M3: Microscopy

Question 1

Micrometer (Mm)

Answer 1

one millionth of meter (10^-6 m)

Question 2

nanometer (mm)

Answer 2

one billionth of meter (10^-9 m)

Question 3

resolution

Answer 3

• distance between two objects at which they can still be seen as separate
• objects that are closer to each other will require a higher resolution to be seen as separate

Question 4

contrast

Answer 4

• difference in light absorbance between 2 objects

- lower contrast –> harder to see object

Question 5

brightfield microscopes overview

Answer 5

• simplest kind of light microscope

- can only reach resolutions of up to 0.3 Mm –> can’t visualize viruses

Question 6

how brightfield microscopes work (5 steps)

Answer 6

1. light (halogen bulb usually) enters microscope from base
2. mirrors reflect light towards sample
3. before light reaches sample, it passes through condenser (converges light beams into focused area on sample)
4. once light passes through sample, iris diaphragm controls how much of that light enters the objective lens (closest lens to sample, highest amount of magnification)
5. light passes through objective lens –> ocular lens (eyepiece) –> eye

Question 7

total magnification

Answer 7

magnification of objective lens x magnification of ocular lens

Question 8

typical magnification of ocular lens

Answer 8

10x

Question 9

phase contrast microscope overview

Answer 9

• uses special condensers and objectives to amplify small differences between cell and background –> can obtain detailed images of specimens without needing to stain samples (so can visualize movement)
• sometimes will dye specific parts of a cell to highlight something

Question 10

dark field microscope overview

Answer 10

• greatly increases contrast, resulting in a dark background and bright objects as light reflects off of specimen
• can’t see inside cells using this method
• often used to find lyme disease pathogen

Question 11

fluorescence microscope overview

Answer 11

• uses fluorophores to see cells on a dark background

- how it works: emits wavelengths to excite fluorophores into producing different colors

Question 12

fluorophores

Answer 12

• fluorescent molecules

- colors most used = green (GFP), yellow (YFP) , and red (RFP)

Question 13

uses of fluorophores in microscopy (3)

Answer 13

1. illuminate cells as a whole
2. couple with normal cellular protein to visualize protein movement and localization
3. tag molecules or antibodies to ID the presence (or absence) of particular proteins

Question 14

confocal microscope overview

Answer 14

• aka laser-scanning microscope
• uses lasers to focus on single place within an object, which increases accuracy of the visualization
• then compiles these images into a single 2D or 3D image

Question 15

electron microscopes overview

Answer 15

• use electron beams instead of light beams
• electron beams have shorter wave lengths –> increased resolution capacity ( >1nm) –> used to visualize super small specimens
• labor intensive process, samples must be fixed
• 3 types: TEM, SEM, STEHM

Question 16

TEM

Answer 16

• aka transmission electron microscope
• thin slices of sample are coated in preservatives and heavily treated, then placed between electron beam source and detector
• image formed from electrons interacting as pass through sample
• can use to visualize subcellular organelles, viral particles, substructures

Question 17

SEM

Answer 17

• sample coated in gold or palladium

- image formed from electrons bouncing off surface of specimen –> image = 3D shell of specimen

Question 18

STEHM

Answer 18

• uses electron beam and holography technique
• can magnify subatomic structures 20 million x naked eye, resolution up to 35pm (10^-12m)
• used to study surfaces of proteins and subcellular structures

Question 19

microscopes to look inside specimen (5)

Answer 19

1. brightfield
2. phase contrast
3. confocal
4. TEM
5. STEHM