FLUORESCENCE

Methods for Cell Analysis Course BioVis – Uppsala, 2015

Matyas Molnar and Dirk Pacholsky

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Information This lecture contains images and information from the following internet homepages http://micro.magnet.fsu.edu/primer/index.html http://www.microscopyu.com/ http://www.olympusmicro.com/primer/lightandcolor/index.html

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Light phenomenon

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Wave or particle? ... Wave!

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Electric compound

Magnetic compound

Wave or particle? ... Wave!

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Wave or particle? ... Wave!

• Light travels in vacuum C = 300.000 km/sec • Seems to slows down in denser matter. • Refractive index n = C/v (v= speed in matter) • light is 1.5 x slower at n=1.5 • Light changes direction between different dense materials : shorter λ refract more

than longer λ Optic lenses, effects in sample... shorter wavelength (blue) has higher energy

E

λ

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Wave or particle? ... Particle!

Light behaves sometimes as quantized energy pockets, light has particle behavior.

Principle of fluorescence

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Wave-particle model When we want to model light when it propagates we use the wave model When we want to describe the properties of light when interacts with material we use quantum theory, and define light as particles (photons). Read more: http://www.livescience.com/24509-light-wave-particle-duality-experiment.html 9

Fluorescence basics

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Fluorescence basics Definition: Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. Usually the emitted light has a longer wavelength, and therefore lower energy than the absorbed radiation. Emission of light happens in time scale of nano second – so to speak immediately Compared to Phosphorescence: - specific type of photoluminescence related to fluorescence. Unlike fluorescence, a phosphorescent material does not immediately emit light. Absorbed radiation may be re-emitted for up to several hours after original excitation. (wikipedia ;) )

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Fluorescence basics Examples of fluorescent probes

Principle of fluorescent microscope

Excitation-Emission filter cube

Principle of fluorescence

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Fluorescence basics 10-15 s : 1 femto sec 10-14 s : 10 fs 10-13 s : 100 fs 10-12 s : 1 pico sec 10-11 s : 10 ps 10-10 s : 100 ps 10-9 s : 1 nano sec 10-8 s : 10 ns 10-7 s : 100 ns 10-6 s : 1 micro sec 10-5 s : 10 µs 10-4 s :100 µs 10-3 s : 1 milli sec 10-2 s : 10 ms 10-1 s :100 ms 100 s : 1 second

http://www.olympusmicro.com/primer/java/jablonski/jabintro/

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Fluorescence: The spectra Normalized Intensity

nm

Alexa 488 QY 0.92 Alexa 555 QY 0.010

Stokes Shift

Alexa 488

X axis: λ in nm Y-axis: Intensity or probability of event that A) fluorophore absorbs the light for excitation (dashed line) and B) Fluorophore emits ligth (full line) • Ex peak at 100% em peak at 100 %, ex 20% em 20 %, same range of emission • Stokes shift: gap between ex-peak and em peak = (loss of energy, dissipation ) important for separation of excitation and emission light in microscope etc Other important features of fluorophores: • Extinction coefficient: absorbtion efficiency of a photon at particular wavelength • Quantum yield: proportion of photons emitted at λ em to those absorbed at λ em 14

Stokes shift Stokes shift is the energy difference between the lowest energy peak of absorbance and the highest energy of emission. The Stokes shift is an extra for observing fluorescence; without it there would be (almost) no way to distinguish between excitation and emitted light Probes with varying Stokes shifts are very useful for multicolor applications

Wavelength

495 nm 520 nm

Stokes Shift is 25 nm Fluorescein molecule

Fluo

resc

nece

Inte

nsity

Excitation Emission

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Parameters for fluorescence efficiency

• Extinction Coefficient • ε refers to a single wavelength (usually the absorption

maximum) • ε measure of how efficiently a substance absorbs light of

a certain wavelength

• Quantum Yield – Qf is a measure of the integrated photon emission over

the fluorophore spectral band – The ratio of photons emitted to photons absorbed

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Excitation Saturation • excited state lifetime τf leaves fluorophore in a

state where MORE photons might damage the fluorophore (triplett state – highly reactive… radicals)

• when the rate of excitation exceeds the reciprocal of τf Saturation occurs

• Emission can then not be further increased by increasing the exitation; instead it may lead to the destruction of the fluorescing molecule (photobleaching - not reversible!)

Usually, increasing dye concentration can be

the most effective means of increasing signal when energy is not the limiting factor (ie laser based confocal systems)

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Photobleaching • Defined as the irreversible destruction of an excited fluorophore • Methods for countering photobleaching

– Illuminate for shorter times – Use high magnification, high NA objective – Use wide emission filters – more signal to capture (may create

problems with multiple probes) – Reduce excitation intensity – Use “antifade” reagents (not compatible with viable cells)

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Filter and dichroic mirror

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Filter and dichroic mirror

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Filter and dichroic mirror in real

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Dealing with fluorescence Cell sample Cell images merged RGB

image

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Dealing with fluorescence ex

Excitation 350 nm excitates Blue and Green, using BP filter 400-550 collects them both. *Remember: the camera is color blind. You decide with your choice of filter what it will see.

