Microscopy

Question 1

briefly describe optics and condenser lenses

Answer 1

-condenser lenses collect and refract light so it converges at a focal point (this is where specimen sits)

-the light passes thru the specimen and creates a larger upside down image
-the microscope converts this via an additional lenses to make a right side up image

Question 2

magnification vs resolution

Answer 2

magnification = an image is made larger

resolution = the extent to which two objects that are close together can be distinguished

Question 3

can an object be high resolution but low mag? what about the other way around?

Answer 3

yes high res and low mag

not really no low res and high mag as wont have usable images

Question 4

bright field light microscopy (5)

Answer 4

• most common
• known as compound microscope
• mag = 1000x resolution = 0.2 um
• may need staining to see
• specimens appear darker/colored against a bright background

Question 5

dark field light microscopy (5)

Answer 5

• light does not enter the object directly
• only light reflected by the specimen is transmitted thru the microscope
• specimen appears light against a dark background
• can be observed without staining
• can see live cells/motility

Question 6

state the 3 types of stains and what must happen to them before the stain

Answer 6

• cells are fixed before staining (usually kills them) to ensure they don’t move around the slide

1. simple
2. negative
3. differential

Question 7

simple stain

Answer 7

cells or structures are stained by a single dye

Question 8

negative stain

Answer 8

cells or structures repel a single dye, staining the background

Question 9

differential stain

Answer 9

cell or structures can be distinguished based on their reaction to the stain

Question 10

describe florescence microscopy

Answer 10

specimens are seen because they emit light (at a higher/longer wavelength)
~uses fluorochromes/phores

• they excite and omit at 2 DIFFERENT wavelengths

Question 11

what does the microscope have to do in florescence microscopy

Answer 11

needs to produce light at the excitation wavelength and detect light at the emission wavelength

Question 12

epiflourescence microscopy

Answer 12

light is emitted by the specimen NOT transmitted through it

Question 13

natural autofluorescence

Answer 13

some cells fluoresce naturally

Question 14

fluorescent dyes

Answer 14

some cells or structures fluoresce when the cell is stained

Question 15

fluorescent tags & 2 examples

Answer 15

attached to cell components

ex.
-antibodies help to bind (immunofluorescence)
-gene fusion proteins

Question 16

vital dye

Answer 16

distinguishes living cells from dead cells

• calcein AM = can pass thru living & dead cells & will go green
• propidium iodide = only pass thru damaged/dead cells & will go red

Question 17

immunofluorescence

Answer 17

uses antibodies + fluorochrome to attach to cell surface illuminating it / then use fluorescent microscopy

Question 18

confocal microscopy

Answer 18

scans thru different planes of focus in the specimen to construct a 3D image from multiple 2D images

-non focused light is blocked out (less damaging to specimen) & can be used on live specimens
-used in combo with fluorescent tagging (live) or dyes (dead)

Question 19

3 types of electron microscopes and what are EMs

Answer 19

EMs use electrons to visualize an image

1. transmission electron microscope (TEM)
2. scanning electron microscope (SEM)
3. cryo-electron microscope (cryo-EM)

Question 20

resolution of an electron microscope and why is it better than light

Answer 20

limit of 0.1-0.2nm
unlike light which is 200nm

Question 21

ultrastructure

Answer 21

structures that are too small to see with a light microscope (less than 200nm)

Question 22

TEM (5)

Answer 22

transmission EM

• must be fixed
• sliced extremely thin
• stained with heavy metals & so kills cells
• electrons pass THRU specimen
• under vacuum

Question 23

how is TEM better than a light microscope / comparison

Answer 23

TEM:
100,000x mag
less than 0.5nm res

Question 24

how does a TEM image appear in regard to regions

Answer 24

electrons pass thru less dense regions = areas appear brighter

dense regions scatter more electrons = appear darker in image

Question 25

shadowing

Answer 25

adds a shadow to specimen by adding heavy metals at an angle

Question 26

freeze etching

Answer 26

rapidly chills and shatter cells forming jagged edges which show partial 3D structure

Question 27

immunolabelling

Answer 27

heavy metals (ex.gold) are bound to antibodies specific to a protein of interest

Question 28

SEM (6)

Answer 28

scanning EM - fixed - coated with heavy metals - scanned over surface of specimen - mag 10,000x 5nm res - easy 3D whole image is obtained - no slices need

Question 29

why might it be good to visualize the outside surface of a virus?

Answer 29

to see surface proteins / detection of virus particles

Question 30

are EM images produced in color ?

Answer 30

NO all start out grey but color can be added

Question 31

cryo EM

Answer 31

- uses a combo of rapid cooling & packing proteins tightly into a sample to allow TEM to visualize individual 3D protein structures

Question 32

what would i use to view a live specimen (2)

Answer 32

- dark field - fluorescence

Question 33

what would i use if i want a 2D image (3)

Answer 33

- bright/ dark field - epiflourescence - TEM

Question 34

what would i use if i want a 3D image (4)

Answer 34

- confocal - TEM with shadowing or freeze etching - SEM - cryo EM

Question 35

Is the thing I want to look at hard to distinguish (e.g. a protein of interest)? (2) what could you use

Answer 35

- fluorescence tagging -immunogold labelling