Microscopes, Basic Cell Structure (ch.2, Lec. 2)
Monday, October 5, 2020 12:07 PM
• Microscopy thanks to Hooke and Leeuwenhoek. Microbes usually 10x smaller than a RBC, can be seen over a range of magnitudes.
• Prokaryotes (bacteria, archaea) are much smaller than eukaryotic microbes -> substructures too small to be resolved by light microscopy.
• Light microscopy (LM): shows the outline/shape of the microbe, but structures/surface detail cannot be seen.
• Ex. Bacillus: forms intricate structures to INC surface area to gain more access to O2.
• We can detect presence of objects whose size we cannot resolve. ->
• Resolution: smallest distance by which 2 objects can be separated and still be distinguished.
○ To resolve involves magnifying, having good contrast, and is limited by wavelength of radiation used. How small are the wavelengths compared to what you are trying to view?
○ Absorption: light's photon is acquired and heats up a dark surface.
○ Reflection: wavelength bouncing off the surface of the object.
○ Refraction: bending of light as it enters a surface that slows its speed.
○ ?Scattering: wave front interacts w/ an object that is smaller than it's wavelength.
• Bright field/compound microscope: magnification depends on objective lens and ocular (eyepiece) lens used.
• Why do we need to use oil for 100x lens? Immersion oil has a refraction index comparable to the lens (n=1.5). Minimizes loss of light rays by refraction, and makes it possible to reach 100x
magnification w/ minimal distortion.
• Ways to observe microbes:
○ Wet mount: place specimen in a drop of water on a slide w/ a coverslip. Good to observe cells in natural state, yet little contract b/w cell and background and can dry out quickly.
○ Simple stain: adds dark colors specifically to cells not the medium/surrounding tissue. Ex. Methylene blue.
○ Differential stain: stains one kind of cell, but not the other. Ex. Gram stain-- flood heat-fixed specimen w/ purple dye, add iodine soln, briefly add alcohol to decolorize them(gram-positive remain
purple), and usually add a counterstain to add pink hue to gram-negative stain.
• 2.5: Dark-field and Phase-Contrast Microscopy
• Dark-field microscopy allow microbes to be visualized as halos. Only allows small wavelength, scattered light to reach the objective lens. -> detected as a halo of light!
○ Illuminates substructures (ie flagella).
• The ONLY light that makes it into an objective lens is light scattered by a specimen.
• Phase-Contrast Microscopy (PCM): contrasts the different refractive indexes b/w the cytoplasm and surrounding medium. -> Used to view live cells and cellular organelles.
○ Specimen refracts/retards light from light source below, altering the light wave phase. -> Specimen appears dark in phase plate.
• Excitation Wavelength: specimen absorbs light of a specific wavelength.
• Emission Wavelength: specimen emits light at a longer wavelength.
○ Fluorescence microscopy uses color filters and requires a high-intensity light source.
○ Light source is put thru an excitation filter to isolate one color of light. -> Dichroic mirror directs excitation light thru objective lens. -> Comes into contact w/ specimen to form a green light. ->
Green light is directed back up thru the mirror, thru the emission filter and ocular lens to the camera.
• Electron microscopy: electrons behave like light waves (very high freq, allow very great resolution). Sample must absorb electrons -> are coated in heavy metal. Electron beam and sample are in a
vacuum, and lens are in magnetic fields.
○ Transmission electron microscopy (TEM): electrons are passing thru the specimens, revealing internal structures.
• Closely parallels design of the bright-field microscope, except uses an electron source and electron magnet focusing the electrons.
○ Scanning electron microscopy (SEM): electrons scans the surface of microbes in detail, revealing external structures in 3D.
• Heavy-metal coating allows electrons to interact w/ specimen surface.
• Most bacteria share fundamentals: thick complex outer envelope, compact genome, and tightly-coordinated cell functions.
• Archaea are prokaryotes: single cell organisms w/ no nucleus. Eukaryotes have nucleus and extensive membranous organelles.
• All cells composed of : water essential ions, small organic molecules, macromolecules. Yet composition varies w/ species, growth phase, and environmental conditions.
