Lecture 2 - Observing Microorganisms

Question 1

List the scale of microscopy from largest to smallest optical resolution

Answer 1

Human eye > light microscopy > scanning electron microscopy > transmission electron micro > atomic force microscopy > x-ray crystallography

Question 2

What is optical resolution?

Answer 2

Distance at which two objects can be distinguished

Question 3

What is optical resolution dependent on?

Answer 3

1. Contrast between object and surroundings
2. The wavelength of the illuminating electromagnetic radiation (Abbe diffraction limit)
3. Density of light detectors/photoreceptors

Question 4

Why is resolution in retina limited by quality and density of photoreceptors?

Answer 4

• 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.

Question 5

Detection is considered?

Answer 5

the ability to discern the presence of an object

Question 6

What is the equation for electromagnetic radiation ? Of light?

Answer 6

E=hv & c= (lambda)v

Question 7

Why can we observe visible light best?

Answer 7

thats the spectrum that reaches sea level the most

Question 8

Magnification increases the _____ of an object (sometimes revealing more detailed info about object)

Answer 8

apparent size

Question 9

What is absorption?

Answer 9

photon energy is captured and stored by object

Question 10

Reflection ?

Answer 10

photon bounces off surface of object

Question 11

Refraction?

Answer 11

speed and direction of photon is altered by the medium

Question 12

Scattering?

Answer 12

deflect from straight path bc object dimensions is similar size to wavelength of incident light eg more bact = inc scatter

Question 13

What do high refractive indexes cause?

Answer 13

change light direction

Question 14

What can limit resolution?

Answer 14

• interference patterns caused by large wavelengths (lamba)
• refractive index (n)
• aperture of lens (sin(theta)) 90*

Question 15

What is best theta for abbe diffraction limit?

Answer 15

90 bc sin 90 = 1 so allow most light to be received and reduce d.

Question 16

Whats equation of abbe diffraction limit?

Answer 16

d=lamba/2nsin(theta)

Question 17

What is considered the numerical aperture?

Answer 17

nsin0

Question 18

Detection and resolution of cells can be enhanced by ____ & _____

Answer 18

staining & fixation

Question 19

Staining works bc

Answer 19

low energy conjugated bonds allow view in visible spectrum

Question 20

Fixation works bc

Answer 20

cells adhere to slide

Question 21

Whats the difference between simple stains and differential stains?

Answer 21

Simple stains moreso to just view and differential stains use to differentiate eg gram - and gram +

Question 22

Stains are typically ____ charge

Answer 22

positive

Question 23

What are the three differential staining methods?

Answer 23

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

Question 24

What is key about incident light in fluorescence microscopy?

Answer 24

incident light is absorbed at shorter wavelength but emitted at longer wavelengths by fluorophore.

Question 25

What use for fluorescent microscopy?

Answer 25

- 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.

Question 26

What does DAPI illuminate?

Answer 26

DNA

Question 27

How does electron microscopy work?

Answer 27

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

Question 28

What are the two types of electron microscopy?

Answer 28

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