Microscopy

Question 1

brightfield microscopy components

Answer 1

magnification and resolution, specimen fixation/staining

Question 2

main components of brightfield microscopy

Answer 2

light source
condenser lens
stage
objective and ocular lenses
detector

Question 3

how is the image captured with brightfield microscopy

Answer 3

light diffracted by specimen and undiffracted light focused by objective lens - captured by video camera

Question 4

deconvolution

Answer 4

designed to remove background and out-of-focus light

Question 5

what is the primary purpose of microscopy

Answer 5

magnification - generate magnified high quality view of specimen

Question 6

what is the most important aspect of todays microscope

Answer 6

resolution

Question 7

what is resolution

Answer 7

minimum distance that can separate 2 points that still remain identifiable as separate points

Question 8

how to calculate overall magnification

Answer 8

objective lens x ocular lens

Question 9

what does the resolving power of microscope depend on (resolution)

Answer 9

wavelength of illumination light
numeral aperture

Question 10

what is numerical aperture

Answer 10

light gathering qualities of objective lensed specimen mounting medium

Question 11

how to maximize resolution

Answer 11

short wavelengths of illuminating light
increase NA

Question 12

how to calculate resolution distance in nm

Answer 12

(0.61 x wavelength)/NA

Question 13

what is the limit of resolution for most standard brightfield (and CLSM)

Answer 13

200nm

Question 14

limits of resolution for human eye

Answer 14

0.1mm

Question 15

what is the resolution limit for super resolution CLSM

Answer 15

20nm (5000X)

Question 16

what is the resolution limit for electron microscope

Answer 16

0.2nm (500,000X)

Question 17

why does electron microscope (EM) use electrons rather than photons

Answer 17

lesser wavelength yields higher resolution

Question 18

what is a major limitation of brightfield microscopy

Answer 18

specimens poor contrast

Question 19

what is usually done to specimen to increase contrast for brightfield microscopy

Answer 19

fixed
embedded
sectioned
stained

Question 20

formaldehyde fixation

Answer 20

cross-links amino group on adjacent proteins/nucleic acids

Question 21

what does the specimen usually get embedded in and why

Answer 21

plastic or wax for support

Question 22

what does the specimen get sectioned with

Answer 22

microtome (cuts specimen into sections)

Question 23

what does the specimen get stained with

Answer 23

molecule specific dyes

Question 24

what are the cons of fixation

Answer 24

results in cell death

Question 25

what are cons of embedding and sectioning

Answer 25

can lead to structural artifacts

Question 26

cons of staining

Answer 26

limited molecule-specific dyes can lead to poor contrast

Question 27

what is fluorescence microscopy

Answer 27

technique for visualizing fluorescent molecules in living or fixed specimens

Question 28

autofluorescence

Answer 28

endogenous fluorescence in specimen

Question 29

immunofluorescence

Answer 29

applied fluorescent dyes or dye-conjugated antibodies

Question 30

what does fluorescence microscopy rely on

Answer 30

autofluorescence immunofluorescence autofluorescent proteins

Question 31

pros of fluorescence microscopy

Answer 31

increased contrast and allows study of structures and (when not fixed) dynamic processes in living cells and in 3D

Question 32

cons of fluorescence microscopy

Answer 32

out-of-focus fluorescence from thick specimen results in blurred image

Question 33

what is an example of an autoflourescent protein

Answer 33

GFP - guanadenosine fluorescent protein

Question 34

what does confocal laser-scanning microscopy (CLSM) allow for

Answer 34

viewing dynamic biological/cellular processes live - specimen is usually living

Question 35

what does CLSM yield

Answer 35

individual 2D z-section (optical slice) of specimen that is less blurry than images from standard fluorescence microscopy

Question 36

what is done to the individual z-section focal planes

Answer 36

collected at different depths in sample and combined

Question 37

what does the combination of the Z-sections form

Answer 37

z-stack - generates 3D image

Question 38

limitations/cons to CLSM

Answer 38

rapid but cannot capture very dynamic cellular processes photobleach phototoxicity not efficient for thick specimens/tissues limited spatial resolution

Question 39

what is photobleach

Answer 39

when the point laser photobleaches fluorescent molecules, they are no longer fluorescent

Question 40

what is phototoxicity

Answer 40

excited fluorescent molecules react with molecular oxygen and produces free radicals which can damage/kill live cells

Question 41

is super-resolution CLSM better or worse than standard CLSM

Answer 41

10x better (20nm)