• Bacterial cells is 70% water weight, then 16% proteins, etc.
Monday, October 5, 2020 12:07 PM
• Microscopy thanks to Hooke and Leeuwenhoek. Microbes usually 10x smaller than a RBC, can be seen over a range of magnitudes.
• Prokaryotes (bacteria, archaea) are much smaller than eukaryotic microbes -> substructures too small to be resolved by light microscopy.
• Light microscopy (LM): shows the outline/shape of the microbe, but structures/surface detail cannot be seen.
• Ex. Bacillus: forms intricate structures to INC surface area to gain more access to O2.
• We can detect presence of objects whose size we cannot resolve. ->
• Resolution: smallest distance by which 2 objects can be separated and still be distinguished.
○ To resolve involves magnifying, having good contrast, and is limited by wavelength of radiation used. How small are the wavelengths compared to what you are trying to view?
○ Absorption: light's photon is acquired and heats up a dark surface.
○ Reflection: wavelength bouncing off the surface of the object.
○ Refraction: bending of light as it enters a surface that slows its speed.
○ ?Scattering: wave front interacts w/ an object that is smaller than it's wavelength.
• Bright field/compound microscope: magnification depends on objective lens and ocular (eyepiece) lens used.
• Why do we need to use oil for 100x lens? Immersion oil has a refraction index comparable to the lens (n=1.5). Minimizes loss of light rays by refraction, and makes it possible to reach 100x
magnification w/ minimal distortion.
• Ways to observe microbes:
○ Wet mount: place specimen in a drop of water on a slide w/ a coverslip. Good to observe cells in natural state, yet little contract b/w cell and background and can dry out quickly.
○ Simple stain: adds dark colors specifically to cells not the medium/surrounding tissue. Ex. Methylene blue.
○ Differential stain: stains one kind of cell, but not the other. Ex. Gram stain-- flood heat-fixed specimen w/ purple dye, add iodine soln, briefly add alcohol to decolorize them(gram-positive remain
purple), and usually add a counterstain to add pink hue to gram-negative stain.
• 2.5: Dark-field and Phase-Contrast Microscopy
• Dark-field microscopy allow microbes to be visualized as halos. Only allows small wavelength, scattered light to reach the objective lens. -> detected as a halo of light!
○ Illuminates substructures (ie flagella).
• The ONLY light that makes it into an objective lens is light scattered by a specimen.
• Phase-Contrast Microscopy (PCM): contrasts the different refractive indexes b/w the cytoplasm and surrounding medium. -> Used to view live cells and cellular organelles.
○ Specimen refracts/retards light from light source below, altering the light wave phase. -> Specimen appears dark in phase plate.
• Excitation Wavelength: specimen absorbs light of a specific wavelength.
• Emission Wavelength: specimen emits light at a longer wavelength.
○ Fluorescence microscopy uses color filters and requires a high-intensity light source.
○ Light source is put thru an excitation filter to isolate one color of light. -> Dichroic mirror directs excitation light thru objective lens. -> Comes into contact w/ specimen to form a green light. ->
Green light is directed back up thru the mirror, thru the emission filter and ocular lens to the camera.
• Electron microscopy: electrons behave like light waves (very high freq, allow very great resolution). Sample must absorb electrons -> are coated in heavy metal. Electron beam and sample are in a
vacuum, and lens are in magnetic fields.
○ Transmission electron microscopy (TEM): electrons are passing thru the specimens, revealing internal structures.
• Closely parallels design of the bright-field microscope, except uses an electron source and electron magnet focusing the electrons.
○ Scanning electron microscopy (SEM): electrons scans the surface of microbes in detail, revealing external structures in 3D.
• Heavy-metal coating allows electrons to interact w/ specimen surface.
• Most bacteria share fundamentals: thick complex outer envelope, compact genome, and tightly-coordinated cell functions.
• Archaea are prokaryotes: single cell organisms w/ no nucleus. Eukaryotes have nucleus and extensive membranous organelles.
• All cells composed of : water essential ions, small organic molecules, macromolecules. Yet composition varies w/ species, growth phase, and environmental conditions.
• Bacterial cells is 70% water weight, then 16% proteins, etc.
