Beware of “Empty” Magnification

Anja Schué, Leica Microsystems

In the simplest case, a microscope consists of one lens close to the specimen (objective) and one lens close to the eye (eyepiece).The magnification of a microscope is the product of the factors of both lenses. A 40x objective and a 10x eyepiece, for example, providea 400x magnification. However, it is not only the magnification but also the resolution that indicates the performance capacityof a microscope.Resolution is the ability to render two closely adjacentdots separately. According to the Rayleighcriterion, the minimum distance between two dotsable to be separately imaged corresponds to approximatelyone-half the wavelength of the light.

d = 0.61 ∗(λ/n ∗ sinα)
λ = light wavelength
n = refractive index of the medium betweenspecimen and objective
α = half the aperture angle of the objective

Therefore, with blue light, the resolution limit isapproximately d = 0.2 μm; with red light, aroundd = 0.35 μm. UV objectives attain a resolution justunder 0.2 μm. With the naked eye, we are not ableto differentiate structures smaller than 0.2 millimetres.The value n × sin α corresponds to the numericalaperture (NA ), the measure of the light gatheringcapacity and the resolution of an objective. Becausethe aperture angle cannot exceed 90° and therefractive index is never less than 1 (nair = 1), NA isalways below 1 for air. When immersion oil is used (n > 1), the numerical aperture increases (to up toapprox. 1.45) and, along with it, the resolution.

Immersion oil increases resolution

To make the microscopic resolution detectable tothe eye, the image appears in the eyepiece with correspondingmagnification. The resolution and magnificationare always directly interdependent. Anobjective with low magnification has a low numericalaperture and thus a low resolution. For a highmagnification objective, the numerical aperture isalso high, typically 0.8 for a 40x dry objective. However,because the numerical aperture cannot beincreased beyond a certain point, the usable magnificationrange is also limited in classic light microscopes.The “useful” magnification is between 500 × NA and 1,000 × NA.

Everything beyond the “useful” magnification iscalled “empty” magnification. Though structuresappear larger, no additional details are resolved.For high-resolution analysis of microstructures,empty magnification is not desirable. Nevertheless,empty magnification can sometimes be quite usefulfor making details more easily visible for the humaneye. Examples of this are video microscopy and alsodigital microscopy, where greatly enlarged imagesare displayed on a monitor.