| Consider the ideal fluorescent atom.The image of this atom in a microscope (confocal orregular optical microscope) is a spot, more technically, an Airydisk, which looks like the picture at right. This is due todiffraction effects. |  |
The size of the spot is related to your resolution.Resolution is being able to tell the difference between twoclosely positioned bright objects, and one big object.If two objects are closer together than your resolution,then they blur together in the microscope image and it'simpossible to tell that they are two points (except maybethe combined image is twice as bright as one object: but still,you can't measure their separation). The best resolution for anoptical microscope is about 0.2 microns = 200 nm.
The good news is, there's a difference between resolution and"ability to locate the position".
If you have one tiny and isolated fluorescent object, youcan often locate the position of that object to better thanyour resolution. The image of the object will show up as anextended blob, and you can find the "center of mass" of thatblob-shaped image. If the blob is N pixels wide and each pixelis M microns across, you can estimate the center of the blob toabout M/N accuracy, which often beats the optical resolution.This is a useful trick, but not solving the same problem asresolution. In some cases you can do various tricks to makethe spot size bigger (increase N) so that you can locate thecenter even better. Various experiments I've heard of haveclaimed to be able to locate the centers of spots to within10-30 nm using this sort of method. You may beinterested in some software available for identifying particlepositions, which implements this center-of-mass method.
The magnification is something different altogether. There's atechnical definition which compares the apparent angular size of theimage, to the actual angular size of the object as it wouldappear if it were 25 cm away from your eye. This is a somewhatarbitary definition and in my opinion is mainly relevant fordevising problems when I teach optics in my introductory physicsclasses. In real life, one often takes pictures using a CCDcamera on a microscope, and projects them on a monitor. Using alarger monitor certainly can magnify the image further. But, itwill still be just as blurry or sharp as the resolution.
Fortunately, in general higher magnification lenses also havebetter resolution. In our lab a 10x objective has a resolutionof 0.7 microns and a 100x objective has a resolution of 0.2microns. One other tradeoff to consider: higher magnificationlenses look at smaller fields of view, in proportion to theirmagnification. A 100x objective that sees a field of viewof 100 x 100 micron^2 can be contrasted with a 10x objectivelooking at a 1000 x 1000 micron^2 field of view.
So, when worrying about how good a microscope is, the mostimportant question is what the resolution is. And in somescience applications (such as my work) youcare a lot about how well you can locate the centers of objects,and hopefully you can beat the resolution. Magnification is amuch less useful specification (in my opinion).
Technical note
More technically, a microscope objective's resolution isquantified by the Numerical Aperture.This webpage has great description of this as well as a morein-depth discussion of resolution. I'll note here that thewavelength of light you use makes a difference; shorterwavelengths improve the resolution.
Links
- How a confocal microscope works
- This explanation was written by Eric Weeks
- Send me email: weeks@physics.emory.edu. Let me know if you havefurther questions, or if there are parts of this explanation thatare confusing.
