
>shrinking in the processing (and sometimes expansion on the water bath).
>Be that all as it may, I was trying to get a problem solved, which was
>to get over the requirement of a scale bar without known magnification
>Nobody surely is going to be in a position to argue unless the scale is
>obviously absurd.
>Of course, we don't know (I think) the nature of the photographed
>material. It might be something in which size is considered to be
>crucial, but I doubt it, as this would have been built into the photos
>in the first place.
>Terry (wishing, as usual I had stayed out of it) Marshall
>-----Original Message-----
>From: Philip Oshel []
>Sent: 19 June 2007 14:46
>To: Marshall Terry Dr, Consultant Histopathologist
>Subject: RE: [Histonet] quick and dirty scale bar
>Scale bars may be meaningless to a pathologist, but not to any
>microscopist I ever met. Or any biologist or materials scientist working
>with micrographs. We certainly don't spend hours in every course
>pounding scale bars and plate making into our microscopy majors' heads
>because its useless.
>Magnification, yeah. Only useful in certain narrow instances.
>Not wanting the arrows is more than a tad bizarre, though. What did they
>want? Flashing LEDs and hyperlinks to websites?
>>This is a tad bizarre. I had this problem recently, spending hours
>>getting pictures just right for a junior's paper, then finding after
>>submission, that they wanted magnification (useless information) and
>>didn't want the helpful arrows which would point the uninitiated in the
>>right direction.
>>There was nothing for it but to start again.
>>Proper pathology journals gave up putting magnifications or scale bars
>>years ago (EM pictures excepted).
>>It's just the  other non-pathology journals which want it, and it's
>>totally meaningless, empty information.
>>I suggest you bullshit. Take a few red cells, call them 6mu, and put on
>>a scale from that. Who's to know the difference?
>>-----Original Message-----
>>[] On Behalf Of Philip
>>Sent: 19 June 2007 14:02
>>Subject: Re: [Histonet] quick and dirty scale bar
>>No, you can't use the 40X scale bar, and yes, it's not that simple.
>>Even if the image with the 40X scale bar were taken on the same
>>microscope with the same camera and the same settings, it wouldn't be
>>in an exact 1:4 relation with the 10X objective.
>>Further, you don't know what if any zoom your collaborator used. If the
>>image was taken with a standard digital camera (e.g., a Nikon
>>Coolpix) through an eyepiece adapter, there is often vignetting around
>>the edges. This prompts people to use the zoom function to eliminate
>>this. Which of course means the image isn't "10X".
>>Which it may not have been anyway, depending on the lens in the
>>eyepiece adapter.
>>The only way to get images with known scale bars is to take a
>>micrograph of a stage micrometer **without changing the camera**,
>>especially the zoom, for each objective used, *at the time the
>micrographs are taken*.
>>Otherwise it is nearly impossible to replicate the zoom used.
>>Calculations/measurements based on field of view aren't trustworthy for
>>the same reasons -- you don't know the true field of view.
>>If no zoom was used, then it may be possible to take a photo of a stage
>>micrometer with the same objective, etc., and be OK. But I wouldn't
>>trust it.
>>Given what you have, there are really only 2 courses: first, is there a
>>structure in the micrograph you have that has a well know  size?
>>RBCs don't count, they change size by a micrometer or more depending on
>>how they're treated (and yes, still look like nice, biconcave discs).
>>If there is, you can calculate magnification and therefore a scale bar
>>from it.
>>But likely not. The other recourse is to have you collaborator take the
>>image again, and this time also take an image of a stage micrometer.
>>Anything else is just guessing.
>>>Hey Guys,
>>>I'm trying to finish up some figures for a paper.  One collaborator
>>>given me 10X images that appear to have been taken from a standard
>>>digital camera.  Can anyone suggest a quick and easy way to add a
>>>bar?  Unfortunately the journal requires it and I just don't have it.
>>>I have a scale bar for a 40X picture from a different
>>>microscope/experiment, any chance that I can use this?
>>>I'm guessing it is not as simple as my 40X scale bar being 1/4 of the
>>>Sorry, but I am clueless about this...  any suggestions would be
>>>Histonet mailing list
>>Philip Oshel
>>Microscopy Facility Supervisor
>>Biology Department
>>024C Brooks Hall
>>Central Michigan University
>>Mt. Pleasant, MI 48859
>>Histonet mailing list

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