Lecture 2 - Observing Microorganisms Flashcards
-Properties of electromagnetic radiation -Magnification, resolution, light microscopy -Differential staining techniques -Fluorescence and electron microscopy
List the scale of microscopy from largest to smallest optical resolution
Human eye > light microscopy > scanning electron microscopy > transmission electron micro > atomic force microscopy > x-ray crystallography
What is optical resolution?
Distance at which two objects can be distinguished
What is optical resolution dependent on?
- Contrast between object and surroundings
- The wavelength of the illuminating electromagnetic radiation (Abbe diffraction limit)
- Density of light detectors/photoreceptors
Why is resolution in retina limited by quality and density of photoreceptors?
- more acute vision = capture the different wavelengths of light, so more rods/cones capture more.
- want pack together bc even if one rod capture two wavelengths it just register as 1 pixel so if squeeze two rods so each can capture one wavelength and produce two pixels.
Detection is considered?
the ability to discern the presence of an object
What is the equation for electromagnetic radiation ? Of light?
E=hv & c= (lambda)v
Why can we observe visible light best?
thats the spectrum that reaches sea level the most
Magnification increases the _____ of an object (sometimes revealing more detailed info about object)
apparent size
What is absorption?
photon energy is captured and stored by object
Reflection ?
photon bounces off surface of object
Refraction?
speed and direction of photon is altered by the medium
Scattering?
deflect from straight path bc object dimensions is similar size to wavelength of incident light eg more bact = inc scatter
What do high refractive indexes cause?
change light direction
What can limit resolution?
- interference patterns caused by large wavelengths (lamba)
- refractive index (n)
- aperture of lens (sin(theta)) 90*
What is best theta for abbe diffraction limit?
90 bc sin 90 = 1 so allow most light to be received and reduce d.
Whats equation of abbe diffraction limit?
d=lamba/2nsin(theta)
What is considered the numerical aperture?
nsin0
Detection and resolution of cells can be enhanced by ____ & _____
staining & fixation
Staining works bc
low energy conjugated bonds allow view in visible spectrum
Fixation works bc
cells adhere to slide
Whats the difference between simple stains and differential stains?
Simple stains moreso to just view and differential stains use to differentiate eg gram - and gram +
Stains are typically ____ charge
positive
What are the three differential staining methods?
Acid-fast stains - stain Mycobacterium
Spore stain- stain endospores of Bacillus and Clostridium
Negative stain - adds contrast to media to make cells and other structures (capsules) more visible
What is key about incident light in fluorescence microscopy?
incident light is absorbed at shorter wavelength but emitted at longer wavelengths by fluorophore.
What use for fluorescent microscopy?
- can observe processes in real time eg FtsZ GFP constriction
- can show how toxin/drug can affect different bacterial chromosomes eg GFP bacteria release toxin to target (dsRed) bacteria and note in DAPI (fluoresces DNA) if target survived by presence of chromosomes.
What does DAPI illuminate?
DNA
How does electron microscopy work?
e- with its wavelike properties as 1nm wave that can be absorbed by heavy metal stains and focused by magnetic fields rather than glass lens
What are the two types of electron microscopy?
transmission- look at internal structures in 2D, e- pass through specimen
scanning - look at external structure in 3D, must be coated with electrically conductive material
