Lecture 6 fluorescence microscopy

Question 1

Defenition of fluorescence (= type of luminescence)?

Answer 1

“luminescence that is caused by the absorption of radiation at one wavelength followed by nearly immediate reradiation usually at a different wavelength and that ceases almost at once when the incident radiation stops”

Question 2

History of fluorescence: who discovered it and the name is derived from..?

Answer 2

erm coined by Sir George Gabriel Stokes (1852) “to denote the general appearance of a solution of sulphateof quinine and similar media”Name derived from fluorite(Calcium Fluoride), which sometimes contain fluorescent divalent Europium

Question 3

What exactly happens during fluorescence?

Answer 3

1) Absorption of light: photon is taken, energy is taken out.
2) This will promote the electron to higher energy: from the ground state -> excited state
3) At first, no light is emitted but there is a thing called ‘internal conversion’, where vibration results into heat and the electron falling into a lower state (no light)
4) Then, the electron falls back into the ground state, = light (fluorescence)

Question 4

What is stokes’ shift and why is it crucial for the use of fluorescence in a microscope?

Answer 4

In most cases there is an energy difference between taken and emitted light. Fluorescence light compared to the absorption light = stokes’ shift.

–> Stokes’ shift allows spectral separation of excitation light from fluorescence light,crucial for use of fluorescence in a microscope

Question 5

What kind of light (absorption/emitted fluorescence) has a lower energy?

Answer 5

Fluorescence has a lower energy = longer wavelength. Absorption = shorter wavelength = higher energy.

Question 6

What are important optical properties for dyes in fluorescence microscopy?

Answer 6

•optical properties

• color
• fluor quantum yield
• stable fluorescence
• low photobleaching

Question 7

What is an important physical property for dyes in fluorescence microscopy?

Answer 7

physical properties: not too large

Question 8

What are important (bio)chemical properties for dyes in fluorescence microscopy?

Answer 8

•(bio)chemical properties:

• not cytotoxic
• specific (localization)
• environmental probe (pH, Ca2+)

Question 9

Tetramethylrhodamine (TMR) is a synthetic dye. What are its properties? What is it used for/what are similar dyes?

Answer 9

• can be attached covalently to proteins or DNA
• pretty stable (also pH etc.)
• for example used for antibody labeling
• similar dyes: Alexa, Cy3, Cy5, Texas Red,Fluorescein

Question 10

Another synthetic dye is YOYO®-1. What does it specifically attach to? When is it hardly fluorescent?

Answer 10

It intercalates in the DNA. Hardly fluorescent in solution: it is flexible then.

Question 11

Another synthetic dye is indo-1. What is it used for and why?

Answer 11

Sensitive to the environment (indo-1: binds calcium: different emission spectrum (intensity) upon ion binding): for determination of calcium concentration.

Question 12

What is also used as a dye?

Answer 12

Something new: quantumdots

• Made of semiconductor material: often CdSe
• Broad absorption, sharp emission (can therefore be used for multiple colours)
• Emission wavelength depends on size
• Very bright, very photostable, only little bit is needed
• But: blinking, big, attachment complex & toxic?

Question 13

Intrinsic fluorophores are those that occur naturally. What is a problem with common intrinsic fluorophores? What needs to be done most of the time?

Answer 13

Common intrinsic fluorophores like tryptophan, NAD(P)H are not good enough

•chlorophylls & flavins work

-> in most cases extrinsic fluorophores have to be added: synthetic or genetically encoded:
GFP

Question 14

How is fluorophore formed with GFP?

Answer 14

Fluorophore formed autocatalytically(oxygen required!) from 3 of GFP’s amino acids!
Can be used to make genetic fusions of gene of protein of interest & GFP

Question 15

What are the 4 major components of fluorescence microscopy?

Answer 15

• excitation source
• objective for collection offluorescence
• filters / dichroic mirrors for separation of excitation and fluorescence light
• detector

Question 16

Excitation sources: lamps (Arc-discharge (Hg or Xe) are used bc of brightness). Adv/disadv?

Answer 16

advantages:

• relatively cheap, many colors.
• disadvantages: highly inefficient, temperature, no sharp lines, cannot be focussed to difraction limited spot

Question 17

Excitation sources: lasers. Adv/disadv?

Answer 17

advantages: very high brightness, can be focused to tiny spot
(parallel beam), spectrally very sharp & pure
disadvantages: often not tunable (= can’t change the color)

Question 18

What should the exciter/excitation filter do?

Answer 18

should only transmit wanted exc. color & block the rest (= first ‘peak’, blue line)

Question 19

What should the ichroicmirror / beam splitter do?

Answer 19

should reflect the excitation colors and transmit fluorescence (Green line in spectrum, schuin)

Question 20

What should the emission/barrier filter do?

Answer 20

Should only transmit fluorescence and block the rest (Red line, final peak)

Question 21

what kind of filters are available?

Answer 21

1) Long pass: lets through long wavelengths
2) Short pass: lets through short wavelengths
3) Band pass: specific band
4) Dichroic long-pass: reflects and transmits

Question 22

Detector for flurescence microscopy: the eye. Characteristics?

Answer 22

• rods more sensitive than cones: no color vision at low light
• threshold of vision is the detection of only ~100 photons!!!
• the eye has an incredible dynamic range of over 10 decades(> 30 bits)

Question 23

what else is used as detectors of fluorescence microscopy? Characteristics?

Answer 23

CCD cameras.
- array of detectors (megapixels)
•very sensitive, very low noise
•rather slow (say up to 100 frames/sec)

Question 24

Other detectors for fluorescence microscopy are point detectors: photomultipliers (PMT) & avalanche photodiodes. (APD) Characteristics?

Answer 24

point detectors: no spatial information
•area from cm2 (PMT) to << mm2 (APD)
•APD more sensitive, less noise than PMT
•PMT more blue sensitive
•time resolution

Question 25

WIDE-FIELD EPI uses cameras. What is required? disadvantages?
(“How should one excite of one uses a camera?”)

Answer 25

WIDE-FIELD EPI uses cameras. Illumination of the whole area of the sample is required. Image using objective and tube lens. Disadvantage: same intensity everywhere on the sample, blurry background, makes it difficult for thick samples.

Question 26

CONFOCAL. “How should one excite when using a point detector?” O

Answer 26

Confocal fluorescence microscopy:
- parallel beam of laser is used. Focussed in one spot, collect the fluorescence of one spot. Do not shine it directly on the detector, it goes through the pinhole.
- Out of focus light will be blocked this way
- optical sectioning
•one voxel measured at a time: laser or sample scanning needed