Basics of fluorescence
Microscopy Course IST Austria, 24.09.- 1.10. 2019 Anna Hapek-Sikora
Emission of light- LUMINESCENCE
Luminescence is the emission of light from any substance not resulting from heat and occurs from electronically excited states.
According to excitation mechanisms: - chemiluminescence - thermoluminescence - electroluminescence
and... PHOTOLUMINESCENCE
PHOTOLUMINESCENCE
fluorescence phosphorescence occures from excited singlet states occures from excited triplet states emission rates slow 103 – 100 s-1 emission rates very fast 109 s-1 lifetimes miliseconds to seconds lifetimes 10 ns
Common fluorophores
Common fluorophores
• organic molecules with aromatic ring structures • delocalized electrones in binding π-orbitals • π-electrons interact with the environment What we see, what we cannot see
E = hν = hc/λ
Few people you should know
Sir George Stokes
Max Planck Nobel prize in 1918
Sir John Herschel Aleksander Jabłoński But we can try
Good enough ;)
Jabłoński Energy Diagram
Stokes Shift
Different deactivation pathways, through which an excited molecule can return to its ground-state. Characteristic of fluorescence emission
● the Stokes' shift
● emission spectra are independent of excitation λ
● fluorescence quantum yield
● fluorescence lifetime
● fluorescence quenching
Stokes' shift
Absorption and emission spectra of Rhodamine with ~25 nm Stokes shift Emission spectra are independent of excitation λ
The emission spectrum is independent of the excitation energy as a consequence of rapid internal conversion from higher initial excited states to the lowest vibrational energy level of the S(1) excited state. Quantum yield
Ratio of the number of emitted to absorbed photons
S1 Relaxation (10 -12s) S1 Q= Γ • ᴦ - emissive rate • knr - non radiative decay rate to S0 hϑA hϑA ᴦ knr Γ +k nr
S0
• Q is close to unity when knr ˂ ᴦ • substances with largest quantum yields (rhodamins) display brightest emissions • Q is highly dependent on the solvent of the fluorophore
Substances with the largest quantum yield display the brightest emissions. Quantum yield of different fluorophores
Fluorescence lifetime (τ)
- τ is the average value of the time spent in the excited state
- formally, the fluorescence lifetime is defined as the time in which the initial fluorescence intensity of a fluorophore decays to 1/e (approximately 37 percent) of the initial intensity
- determines time avaiable for molecular interactions, diffusion into environment and hence the info avaiable from its emission
F(t) = F0 • exp(-t/τ) 1 τ= ᴦ + knr
• ᴦ - emissive rate
• knr - non radiative decay rate to S0
τ Fluorescence quenching
Refers to any process which decreases the fluorescence intensity of a given substance.
Different mechanisms:
Collisional quenching (dynamic) - excited state fluorophore is deactivated by other molecules (quenchers) - - - - O2; I , Cl (halogens); amines, e deficient molecules - mechanisms: e- transfer ( Indole → Acrylamide) - intersystem crossing to triplet state (halogens, heavy atoms)
Static quenching - fluorophore forms nonfluorescent complexes with other molecules - occures in the ground state independent of diffusion or molecule collision (dye aggregation )
Nonmolecular mechanisms - attenuation of the incident light Fluorescence Anisotropy
Effects of polarized excitation and rotational diffusion on the polarization or anisotropy of the emission.
Photoselected Randomized fluorophores fluorophores Rotational diffusion
I
III polarized polarized unpolarized excitation emission emission (laser light)
Resonance energy transfer
- occurs in excited state - whenever emission spectrum of fluorophore called DONOR overlaps with absorption spectrum of another molecule called ACCEPTOR
Förster resonance energy transfer FRET
FRET occurs when DONOR and ACCEPTOR are within the Förster distance (0.5-10 nm) Donor-Acceptor spectral overlap
FRET efficiency
The FRET efficiency (E) is the quantum yield of the energy transfer transition, i.e. the fraction of energy transfer event occurring per donor excitation event. 1 EFRET – efficiency EFRET = 6 r – distance donor-acceptor R - the distance at which the energy transfer efficiency is 50% 1+(r /R0) 0
R0 50% of E
FRET efficiency
The efficiency of the energy transfer decreases to the 6th power of the distance between donor and acceptor.
