Biophotonics_Concepts_to_Applications

• Dark field microscopyemploys oblique illumination to enhance contrast in
  specimens that are not imaged well when using brightfield illumination. In this
  observation method an opaque ring-shaped diaphragm with a small central
  opening is used together with the condenser lens to direct a hollow inverted cone
  of light onto the specimen at high azimuthal angles. Thus,first-order wave
  fronts do not directly enter the objective lens, which creates an artificial dark
  background in the microscope. The result is that the specimen features are seen
  as bright objects on a black background. Darkfield microscopy is a popular tool
  for biophotonics and medical investigations, such as viewing and imaging living
  bacteria, cells, and tissues.


• Phase contrast microscopy makes use of tiny refractive index differences
  between cells and their surrounding aqueous solutions and within the cells
  between the cytoplasm and the cell nucleus. This observation method is suitable
  for viewing very thin colorless and transparent unstained specimens (e.g., cul-
  ture cells on glass) and for live cells. Typically these specimens are approxi-
  mately 5– 10 μm thick in the central region, but less than 1μm thick at the
  periphery. Such specimens absorb extremely little light in the visible portion of
  the spectrum and they cannot be viewed with the human eye when using bright
  field and darkfield illumination methods. The phase contrast method translates

Table 8.1 Summary offive different microscopy observation methods

Microscopy

method

Features General applications

Brightfield

microscopy

• Common observation method


• Light illuminates the entirefield
  of view


• Cell staining adds color

Used to observe color and light

intensity information from a

stained specimen

Darkfield

microscopy

• Oblique illumination enhances
  contrast


• Transparent specimens seen as
  bright objects on a black
  background

Used for viewing and imaging

living bacteria, cells, and tissues

Phase contrast

microscopy

Uses tiny refractive index

differences between cells and their

surrounding aqueous solutions and

within cells

Suitable for viewing very thin

colorless and transparent unstained

specimens (e.g., culture cells on

glass) and for live cells

Differential

interference

contrast

microscopy

Uses plane-polarized light and

light-shearing prisms to exaggerate

minute differences in thickness

gradients and in refractive indices

of a specimen

Suitable for viewing very thin

colorless and transparent unstained

specimens and for live cells

Polarized light

microscopy

Uses crossed polarizing elements to

dramatically improve the quality of

an image obtained from

birefringent materials

Used for specimens such as plant

cell walls, starch granules, and

protein structures formed during

cell division

8.1 Concepts and Principles of Microscopy 239