Microscopy

Question 1

2 types of microscopes

Answer 1

1. light

2. electron

Question 2

magnification of a light microscope

Answer 2

~ 1000X

Question 3

magnification of an electron microscope

Answer 3

20 000X for SEM and 100 000X for TEM

Question 4

illumination of light microscope

Answer 4

visible or UV light

Question 5

illumination of electron microscope

Answer 5

beam of electrons

Question 6

lenses on light microscope

Answer 6

glass or quarts (for UV)

Question 7

lenses on electron microscope

Answer 7

electromagnets

Question 8

detection for light microscope

Answer 8

eye or light sensor

Question 9

detection for electron microscope

Answer 9

monitor or light sensor

Question 10

7 types of light microscopy

Answer 10

1. brightfield
2. phase contrast
3. differential interference contrast
4. widefield fluorescence
5. composite images
6. confocal fluorescence
7. linescans

Question 11

brightfield

Answer 11

light passes through specimen

Question 12

example of brightfield microscopy

Answer 12

compound microscope

Question 13

con of brightfield

Answer 13

stains often needed, therefore specimen must be killed

Question 14

phase contrast

Answer 14

light passes through specimen, complex lenses make lights lighter and darks darker

Question 15

why is phase contrast better than brightfield

Answer 15

no stain required

Question 16

differential interference contrast (DIC)

Answer 16

light passes through specimen, complex lenses make darks darker and lights lighter

Question 17

difference between DIC and phase contrast images

Answer 17

phase contrast = small halos

DIC = artificial shadows

Question 18

widefield fluorescence

Answer 18

short wavelenght light excites fluorescent molecules and long wavelength light is emitted and detected

Question 19

3 stains for widefield fluorescence

Answer 19

1. DAPI
2. fluorescein
3. rhodamine

Question 20

composite images

Answer 20

2 or more monochromatic images given artificial colors and superimposed

Question 21

who are composite images helpful for

Answer 21

people with red/green color blindness

Question 22

confocal fluorescence

Answer 22

uses lasers to deliver excitation light to a single point at a time, revealing a 2D plane of a 3D specimen

Question 23

linescans

Answer 23

graph showing a change of intensity of a specific color, along a line through an image

Question 24

2 steps of preparing a specimen

Answer 24

1. getting the specimen on the slide

2. adding stains, probes, or antibodies

Question 25

how do you put whole cells onto a slide

Answer 25

put cell culture on a slide and cover with a coverslip

Question 26

how do you put sections of cells or small organisms on a slide

Answer 26

immobilize in wax or plastic and section with a microtome

Question 27

how do you put large molecules on a slide

Answer 27

put isolated molecules on slide

Question 28

4 types of identifiers

Answer 28

1. regular stains
2. fluorescent stains
3. DNA probes
4. antibodies

Question 29

orcein

Answer 29

a regular stain with an affinity for DNA

Question 30

DAPI

Answer 30

fluorescent stain with an affinity for DNA

Question 31

DNA probe

Answer 31

single stranded DNA with an affinity to a target DNA molecule, attached to a fluorescent molecule

Question 32

antibodies

Answer 32

small proteins made by WBCs with affinity for target molecules

Question 33

2 types of fluorescent antibody methods

Answer 33

1. direct immunofluorescence

2. indirect immunofluorescence

Question 34

direct immunofluorescence

Answer 34

antibodies with fluorescent dye bind to protein

Question 35

indirect immunofluorescence

Answer 35

antibodies bind to a protein and then secondary antibodies with dye bind to the primary antibodies

Question 36

which method of immunofluorescence is more common and why

Answer 36

indirect because:

1. each primary antibody is binded to several secondary ones, increasing fluorescent signal
2. cheaper to buy secondary antibodies

Question 37

how do you make antibodies against a protein?

Answer 37

1. inject foreign protein in animal

2. isolate antibodies from blood of animal (~3 months later)

Question 38

isolating antibodies from blood

Answer 38

1. add blood to a column with the protein in it
2. mild conditions antibody will bind to protein
3. harsh conditions to unbind it after other contents are removed

Question 39

where are primary antibodies more frequently produced

Answer 39

rabits

Question 40

where are secondary antibodies more frequently produced

Answer 40

goats

Question 41

difference between DNA probes and antibodies

Answer 41

DNA probes show DNA

antibodies show proteins