Donor Acceptor Donor Acceptor Förster Fluorescent Excitation Emission Quantum Extinction Distance Protein Maximum Maximum Yield Coefficient (nm) Pair (nm) (nm)
EBFP2-mEGFP 383 507 0.56 57,500 4.8
ECFP-EYFP 440 527 0.40 83,400 4.9
Cerulean-Venus 440 528 0.62 92,200 5.4
MiCy-mKO 472 559 0.90 51,600 5.3
TFP1-mVenus 492 528 0.85 92,200 5.1
CyPet-YPet 477 530 0.51 104,000 5.1
EGFP-mCherry 507 510 0.60 72,000 5.1
Venus-mCherry 528 610 0.57 72,000 5.7
Venus-tdTomato 528 581 0.57 138,000 5.9
Venus-mPlum 528 649 0.57 41,000 5.2 FRET applications
● can be used to measure distances between domains in a single protein and therefore to provide information about protein conformation
● detect interaction between proteins
● applied in vivo, FRET has been used to detect the location and interactions of genes and cellular structures including intergrins and membrane proteins
● is also used to study lipid rafts in cell membranes
Summary Molecular information from fluorescence
Emission spectra and Stoke’s shift - location of the fluorophore - binding to macromolecules (DNA, membrane) - chemical sensing dyes Quenching of fluorescence - accessibility of fluorophores to quenchers (location on macromolecule, porosity of proteins) FRET - distance measurment between sites on macromolecules. Protein-protein interaction. Fluorescence polarization and anisotropy - used to measure molecular weight of proteins or protein binding
10 minutes break!
Fluorophores
Intrinsic Extrinsic
natural added to the sample
aromatic aa fluorescein NADH rhodamine, flavins DAPI chlorophyll
Cells are autofluorescent!
Important characteristics of fluorophores
● Absorbtion and emission maxima (nm)
● Fluorescensce intensity of the emitted light
● Molar Extinction Coefficient
● Quantum Yield (0-1)
● Solubility in water or other substances
● Lifetime of the excited state
● Photostability
Absorbtion and emission maxima
Molar extinction coefficients M-1 cm-1
The extinction coefficient is determined by measuring the absorbance at a reference wavelength (characteristic of the absorbing molecule) for a one molar (M) concentration (one mole per liter) of the target chemical in a cuvette having a one-centimeter path length.
Extinction coefficients are a direct measure of the ability of a fluorophore to absorb light, and those chromophores having a high extinction coefficient also have a high probability of fluorescence emission.
Molecules exhibiting a high extinction coefficient have an excited state with a short intrinsic lifetime.
Photostability and photobleaching
Fluorophores permanently loses ability to fluorescence. Why?
The average number of excitation and emission cycles that occur for a particular fluorophore before photobleaching is dependent upon the molecular structure and the local environment.
- due to photon induced chemical damage - covalent modifications - transition from singlet to triplet state (longer time in excited state to interact with others molecules)
Dye + dye photobleaching Dye+light+oxygen
How to control photobleaching
• reduction of light intensity and time span of light exposure
• increase of fluorophore concentration
• remove oxygen
• stop (compete with) photooxidation
use antioxidants „antifade reagents“ (e.g. DABCO)
• 2-photon excitation
• employment of more robust fluorophores (Alexa Fluor dyes)
• employment of dyes with longer excitation wavelength
FRAP – application of photobleaching
Used to investigate the diffusion and motion of biological macromolecules. Phototoxicity
Cell damage as a result of exciting exogenous or endogenous fluorophores with lasers or high-intensity arc-discharge lamps
Reasons:
• In their excited state, fluorescent molecules tend to react with molecular oxygen to produce free radicals that can damage subcellular components
• particular constituents of standard culture media, including the vitamin riboflavin and the amino acid tryptophan, may also contribute to adverse light-induced effects on cultured cells
• interaction between light and DNA intercalating dyes causes denaturation of the polymer, Bernas T. et al. 2005
How to fight with it?
Fighting with photoxicity
Fighting with photoxicity
• reduction of light intensity and time span of light exposure
• addition of scavengers into the medium (ascorbic acid)
• fluorescent proteins are generally not phototoxic
• fluorophores that exhibit the longest excitation wavelengths possible should be chosen to minimize damage to cells by short wavelength illumination
• many synthetic fluorophores, such as the MitoTracker and nucleic acid stains (Hoechst, SYTO cyanine dyes, and DRAQ5) are highly toxic to cells.
Good to read
And many more, just google it Antibody labeling
Covalent binding: Noncovalent binding:
• free NH2-groups • Fc-portion of antibodies • free SH-Groups ( Zenon® labeling system) • carbohydrate side chains
Antibody labeling
Protein labeling reagents
Rhodamine FITC 600/615 480/510
Why this two?
- high quantum yield (0.90, 0.95) - display longer excitation and emission λ than aa - high molar extinction coefficient (80 000 M-1 cm-1) - lifetime ~ 4ns - good for antibody labeling
Stokes shift in protein labeling
FITC 494/519
Small stoke shift leads to self-quenching events FRET can occur between two molecules attached to the same protein if they are within Förster distance
More fluoresceine molecules attached to protein ≠ brighter signal
DNA probes
Very weak intrinsic emission is observed from unlabeled DNA
Number of probes bind spontaneously to DNA and display enhanced emission up to 1000 fold upon binding
Ethidium bromide, PI, 7-AAD
DAPI, Hoechst
TOTO familly DNA staining
DAPI stained PI stained NIH 3T3 mouse fibroblast HEK 293
Applications: counterstains for nucleic acids and chromosomes, cell cycle analysis, dead cell staining.
