Fluorescence Microscopy

Question 1

What is fluorescence?

Answer 1

Fluorescence = property of some atoms or molecules to absorb light as a wavelength and subsequently emit light of longer wavelengths (stokes shift) after a brief time interval (fluorescence lifetime)

Is based on the ideas of excitation and emission

(Stokes shift basically = excitation has higher greater energy than emission because some energy is lost as heat)

Question 2

Fundamentals of excitation and emission

Answer 2

Excitation light is of a lower wavelength (higher energy)
Cells/targets/markers absorb photons of excitation light → electrons become excited

Going from excited back to ground state means some energy is lost in the form of emission

Question 3

Emitted light is always of a ______ wavelength than excitation light

Answer 3

always of a LONGER wavelength than excitation light
ex: might absorb blue or violet and emit green

From lowest to highest energy: ROYGBIV
(The smaller the wavelength, the greater the frequency, the greater the energy)

Question 4

Fluorescence microscopy allows us to

Answer 4

• examine morphological structure over time
• observe protein expression (i.e. by tagging w/ GFP)
• study cellular and subcellular function without disturbing membrane
• obtain information about the kinetics and spatial distribution of cell
• optically record from individual synapses

Question 5

What kind(s) of imaging can you do with fluorescence microscopy?

Answer 5

static and dynamic imaging

static: fluorescent antibodies against subcellular structures
dynamic: measure cellular dynamics i.e. by dye loading with patch pipette

Question 6

Basics of IHC

Answer 6

Primary antibody binds antigen
Secondary antibody binds primary antibody
Secondary antibody is labeled with fluorochrome

Question 7

GFP

Answer 7

• bimodal absorption

- absorbs blue/violet light (395 and 475 nm) and emits green (509 nm)

Question 8

Tagging proteins with GFP (how to and why)

Answer 8

• Inject/insert GFP before stop codon for protein

- Can be used for cell lineage/gene expression marking, protein tagging, and protein-protein interaction monitoring

Question 9

Brainbow

Answer 9

Uses stochastic and combinatory expression of a few spectrally distinct fluorescent proteins

• Expresses fluorescent proteins in different ratios within each cell
• Three separate FPs are arranged sequentially in transgene along with a pair of lox sites (i.e. loxP-loxP, lox2272-lox2272)

Question 10

Classes of fluorescent indicators

Answer 10

• antibodies (labeled w/ fluorophore)
• GFP
• Ion/environment-sensitive indicator dyes

Question 11

What is the basic principle of epifluorescence? How many filters are there?

Answer 11

Epifluorescence principle = how light flows through a microscope

Excitation photons flow from light source through excitation filter (short-pass) to dichroic mirror → reflected from dichroic mirror through objective onto specimen → emission photons pass through objective, mirror, and emission filter (long-pass) through detector

Question 12

Parts of microscope for fluorescence imaging

Answer 12

• Light source
• Filter block
• Objective
• Photon detector

Question 13

Light source in different types of imaging

Answer 13

Fluorescence: either tungsten halogen lamp or mercury arc lamp (HBO)

Confocal: Laser (gives off monochromatic light of same wavelength that is coherent and linear polarized)

Two-photon: Ti/Sa laser (gives very short pulses in the fs range)

Question 14

Chromatic aberration?

Answer 14

Light rays passing through lens focus at different points, depending on their wavelength
Colors bend light to different degrees
There are lenses that correct this
Can be axial or lateral
Objective he’ps focus the different wavelengths of light to the same point

Question 15

What are the 2 types of abberrations/aberration correct?

Answer 15

chromatic aberration and spherical aberration

Question 16

What is spherical aberration?

Answer 16

= When light rays enter at different points of a spherical lens are not focused to the same point of the optical axis, creating different focal points
(light that hits at the edge of a lens bends more than light that hits at the center)

Question 17

What is numerical aperature

Answer 17

= how much light you can gather and how much detail you see

= nsine(mu)

Question 18

Which medium is usually used for its high refractive index?

Answer 18

Oil

-refractive index close to glass, meaning the light bends less and more light can reach the objective

Question 19

What is a resolution limit?

Answer 19

The minimum distance 2 points can be told apart as separate

Question 20

Why is the use of filters important?

Answer 20

Excitation light has much greater energy than the secondary fluorescence/emission light, so you need to be able to block the bright excitation light from reaching the detectors

Question 21

What does the filter block contain?

Answer 21

• Excitation filter (short pass filter)
• Dichroic mirror (reflects low wavelengths and allows longer wavelengths to pass through)
• Emission/barrier filter (long pass filter)

Question 22

Photon detectors used in each type of microscopy

Answer 22

Conventional: Photomultiplier tube (PMT) and/or CCD camera

Confocal: PMT (colorblind system based on a pseudo-color look up table)

Two-photon: PMT

Question 23

Basic idea of two-photon microscopy

Answer 23

Two photons enter at same time and same place with doubled wavelength

Uses photons from infrared spectrum (>750 nm)
Have high photon density
Excite w/ two photons of longer λ and less energy

Single photon: E= hv c = λv E ~ 1/λ
two-photon: E* = 1/2E = ~ 1/2λ

Question 24

Confocal vs two-photon

Answer 24

• Confocal has sufficient excitation at any time
• Two photon only has sufficient excitation in the focal plane w/in a laser pulse
• Focal plane in confocal is established by the pinhole, but don’t need pinhole in two-photon
• Both have same general structure, but with different laser(?) and two photon doesn’t require a pinhole
• Several emission signals can be sent, but in confocal microscopy, only the ones that enter through the pinhole are the ones observed
• Two-photon has excitation only within a narrow range of focal range but has better Z resolution
• Less bleaching in two-photon because excitation can only occur in a super narrow focal plane

Question 25

Technique with the best spatial resolution (from highest to lowest)

Answer 25

1. Two-photon
2. Confocal
3. Conventional

Question 26

Ranking of depth of penetration

Answer 26

1. Two-photon
2. Confocal
   (virtually none in conventional)

Question 27

Technique with best temporal resolution

Answer 27

Conventional

Question 28

Which techniques have the most dye availability?

Answer 28

1. COnventional
2. Confocal
3. Two-photon

Question 29

What method would be best for imaging thick preparations like acute and cultured brain slices, or in vivo imaging with an interest in deeper structures?

Answer 29

Two-photon

Question 30

Advantages and disadvantages of confocal fluorescence microscopy

Answer 30

Advantages
+ Improved spatial resolution
+ 3D scanning

Disadvantages

• more complicated imaging control
• low depth of light penetration
• bleaching (photons can only go through so many cycles of excitation and emission)

Question 31

Advantages and disadvantages of two-photon imaging

Answer 31

Advantages
\+ Optimized Z-resolution
\+ Reduced bleaching
\+ More efficient than confocal (removed pinhole)
\+ Higher depth of light penetration

Disadvantages

• Complicated combination of laser and imaging control
• High cost
• Reduced temporal resolution