Microscopy

Question 0

3 things microscope must do:

Answer 0

1. Produce a magnifies image of the specimen
2. Separate or resolve the details in the image
3. Render the details visible to the eye

Question 1

What does a microscope do?

Answer 1

Enhances ability to see objects by making them bigger

Question 2

Magnification

Answer 2

• process of enlarging the physical appearance of something
• achieved by placing a biconvex lens into the light path
• light from object passes through lens and is bent (refracted) towards the eye
• objects seem bigger than they really are

Question 3

Magnification that has no increase in detail is called?

Answer 3

Empty magnification

Question 4

What is magnification a combination of?

Answer 4

All the lenses in the light path

Question 5

Resolution

Answer 5

Shortens the distance between 2 separate pints in the microscopes field of view that can still be distinguished as distinct entities

Question 6

What determines microscope resolution?

Answer 6

Representation of point spread function of single sources of light

Question 7

Microscope resolution of airy disks

Answer 7

Resolvable: 2 pings separated by more than the radii of airy disks

Not resolvable: 2 points separate by less than the radii of airy disks

Question 8

Wave length

Answer 8

Shorter lamda = better resolution

Question 9

Numerical aperture

Answer 9

Higher NA = better resolution

Question 10

Resolution equation

Answer 10

R = 0.61 lamda/ NA

Question 11

What is the resolving power of a light microscope under optimal conditions?

Answer 11

200 nm

Question 12

Increasing magnification means decrease of…?

Answer 12

FOV (field of view)

Question 13

2 types of contrast

Answer 13

1. Phase contrast

2. Differential interference contrast (DIC)

Question 14

Phase contrast

Answer 14

• changes in refractive index causing light rays to shift

- causes a halo around cell

Question 15

Differential interference contrast (DIC)

Answer 15

• light splits into 2 waves so light passes through different parts of sample
• gives a 3D Pseudo effect
• light and dark regions

Question 16

Benefits of using DIC and Phase contrast

Answer 16

• method used living cells and they can be examined at natural state
• no harm, staining, or killing is necessary

Question 17

DIC goods and bads

Answer 17

Goods

• gives 3D image, more detail
• important technique for imaging thick plants and animal tissues

Bads

• more expensive than phase contrast
• some plastics not suitable for DIC imaging

Question 18

Staining

Answer 18

Strain different cellular components different colors to allow for identification of parts

Question 19

Fluorescence Microscopy

Answer 19

Emission of light from a compound following adsorption of light of shorter wavelengths

• fluorescent proteins can be used to tag specific proteins in cells

Question 20

Fluorescent dyes

Answer 20

Can be attached to wide range of antibiotics or phalliodin to allow specific structures in the cell to be visualized by microscopy

Question 21

Epifluorescence microscopy (Epi)

Answer 21

• camera based technique
• fluorophores throughout sample are excited
• images contain interference from out of focus molecules
• good for fixed or live cell imaging and multicolor
• see structures throughout cell

Question 22

2 types of confocal microscopy

Answer 22

1. Spinning disc microscopy

2. Laser scanning confocal microscopy (LSCM)

Question 23

Spinning disc microscopy

Answer 23

• camera based
• fast and suitable for photo sensitive samples
• imaging highly dynamic processes

Question 24

Laser scanning confocal microscopy | LSCM

Answer 24

- point scanner | - slower

Question 25

Advantages of confocal microscopy

Answer 25

- ability to control depth - eliminates out of focus light using a pinhole - optically section though 3D reconstruction - image can be zoomed without loss of resolution

Question 26

Disadvantages of confocal microscopy

Answer 26

- sample still illuminated causing phototoxicity - slower than a camera - resolution limited to 200 nm

Question 27

Total internal reflection fluorescence (TIRF) microscopy

Answer 27

- uses camera to capture image(fast) - reduces phototoxicity - suitable for fixed and live cells - showed skeleton of cell - only see structure at adherent membrane

Question 28

Example applications of TIRF

Answer 28

- time lapse in endocytosis - focal adhesion dynamics - exocytosis

Question 29

Super resolution microscopy

Answer 29

- 2 objects seen as one in diffraction limited microscopy | - Nobel prize winning techniques

Question 30

Direct stochastic optical reconstruction microscopy (dSTORM)

Answer 30

- uses fluorescent antibodies to label proteins - used on dead samples - increased resolution by x10 - randomly switching on and off causing "blinks" and nm precision

Question 31

2 color dSTORM

Answer 31

- increased resolution allows finer detail | - proteins can be seen as distinct structures with no overlap

Question 32

Advantage of dSTORM over EM

Answer 32

Preparation is easy and multi color 3D images can be taken

Question 33

Electron Microscopy (EM)

Answer 33

- original super resolution microscopy - objects as small as 1 atom can be seen - uses a beam of electrons to illuminate sample as electrons have smaller wavelengths - electromagnetic cools used to focus electron beam instead of glass lens - can only be done on dead samples and preparation is complicated

Question 34

What microscope has the highest resolution?

Answer 34

- EM | - behave it uses a beam of electrons instead of light