Resolution vs Visibility Through the Microscope

The resolution of objects viewed through the light microscope is a calculated value based on configuration of the microscope. Visibility is a function of contrast. To take advantage of resolution there must be sufficient contrast to see the subject but objects far below the resolution limit of a specific microscope configuration can be made visible with sufficient contrast. This is the same phenomena that allows us to see the stars in the night sky, all of which are far below the resolution limit of our eyes. A few examples through the microscope are provided below showing methods of improving the resolution and/or visibility of objects viewed through the "standard" light microscope. Click on the photographs below for more information.

Resolution and Ocular Magnification

The resolution of the microscope is determined by the objective and the condenser. Numerical apurture is important but the diffraction maxima collected at the back focal plane of the objective is more important. The eyepiece must provide sufficient magnification for the observer to see what is being resolved. The images below provide a simple example.

$Diffraction Pattern at Back Focal Plane$ $Darkfield Diffraction Pattern at Back Focal Plane$

These two images show the diffraction maxima at the back focal plane of the 10X objective when viewing a diffraction grating with a spacing of 1.9 micrometers. The first is using brightfield illumination. The "0" order maximum at the center provides no information on spacing of the grating. Only higher order maxima at the very edge of the field of view, blue and green in this example, indicate resolving power. The second is using darkfield illumination. Higher order diffraction maxima fill the back focal plane.

Darkfield Image of 1.9 micrometer Diffration Grating

These two images show the effect of the eyepiece magnification. The eyepiece magnification in the first image is sufficient to see the resolved grating. The eyepiece magnification in the second image is not sufficient for the camera to detect the resolved grating.

Brightfield vs Darkfield Illumination

$Diffraction Pattern at Back Focal Plane$

Brightfield Image of 1.9 micrometer Diffration Grating

$Darkfield Diffraction Pattern at Back Focal Plane$

Darkfield Image of 1.1 to 0.32 micrometer rulings

Brightfield vs Oblique Illumination

$Diffraction Pattern at Back Focal Plane$

Oblique Image of 1.9 micrometer Diffration Grating

$Diffraction Pattern at Back Focal Plane$

Phase Contrast vs Visibility Through the Microscope

Phase contrast microscopy makes "phase" objects more visible. Every object is a "phase" object strictly speaking but some objects are primarily phase objects, being nearly invisible using standard brightfield illumination. Phase constrast does not always improve visibility. Small objects with significantly different refractive indices than the medium in which they are mounted will be visible using darkfield illumination even when they are invisible using phase contrast. The "Phase Contrast Test Slide" is a good example, as shown below.

40X 0.65 NA Phase Contrast Image of Phase Contrast Test Slide

10X 0.25 NA Darkfield Image of Phase Contrast Test Slide

Polarized Light vs Visibility Through the Microscope

Objects with more than one refractive index can be made self-luminous by using crossed polarized light. This can create sufficient contrast to see the object.

Chrysotile Field 1 in 1.550 RI Liquid Viewed with Brightfield Illumination

Chrysotile Field 1 in 1.550 RI Liquid Viewed with Polarized Light

Asbestos Fibers and Visibility Through the Light Microscope

Much has been made of the need for transmission electron microscopy (TEM) "because the light microscope can't see asbestos fibers thinner than 0.25 micrometers". This quote is in error on many counts, but is a common misconception. First, it is not the microscope that "sees", but rather the human eye establishes the detection limit. If the light source is not "on" the resolution limit for the microscope has not changed but the eye won't see anything. Second, the ability to "see" (detect) an object is not dependent on resolution. This point has been made for the general case above but will be made again here in the specific case of asbestos fibers. Third, after over thirty years of diligent searching there has been no evidence that asbestos fibers shorter than five micrometers cause any health effect more detrimental than nuisance dust. Fresh crystalline silica is more detrimental in this respect. That challenges the "need" for an instrument that can "see" particles that are not apparently detrimental as a replacement for light microscopy. We will continue monitoring with the TEM because it is required in many governement and industrial regulations, but it is not a "superior" method in the case of hazardous asbestos detection or identification at sites of asbestos removal projects.

Cleared Filter Containing Amosite, Phase Contrast 40X Objective

10X 0.25 NA Darkfield Image of Cleared Filter Containing Amosite

Cleared Filter Containing Chrysotile, Phase Contrast 40X Objective

10X 0.25 NA Darkfield Image of Cleared Filter Containing Chrysotile

40X 0.65 NA Phase Contrast Image of HSE Groups 5, 6, and 7