Notes from the entirety of Chapter 9 from the textbook 'Molecular Biology of the Cell' Sixth edition
About microscopy, including light microscopy, electron microscopy, dark-field microscopy, confocal microscopy, phase-contrast microscopes, differential-interference-contrast microscopes, and imager...
● The objective lens collects a cone of light rays to create an image
● The condenser lens focuses a cone of light rays onto each point of the specimen
● Resolution: the resolving power of the microscope depends on the width of the cone
of illumination and therefore on both the condenser and the objective lens
● Numerical aperture: the higher the numerical aperture the greater the resolution
and the brighter the image
Light Microscope
● Light microscope can resolve details 0.2μm apart
● Bacteria and mitochondria, which are about 0.5μm wide, are typically the smallest
structures that can be seen using a light microscope
● Light waves travel through an optical system by a variety of slightly different routes,
do that they interfere with each other and cause optical diffraction effects
● Two trains of waves travelling two different paths which reach the same point and are
precisely in phase will reinforce each other and increase brightness
● Two trains of waves that are out of phase will interfere with each other and cancel
each other either partly or entirely
● The limit of resolution depends on both the wavelength of the light and the
numerical opening of the lens system used
● The numerical aperture affects light-gathering ability of the lens and is related to both
the angle of the cone of light that can enter it, and the refractive index of the medium
the lens is operating in
● Refractive index is the ratio of the speed of light in a vacuum to the speed of light in
a particular transparent medium
● Although possible to enlarge an image, it is not possible using a conventional light
microscope to resolve two objects in the light microscope that are separated by less
than 0.2μm - they will appear as a single object
Phase-Contrast Microscope/Differential-Interference-Contrast Microscope
● Many ways for contrast to be generated in a specimen - staining a specimen creates
contrast through colour (possibility that some cell components are lost/distorted
during specimen preparation)
● Dark-field microscopy - light rays can be scattered in all directions from objects in
their path. If oblique lighting is arranged and does not enter the objective directly,
focused but unstained components of a cell can scatter the rays, which then create a
bright image against a black background
● When light passes through a living cell, the refractive index of the cell changes the
wavelength of the light (e.g. a thicker part of the cell, like the nucleus, slows the light
passing through it). The phase of light is shifted relative to the light that has passed
through a thinner area of the cell
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