Membrane probes
Mouse fibroblasts Membranes-Texas red DHPE Actin-Fluorescin DNA-DAPI
B. subtilis PY79 Stained with TMA-DPH
Texas Red DHPE Other cell organella stainings
Immunocytochemistry/Immunofluorescence: ABCA7 Antibody. Staining of human cell line A-431 shows positivity in plasma membrane and golgi apparatus
Bovine pulmonary artery endothelial cell labeled with probes to visualize mitochondria, peroxisomes, and the nucleus.
Chemical sensing probes
Wavelength-ratiomeric dyes which allow the analysis of kation concentrations like Ca2+, H+, Na2+, K+, Mg2+ by determining the ratio of intensities at different excitation or emission wavelengths Dyes are trapped in cells by lysis of cell permeable esters or by microinjection.
Example: Ca+2 indicators
chemical indicators genetically encoded indicator small molecules able to chelate ions fluorescent proteins derived from GFP or its variants + calmodulin BAPTA based funcional carboxylix acid gr. genes encoding these proteines transfected to cell lines fura-2, indo-1, fluo-3, fluo-4, Calcium Green-1 Cameleons
GFP- Green Fluorescent Protein
238 aa protein isolated from Aequorea victoria barrel of β-sheets
WT GFP → ex.max. near to UV Mutation S65T → ex.max. shifted to 488 nm (Tsien) extraordinary photostability and high quantum yield Nobel prize in chemistry in 2008 for Martin Chalfie, Osamu Shimomura, Roger Y. Tsien
Pallette of fluorescent proteins
there is a wide variety of fluorescating proteins
availabe (Emmax 448 – 600 nm) derived from modified GFP (YFP, CFP) or corals RFPs.
Proteins can be fused to any protein of interest to study protein geography, movement, lineage and biochemistry in living cells
Roger Y. Tsien Nobel Lecture 2008 www.fpbase.org Phycobiliproteins
Red algae - Rhodophyta Phycoerythrobilin HeLa cells stained with PE labeled antibody
• intensely fluorescent proteins from cyanobacteria and algae • chromophores (bilins) are covalently bound to the protein subunit • very stable (10x more than FITC), water soluble, display high quantum yields • do not form autocatalytically, use them as a chemical tags (bind with antibodies) • conjugatable to other proteins
Quantum dots
- nanocrystals made of semiconductor - small enough to exhibit quantum mechanical properties - coated with a hydrophilic polymer - can be conjugated to antibodies, peptides, carbohydrates - energy of emitted photon depends on the particel size
Significant benefits: long term photostability, high fluorescense intensity levels, multiple colors with single wavelength excitation for all emission profiles, narrow emission profile.
Optical Highlighters fluorescent proteins which undergo a variety of light-induced switching characteristics
photoactivatable photoswitchable
photoconvertable
Photoactivatable
An optical highlighter derived from aceGFP, isolated from Aequorea coerulescens, has been reported to transition from cyan (468 nanometers) to green fluorescence (511 nanometers) upon illumination at 405 nanometers. Used for superresolution techniques like PALM Photoconvertable
One of the most useful classes of optical highlighters encompasses the growing number of fluorescent proteins reported to undergo photoconversion from one emission wavelength to another.
First isolated from the stony Open Brain Coral, Trachyphylla geofrey. Kaede photoconverts from green to red emission by illumination with ultraviolet light. Photoconversion of Kaede is stable and irreversible. Mechanism is the light catalyzed cleavage of the the His 62-Tyr63-
Gly64 backbone Photoswitchable
the ability to switch between fluorescent and dark states - Dronpa is most prominent, the monomeric variant derived from a stony coral tetramer - illumination at 488 nanometers drives Dronpa to the dark species, which can be switched back on by brief illumination at 405 nanometers - this cycle can be repeated several hundred times without significant photobleaching
- used for superresolution techniques like RESOLFT
Take care: excitation efficiency
FITC (Emax 480) excitation with 488 nm laser (87%), 477 nm laser (56%), 514 laser (28%) Alexa Fluor 546 excitation with 568 laser (84%), 543 laser ( 43%), 594 (4%) Take care when double labeling with FITC and Alexa Fluor 546, 7% of Alexa Fluor 456 are excited with 488 nm laser.
Take care: bleedthrough artifacts
- are due to very broad bandwidths and asymetrical spectral profiles exhibited by many common fluorophores which lead to spectral overlap - fundamental problem of both widefield and laser scanning microscopy - manifested by the emission of one fluorophore being detected in the PMT or filter combination reserved for another fluorophore - artifacts can complicate the interpretation of the results especially when investigating the colocalization of fluorophores, quantitative measurements
Scanning of a thin section of mouse intestine Labeled with Alexa fluor 488 Simultaneously and Cy3 with 488 nm argon laser and 543 nm helium-neo laser
Sequentialy
Spectra analyzer
Remember!
Always know properties of your fluorophore. Choose:
● optical filters which can match absorption and emission maxima of your fluorophore optimally!
● fluorophores which have absorption max. which closely match your laser lines
● dyes with high quantum yield for target which you have less
It's good to spend some time to design your experiment properly.
...and good luck!