Lecture 6 fluorescence microscopy Flashcards
Defenition of fluorescence (= type of luminescence)?
“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”
History of fluorescence: who discovered it and the name is derived from..?
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
What exactly happens during fluorescence?
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)
What is stokes’ shift and why is it crucial for the use of fluorescence in a microscope?
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
What kind of light (absorption/emitted fluorescence) has a lower energy?
Fluorescence has a lower energy = longer wavelength. Absorption = shorter wavelength = higher energy.
What are important optical properties for dyes in fluorescence microscopy?
•optical properties
- color
- fluor quantum yield
- stable fluorescence
- low photobleaching
What is an important physical property for dyes in fluorescence microscopy?
physical properties: not too large
What are important (bio)chemical properties for dyes in fluorescence microscopy?
•(bio)chemical properties:
- not cytotoxic
- specific (localization)
- environmental probe (pH, Ca2+)
Tetramethylrhodamine (TMR) is a synthetic dye. What are its properties? What is it used for/what are similar dyes?
- 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
Another synthetic dye is YOYO®-1. What does it specifically attach to? When is it hardly fluorescent?
It intercalates in the DNA. Hardly fluorescent in solution: it is flexible then.
Another synthetic dye is indo-1. What is it used for and why?
Sensitive to the environment (indo-1: binds calcium: different emission spectrum (intensity) upon ion binding): for determination of calcium concentration.
What is also used as a dye?
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?
Intrinsic fluorophores are those that occur naturally. What is a problem with common intrinsic fluorophores? What needs to be done most of the time?
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
How is fluorophore formed with GFP?
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
What are the 4 major components of fluorescence microscopy?
- excitation source
- objective for collection offluorescence
- filters / dichroic mirrors for separation of excitation and fluorescence light
- detector
Excitation sources: lamps (Arc-discharge (Hg or Xe) are used bc of brightness). Adv/disadv?
advantages:
- relatively cheap, many colors.
- disadvantages: highly inefficient, temperature, no sharp lines, cannot be focussed to difraction limited spot
Excitation sources: lasers. Adv/disadv?
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)
What should the exciter/excitation filter do?
should only transmit wanted exc. color & block the rest (= first ‘peak’, blue line)
What should the ichroicmirror / beam splitter do?
should reflect the excitation colors and transmit fluorescence (Green line in spectrum, schuin)
What should the emission/barrier filter do?
Should only transmit fluorescence and block the rest (Red line, final peak)
what kind of filters are available?
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
Detector for flurescence microscopy: the eye. Characteristics?
- 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)
what else is used as detectors of fluorescence microscopy? Characteristics?
CCD cameras.
- array of detectors (megapixels)
•very sensitive, very low noise
•rather slow (say up to 100 frames/sec)
Other detectors for fluorescence microscopy are point detectors: photomultipliers (PMT) & avalanche photodiodes. (APD) Characteristics?
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
WIDE-FIELD EPI uses cameras. What is required? disadvantages?
(“How should one excite of one uses a camera?”)
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.
CONFOCAL. “How should one excite when using a point detector?” O
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
