2.1.1 microscopy

Question 1

how does a light microscope work?

Answer 1

they direct light through a sample on a glass slide
the image is focused using lenses + is visible through the eyepiece

Question 2

explain why the LM has two types of lenses?

Answer 2

the objective lenses will magnify the sample image and the eyepiece lenses magnifies it again
this allows for a higher overall magnification

Question 3

what is the magnification and resolution of the LM?

Answer 3

resolution is about 0.3 micrometers
magnification is about x1500
this has the worst mag and resolution

Question 4

what are the advantages of a LM?

Answer 4

you can view dead and living specimens
it’s good for viewing whole cells or organisms
you can see natural color
they are small and portable
easy use and simple preparation
cheapest equipment

Question 5

what are the disadvantages of a LM?

Answer 5

low magnification and resolution (wavelength of light)
cannot see internal structure

Question 6

how do you calibrate an eyepiece graticule?

Answer 6

1. line up graticule and stage micrometer while looking through eyepiece
2. count the no. of E.G divisions in 1 division of S.M
3. calc the ration {remember 1 S.M division = 1 micrometer}
4. calc how much one division on E.G is worth

Question 7

how do transmission electron microscopes (TEM) work?

Answer 7

electron beams are fired w/ an electron gun through the specimen
some electrons get absorbed; the more that get absorbed the darker it appears
electromagnets used to focus
views through fluorescent screen
done in a vacuum

Question 8

why must TEM specimens be in a vacuum?

Answer 8

to prevent diffraction of electron when they hit air particles
must be viewed on a copper grid (glass = diffraction)

Question 9

what is the mag + resolution of TEM?

Answer 9

they have the BEST mag and res.
you can see internal structure
due to electrons having a shorter wavelength so specimens can be closer before they diffract

Question 10

what are the advantage of TEM?

Answer 10

you can see internal structure
very high resolution and magnification
can be artificially colored

Question 11

what are the disadvantages of TEM?

Answer 11

the image is in 2D and black and white
cannot view live specimens (vacuum)
expensive equipment
need special skill to operate
not portable and heavy
extensive sample prep process

Question 12

how do scanning electron microscopes (SEM) work?

Answer 12

a beam of electrons is fired across the surface of the sample
the electrons scatter off
the different depths in the sample create 3D images

Question 13

what are the advantages of SEM?

Answer 13

can see 3D images
can view thicker specimens

Question 14

what are the disadvantages of SEM?

Answer 14

can only view dead specimens (vacuum)
heavy and not portable
expensive equipment
complex sample prep
samples are artificially colored

Question 15

what is the mag and resolution of the SEM?

Answer 15

they have the 2nd best mag and resolution
not as high as TEM

Question 16

how do you prepare slides for all 3 microscopes?

Answer 16

LM = can have wet and dry mount; squash and smear slides; use glass slide and coverslip; can be stained; extremely thin specimens needed
TEM = stained with heavy metals; set in resin and stained again
SEM = fractured and stained with heavy metals

Question 17

what are artifacts and how are they created?

Answer 17

visible structural detail caused by processing of a specimen and NOT a part of the specimen itself
can occur in light and electron microscopes
ie bubbles, loss of membrane continuity, empty space in cytoplasm

Question 18

how do laser scanning confocal microscopes (LSCM) work?

Answer 18

flood the specimen with a laser (powerful beam of light)
mirrors are used to focus it
the laser will illuminate fluorescent stains on spec allowing them to be seen

Question 19

what is the mag and resolution of a LSCM?

Answer 19

they have a high mag and res. due to the pinhole which blocks out of focus light
res. around 800nm
in between LM and EM

Question 20

what are the advantages of LSCM?

Answer 20

they have a high mag and res (pinhole)
can view thicker specimens
able to create 2D and 3D images (stacking)
can see dead and living specimens

Question 21

what are the disadvantages of LSCM?

Answer 21

laser can damage cells in living specimens
more expensive than LM
complex’s set up and sample prep
not portable
creating stacks is time consuming
special skill required

Question 22

how is staining used in light microscopes?

Answer 22

normally specimens are transparent so dyes absorb light and allow more visibility
dyes absorb some colors + reflect others
certain tissues absorb certain dyes

Question 23

which stains are commonly used in LM?

Answer 23

toluidine blue (blue cells)
phloroglucinol (pink/red cells)

Question 24

how is staining used in EM?

Answer 24

electrons have no color so stains are needed
heavy metal compounds absorb electrons well
makes specimen black and diff shades of grey

Question 25

which stains are commonly used for EM?

Answer 25

osmium tetroxide
ruthenium tetroxide

Question 26

how are stains used in LSCM?

Answer 26

use fluorescent dyes
they’re bound to monoclonal AB which bind to specific proteins
laser causes dye to fluoresce
allows precise staining

Question 27

which stains are used in LSCM?

Answer 27

green fluorescent protein (GFP)
emits green light
can be engineered to fluoresce diff colors allowing diff components to be studied together

Question 28

what is differential staining?

Answer 28

let’s you differentiate between 2 types of organisms/organelles in one sample
done by using multiple stains on one sample

Question 29

how does gran stain technique work?

Answer 29

applying crystal violet and iodine stain + wash with alcohol
gram + = retain purple stain
gram - = stain lost bc of thinner walls; counter safranin dye applied (red)
tells you about types of bacteria
gram + = susceptible to penicillin
gram - = walls too thin; not susceptible

Question 30

how does acid fast technique work?

Answer 30

differentiate diff types of mycobacterium
lipid solvent carry’s carbolfuchsin dye int cells; washed with dilute acid alcohol sol.
mycobac. = remain red
other bac. = lose stain; counter stain by methylene blue

Question 31

define magnification

Answer 31

how many times larger an image is than the object
magnification = image size / actual size
magnification = eyepiece mag. x objective mag.

Question 32

define resolution

Answer 32

the microscopes ability to distinguish two points on an image as separate objects
high resolution = closer objects can still be distinguished