Fig 002 Microscopes

Bright-field Microscopy

Bright-field microscopy is used with specimens stained with colored dyes.

A Microscope magnifies the image of an object so smaller details can be seen by the eye. It forms a magnified image by focusing light through a series of glass lenses on a tissue section.

• Condenser Lens: Focuses light from the illumination source onto the specimen
• Objective Lens: Collects light that passes through the specimen, enlarges and projects an image onto the eyepiece
  • 4x, 10x, 20x, 40x, 60x, and 100x are widely used
  • Inverts the image of the specimen
• Eyepiece lens: Further magnifies the image and projects it onto the retina
  • 10x eyepiece - most widely used
  • Inverts the image of the specimen

Total Magnification is calculated by multiplying the magnifications of the objective lens and the eyepiece. With a 100x objective lens and a 10x eyepiece, the total useful magnification is 1,000x.

Because both the objective lens and the eye piece invert the image, the final image seen through the eyepiece has the same orientation as in the specimen.

The light path in most bright-field microscopes is the opposite of what is shown here. The light source is beneath a stage that holds the specimen. Light passes upwards through the condenser, specimen, objective lens, and eyepiece.

Resolution

Resolution is the smallest distance between two objects that can be distinguished as separate objects. The human eye can resolve objects separated by a least 0.250 mm. A magnification of 1,000x would give a resolution of ~0.25 µm.

However, the actual resolution of an objective lens is determined by:

• Wavelength of Light (λ) that illuminates the specimen
  • Human eye is most sensitive to green light (~555 nm)
• Numerical Aperture (NA) of the objective lens: Measure of how well an objective can gather light
  • Higher NA produces higher resolution
  • Maximum NA is 1.0 with air between the objective and the specimen (i.e., refractive index of air)
  • Higher NA's require immersion oil between the objective and the specimen (i.e., refractive indexes between 1.0 and 1.5)

The maximum resolution of an objective can be calculated using the Abbe Equation (resolution = 0.5 λ / NA).

Lens	NA	Resolution
4x	0.10	2.8 µm
10x	0.25	1.1 µm
20x	0.50	555 nm
40x	0.75	370 nm
60x (oil)	1.20	230 nm
100x (oil)	1.20	230 nm

Observed resolution is usually poorer because of misaligned condenser lens, scattering by the specimen, aberrations in the objectives, and broad spectrum of colors used to stain specimens.