Cell sample

Cell image*

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Dealing with fluorescence ex

Excitation 350 nm excitates Blue and Green, using BP filter 400-480 collects only the blue. *Remember: the camera is color blind. You decide with your choice of filter what it will see.

Cell sample

Cell image*

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Dealing with fluorescence ex

Excitation 480 nm excitates Green and Red, using BP filter 510-530 collects only the green. *Remember: the camera is color blind. You decide with your choice of filter what it will see.

Cell sample

Cell image*

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Probes for Proteins

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Probes for Nucleic Acids • Hoechst 33342 (AT rich) (uv) 346 460 • DAPI (uv) 359 461 • POPO-1 434 456 • YOYO-1 491 509 • Acridine Orange (RNA) 460 650 • Acridine Orange (DNA) 502 536 • Thiazole Orange (vis) 509 525 • TOTO-1 514 533 • Ethidium Bromide 526 604 • PI (uv/vis) 536 620

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Probes for Calcium Ions • INDO-1 Ex350 Em405/480 • QUIN-2 Ex350 Em490 • Fluo-3 Ex488 Em525 • Fura -2 Ex330/360 Em510

Frog olfactory bulb neurons labeled with fluo-3 and Fura Red™ Ca2+ indicators, demonstrating a Ca2+ response to treatment with KCl and nifedipine. Image is a ratio of fluo-3 and Fura Red™ fluorescence images acquired with a Leica confocal laser-scanning microscope The image was contributed by J. Bischofberger and D. Schild, Physiology Institute, University of Göttingen.

http://www.invitrogen.com/site/us/en/home/support/Research-Tools/Image-Gallery/Image-Detail.alternateID.g000410.html

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pH Sensitive Indicators

• SNARF-1 488 575 • BCECF 488 525/620 440/488 525

[2’,7’-bis-(carboxyethyl)-5,6-carboxyfluorescein]

Probe Excitation Emission

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Probes for Oxidation States

• DCFH-DA (H2O2) 488 525 • HE (O2

-) 488 590 • DHR 123 (H2O2) 488 525

Probe Oxidant Excitation Emission

DCFH-DA - dichlorofluorescin diacetate HE - hydroethidine DHR-123 - dihydrorhodamine 123

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Specific Organelle Probes

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Specific Organelle Probes

Gibco® human aortic smooth muscle cells (HASMC, Cat. No. C0075C) were transduced with CellLight™ Golgi-GFP , CellLight™ Mitochondria-RFP Hoechst 33342 Imaging was performed on live cells using a DeltaVision® Core microscope + standard DAPI/FITC/TRITC filter sets.

http://www.invitrogen.com/site/us/en/home/support/Research-Tools/Image-Gallery/Image-Detail.10239.html 32

Fluorescent protein • GFP - Green Fluorescent Protein

– GFP is from the chemiluminescent jellyfish Aequorea victoria

– excitation maxima at 395 and 470 nm (quantum efficiency is 0.8) Peak emission at 509 nm

– contains a p-hydroxybenzylidene-imidazolone chromophore generated by oxidation of the Ser-Tyr-Gly at positions 65-67 of the primary sequence

– Very stable – Major application is as a reporter gene for assay of

promoter activity – requires no added substrates – Now in the enhanced form of eGFP , eYFP, eCFP

http://gfp.conncoll.edu/ http://brainwindows.wordpress.com/category/gfp http://www.biojobblog.net/2008/10/08/gfp-finally-gets-its-due/

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Fluorescence Resonance Energy Transfer - FRET • Effective between 1-10 nm only • Emission and excitation spectrum must significantly overlap • Donor transfers non-radiatively to the acceptor • Examples:

– PE-Cy7™ • BFP-GFP

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Fluorescence Resonance Energy Transfer - FRET

Wavelength

Absorbance1

DONOR

Absorbance 2

ACCEPTOR

Molecule 1 Molecule 2

Emission 1 Emission 2

Overlap

I n c r e a s e d S t o k e s s h i f t

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Fluorescence Resonance Energy Transfer - FRET

PE-Cy7 – wide Stoke´s shift, excitation in range of PE (and CY7), emission at Cy7 range. Why are such fluorophores needed? Multicolor fluorescence application using e.g. Only one Laser e.g. 488 (see slide ”dealing w fluorescence flow cytometer)

PE exitation and emission spectra w 561 nm Laser

Cy7 exitation and emission spectra w 750 nm Laser

Cy7 exitation and emission spectra w 561 nm Laser

PE-Cy7 exitation and emission spectra w 561 nm Laser

Common tandem conjugate probes that rely on FRET

• Probe Ecxitation Emission • PE-Cy5 488, 532, 561 660 • PerCp-Cy5.5 488 695 • Pe-Cy7 488, 532, 561 780 • APC-Cy7 640 780

• Good points:

– large stokes shift, many colors from one laser • Not so good points

– Large overlap between dyes – Sensitive to temperature and light degradation into two separate fluorophores

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Important points • Fluorescence is the primary information source for

confocal microscopes and flow cytometry equipment • Fluorescence emission is longer than the exciting

wavelength • Dye molecules must be close to, but below saturation

levels for optimum emission • Fluorescence probes must be appropriate for the

excitation source and the sample of interest • Correct optical filters must be used for multiple color

fluorescence emission

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THANKS FOR YOUR ATTENTION!

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