Lecture 2 - Microscopy

Question 1

What is used to illuminate specimen in a microscope?

Answer 1

condenser

Question 2

What is used to detect light in a microscope?

Answer 2

objective

Question 3

What is magnification?

Answer 3

the extent to which the image of the specimen (viewed under microscope) is enlarged

Question 4

What happens if specimen is placed in the path of light/electron beam? (beach ball altering the motion of the rope)

Answer 4

specimen changes the physical characteristics of a beam to create an image for human eye/photographic detector

Question 5

Why does phase shift happen?

Answer 5

due to difference in refractive index

phase shift is undetected by eye; but can be converted to amplitude shift in phase-contrast miscroscope

Question 6

Why does amplitude change (change in brightness) happen?

Answer 6

due to light absorbance (for unstained biological specimen - minor)

Question 7

What is refractive index?

Answer 7

measure of change in the velocity (quickness of motion) of light as it passes from one medium (any material substance which can propagate waves or energy) to another

n=c/v
ratio of speed of light in vacuum (c) to the speed of light in that medium (v)

Question 8

What is diffraction?

Answer 8

bending of a wave around the edges of an opening or an obstacle

the image you see when you look at a specimen through a series of lenses is really just a pattern of either additive or canceling interference of the waves that went through the lenses, a phenomenon known as diffraction.

Question 9

What are the 3 elements always needed to form an image?

Answer 9

1. source of illumination
2. specimen
3. system of lenses

Question 10

Why very small objects can be seen only by electron microscopy?

Answer 10

the wavelengths of electrons are very much shorter than those of photons. Thus, objects such as viruses and ribosomes are too small to perturb the waveform of photons, but they can read-ily interact with electrons.

Question 11

What is interference?

Answer 11

the process by which two or more waves combine to reinforce or cancel one another, pro-ducing a wave equal to the sum of the two combining waves

Question 12

What is focal length?

Answer 12

the distance between the mid-line of the lens and the point at which rays passing through the lens converge to a focus

The focal length is determined by the index of refraction of the lens itself, the me-dium in which it is immersed, and the geometry of the lens

Question 13

What is angular aperture?

Answer 13

the half-angle α of the cone of light entering the objective lens of the microscope from the specimen

a measure of how much of the illumination that leaves the specimen actually passes through the lens

Question 14

What is resolution?

Answer 14

the minimum distance two points can be apart and yet still remain identifiable as separate points when viewed through the microscope: the higher the resolution, the smaller the objects that can be distinguished using a particular lens

Question 15

factors affecting resolution:

Answer 15

light properties (the wavelength of the light used to illuminate the specimen)
lens properties (the angular aperture, aberrations)
the refractive index of the medium surrounding the specimen

Question 16

Why some microscope lenses are designed to be used with a layer of immersion oil between the lens and the specimen?

Answer 16

Immersion oil has a higher refractive index than air and therefore allows the lens to receive more of the light transmitted through the specimen.

r=200nm with an oil immersion lens
r=300nm in air

Question 17

How does airy disc appear?

Answer 17

as a bright point of light around which concentric ringes/ripples are seen

Question 18

What determines the diffraction pattern? (airy disc size)

Answer 18

wavelength of light and the size of aperture (circular opening) through which the light passes

Question 19

light waves of the same wavelength and frequency arriving simultaneously at the same point

Answer 19

interfere with one another

Question 20

light waves in phase combine

Answer 20

constructive interference - amplitude increases - perceived brightness increases

Question 21

light waves out of phase

Answer 21

cancel each other partly - destructive interference - amplitude decreases - brightness decreases

Question 22

3 characteristics of Brightfield microscopy

Answer 22

1. transparency of most of biological specimens
2. need to use dyes
3. need to fix (chemically preserve) and stain => dead samples

Question 23

How to observe living cells directly?

Answer 23

1. Phase-contrast microscopy
2. Differential interference contrast microscopy
3. Fluorescence microscopy

Question 24

How does phase-contrast microscopy work?

Answer 24

converts phase difference into brightness difference

Question 25

How does differential interference contrast (DIC) microscopy work?

Answer 25

polarized light is used

Question 26

How does fluorescent dye work?

Answer 26

the absorption of light by a mol-ecule and ends with emission of light with a longer wavelength

Question 27

What is fluorescent probe?

Answer 27

molecule capable of emitting fluorescent light to indicate the presence of a specific molecule/ion

used for immunostaining

Question 28

What is immunostaining?

Answer 28

technique based on ability of antibodies to recognize and bind to specific molecules

Question 29

How does indirect immunofluorescence happen?

Answer 29

a tissue or cell is treated with an antibody (primary) that is not labeled with dye; this antibody attaches to specific antogenic sites within tissue/cell; then the secondary antobody (labeled with a fluorescent dye) is added

Question 30

The difference of confocal miscroscopy

Answer 30

light miscroscope that employs a laser beam minimizing blurring excluding out-of-focus light from an image of a single plane

before the detector - second pinhole - at a position where rays from illuminated point come to focus

Question 31

How does stimulated emission depletion (STED) work?

Answer 31

uses very short pulses of laser light to cause molecules in a specimen to fluorescence.
The 1st pulse -> ring-shaped depletion pulse (moves electrons from the excited state to a lower energy state before fluorescencing, thus reducing the amount of spontaneous fluorescent emission)

Question 32

What is the difference between PALM and STORM?

Answer 32

PALM uses photo switchable/convertible fluorescent proteins (FPs), whereas STORM uses organic dyes as fluorescent probes for imaging.

Question 33

How does STORM microscopy work?

Answer 33

single fluorophores are being imaged (when it is important to count the exact number of molecules in a given structure)

a target structure is activated by a subset of probes (fluorescent labels). After enough fluorophors have been localized, high-resolution image occures

Question 34

What is the practical resolution limit of electron microscopy?

Answer 34

0.1 nm