Module 1- light microscopy

Question 1

what was the first created microscope

Answer 1

light microscope

Question 2

how do light microscopes work

Answer 2

they utilize lenses to bend light beams to focus and magnify images of the sample

Question 3

what causes the different magnifications on light microscopes

Answer 3

different lenses have different focal lengths and thus different magnifications

Question 4

how do modern LM increase there magnification ability

Answer 4

they combine two lenses in series to increase magnification ability. they combine the power of ocular lenses and the objective lends

Question 5

what is the most common type of light microscope

Answer 5

bright field

Question 6

how do bright field microscopes work

Answer 6

a light source shines through the sample and an image of the sample is made visible by its ability to bee detected as different from the clear background of the glass slide

Question 7

what is resolution

Answer 7

the ability to distinguish a point form a neighbouring point. poor resolution is when objects cant be distinguished form each other and blur together

Question 8

for light microscopes what is the physical resolution limit

Answer 8

it is dictated by the wavelength of light. for 0.2 micro meters

Question 9

what things does the ability of being able to see small things depend on

Answer 9

it is determined on the resolution of the system being used. magnification is required but is not the limiting factor

Question 10

what does immersion oil do

Answer 10

when light passes from one medium to another it can be “bent” this oil has the same refractive index as both slide and the lens to maximize capture of all light passing through the sample

Question 11

what is contrast

Answer 11

being able to see the specimen and distinguish it from the background

Question 12

what are ways scientists have been able to increase contrast of cells

Answer 12

by treating the sample with a stain of combination of stains

Question 13

what do slides treated with a stain need to do inorder to be successful

Answer 13

it needs to differentially bind to some components greater then others

Question 14

what is differential staining

Answer 14

various combinations of stains to specifically stain some types of cells or structures one color while staining cells or structures with different biological properties a separate color

Question 15

what is a downside to staining slides

Answer 15

it often kills the organism in the sample. this isnt ideal for application where a living cell is desired

Question 16

what is dark field microscopy

Answer 16

imaging on a dark field microscope results in a dark background

Question 17

how does the lighting work to achieve dark field microscopy

Answer 17

light shines at the side of the sample instead of through the bottom. the only light that reaches the viewer are the beams that hit an object on the side and been reflected up through the lens

Question 18

what is an alternative type of microscopy for staining

Answer 18

darkfield

Question 19

what principal does phase contrast microscopy take advantage of

Answer 19

-it take advantage of the fact that light traveling through different media travels speed dependent on the refractive index of that media

Question 20

how does phase-contrast microscopy work

Answer 20

-light passing through a living cell will thus be slightly delayed relative to a beam not passing through the cell
-it amplifies these differences so that light passing through an object will be offset sufficiently to cancel out light that did not pass through

Question 21

how does differential microscopy work (DIC)

Answer 21

-it uses two beams of plane polarized light arranged at right angles to each other
-a reference beam passing through an adjacent area of the slide is combined with that flowing through the sample and delayed beams cancel out as they are out of phase and form the contrast of the image
-light is gathered and passed through a set of prisms to maximize contrast and create and image

Question 22

what principal does florescent microscopy use

Answer 22

-that an object can be detected by its ability to emit light
-and the property that some materials can absorb energy form one source become excited and emit visible light of a certain wavelength

Question 23

what do useful compounds called fluorophores have the ability to do

Answer 23

to absorb energy form a provided light source (usually form a shorter WL) and emit visible light (usually form a lower WL)

Question 24

what kind of images does confocal microscopy make

Answer 24

it creates sharp, composite three dimensional images

Question 25

how does confocal microscopy work

Answer 25

-a computer guides and collects the image data and combines to create the view image -the scanning laser is guided to precisely illuminate discrete small points within the sample in a sequential scan of the sample allowing the computer to capture image info at precise points in the x and y axis and also in the vertical z axis -then it stacks the flat images to create 3d images

Question 26

how must microorganisms be in order to be flood and washed in the staining process

Answer 26

suck to the slide via fixation

Question 27

what are two general methods of fixation

Answer 27

heat and chemical fixation

Question 28

what is heat fixation often used for

Answer 28

with bacteria and archaea

Question 29

how is heat fixation done

Answer 29

first you place a drop of cells suspended in liquid on the slide. allow sample to air dry. them place over a bunsen burner. the heat form the flame will cook the sugar on the surface of the cells to the glass, premaritally attaching the cells

Question 30

what happens if you use to much heat on heat fixaition

Answer 30

it can destroy the structures

Question 31

how does chemical fixation work

Answer 31

it immobilizes cells by using chemical agents such as formaldehyde that promotes extensive cross linkage between biological molecules and the glass slide. these reagents also crosslink internal structures to preserve their structure during staining

Question 32

what is an example of things we may see chemical staining in

Answer 32

more complex samples such as human tissue

Question 33

what are staining of cells done with

Answer 33

dyes

Question 34

how do dyes work

Answer 34

selective use can highlight desired cellular features. they differentially bind to cells and/or structures and improve the contrast with the background

Question 35

how may one select dies based off of

Answer 35

if they want to highlight a positively charged cell structure they may choose a negatively charged dye to stick

Question 36

what is the use of dyes called

Answer 36

simple staining

Question 37

what is simple staining useful for

Answer 37

to simply visualize cells present for determining size, shape, and arrangement of bacterial and archaeal cells

Question 38

what is the most widely used differential stain

Answer 38

gram stain

Question 39

what is differential staining

Answer 39

the use of one or more stain. it allows for visualization and distinction of a mixture of organisms that stain differently with the dies used

Question 40

how does gram stain work

Answer 40

-it has the ability to differentiate between two different classes of bacteria, gram + and gram -, which stain differently based on the physiology of their outer surface -as the two classes of bacteria differ in their physiology and response to antibiotics and disease course infections. staining helps as an early step for determining treatments in med microbiology