Topic 6: Use and Care of the Microscope

• Microscope was invented shortly before 1600 by Zacharias Janssen.
  • Used to examine microorganisms in 1680s by Antoni van Leeuwenhoek
• Simple microscopes: early microscopes that consisted of biconvex lenses (essentially magnifying glasses)
• In order to see microbes, a compound microscope is used.
  • Has two lenses between the eyes and the object
• Brightfield compound microscope: shows dark objects in a bright field.
• Basic frame of the microscope consists of a base, a stage to hold the slide, an arm for carrying the microscope, and a body tube for transmitting the magnified image.
• Condenser: consists of several lenses that concentrate light on the slide by focusing it into a cone; contains iris diaphragm
• Iris diaphragm: controls the angle and size of the cone of light - ensures that optimal light will reach the slide
• Two types of lenses → objective and ocular
• Monocular microscope: has only one ocular lens
• Binocular microscope: has two ocular lenses
• Course adjustment: used for focusing with low-power objectives
• Fine adjustment: used for focusing with the high-power and oil immersion lenses
• Field of vision: area seen through a microscope
• Magnification of microscope depends on the type of objective lens used with the ocular lens
  • Total magnification of object is calculated by multiplying the magnification of ocular by objective lens
• Resolution, or resolving power: ability of lenses to reveal fine detail or two points distinctly separated
  • function of the wavelength of light used and a characteristic of the lens system called numerical aperture
  • resolving power = wavelength of light used / (2 X numerical aperture)
  • small wavelengths increase resolving power
• Electron microscopes use electrons as the source of “light”
  • electrons have a short wavelength therefore excellent resolving power
  • light microscope has ~200 nm resolving power
  • electron microscope has ~0.2 nm resolving power
• Increase of numerical aperture increases resolving power
• Numerical aperture is dependant on maximum angle of the light entering the objective lens and on the refractive index of the material between the objective lens and the slide.
  • Refractive index: the amount of light bends
  • NA = N sin θ
• Light is refracted when it emerges from the slide because of the change in media as the light passes from glass to air.
  • For immersion oil, light ray continues without refraction because immersion oil has same refractive index as glass
• Focal point: light rays bent to converge, where image is formed
  • multiple focal points are a result of curvature of the lens → called spherical abberration
• Spherical abberation can be minimized by use of iris diaphragm - eliminates light rays to the periphery of the lens or by a series of lenses resulting in essentially a flat optical system
• Chromatic abberration: multitude of colors in field
  • caused by prismlike effect of the lens as various wavelengths of white light pass through to a different focal point for each wavelength.
  • can be minimized by the use of filters (like blue) → called achromatic lenses
  • can be minimized by lenses corrected for red, blue, and other wavelengths → called apochromatic lenses
  • can be minimized by using a light source of one wavelength (monochromatic light)
• Compound microscopes require a light source
• Intensity of light can be adjusted with a rheostat

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