What is luorescence Coil線圈是熒光

1、how fluorescence worksadele marstontopics coveredx the nature of light and colourx colour detection in the human eyex the physical basis of fluorescencex fluorescent probes and dyesx dyes that bind organellesx chemical dyesx fluorescent proteinsx photobleaching and quenchingthe nature of lightthe en

2、ergy of light is contained in discrete units or quanta known as photons light is a form of electromagnetic radiationphotons have the property of both particles and wavesfor simplicity, usually only the electrical component is drawnlight as a wave: the nature of light and colour - 1the electromagneti

3、c spectrumwavelengths 400nm-750nm are visible to the human eye the nature of light and colour - 2the human eyex sensitivitypeak sensitivity is at 555nm (yellow-green)in bright light, 3 orders of magnitudeafter time to accommodate, 10 orders of magnitude!x resolution0.1mm for an object 25mm from the

4、eyex composed of rod and cone cells can detect differences in light intensity and wavelength (colour)colour detection in the human eye - 1rod cell photoreceptorsx comprise 95% of photoreceptors in the retinax active in dim light but provide no colour sensex peak sensitivity at 510nm (blue-green)x co

5、ntain rhodopsinx bright light temporarily bleaches rhodopsin(20-30 min recovery time)x best high visual sensitivity in a darkened roomretinalcolour detection in the human eye - 2cone cell photoreceptorsx comprise only 5% of photoreceptors in the retinax contained nearly exclusively in fovea (0.5mm s

6、pot)x 3 types: red, green and bluex action spectra differ for the different cone cells colour detection in the human eye - 3positive and negative coloursx positive colours are generated by combining different colour wavelengths- yellow perceived by stimulating red and green cones individually with 2

7、 different wavelengthsx negative colours are generated by the subtraction (absorption) of light of a specific wavelength from light composed of a mixture of wavelengths- yellow perceived because a single wavelength stimulates both red and green cones colour detection in the human eye - 4fluorescence

8、x occurs following excitation of a fluorescent molecule upon absorption of a photonx energy is released as light as the molecule decays to its ground state the physical basis of fluorescence - 1absorptionemissiontypical fluorochrome:100,000 cycles per second for 0.1-1 secondsexcitationenergy loss (r

9、apid 10-9-10-12s) excited statesground stateemitted light (longer wavelength)jablonski diagramfluorochrome “a molecule that is capable of fluorescing”excitation and emission spectrastokes shiftfor fitc (fluorescein-5-isothiocyanate) coupled to iggwavelengththe physical basis of fluorescence - 2filte

10、r setemissionexcitationdichromatic mirrorfitc filter set (chroma)light into detector (eyepiece/camera)to objectiveemission intensity depends on the excitation wavelengththe physical basis of fluorescence - 3properties of fluorophoresx stokes shift - difference between excitation and emission maxima

11、(large advantageous)x molar extinction coefficient - potential of a fluorophore to absorb photonsx quantum efficiency (qe) of fluorescence emission -fraction of absorbed photons that are re-emittedx quantum yield - how many photons emitted by a fluorophore before it is irreversibly damagedx quenchin

12、g - quantum yield (but not emission spectrum) altered by interactions with other moleculesx photobleaching - permanent loss of fluorescence by photon-induced chemical damagefluorescent probes and dyes - 1choice of fluorophore will depend on the applicationx protein localization (immunofluorescence m

13、icroscopy or gfp-tagging).x organelle marking (e.g. dapi to label nucleus)x protein dynamics (frap )x protein interactions (fret)x ion concentration (using ratiometric dyes)x enzyme reactions (“caged” fluorescent compounds)x cell viability (viability-dependent permeabilization)fluorescent probes and

14、 dyes - 2some applications of fluorescence microscopyfluorochromes in microscopyx biologically active fluorescent compounds - bind directly to cellular structuresx chemical dyes - most need to be coupled to a macromolecule to be useful in microscopyx fluorescent proteins - can be fused genetically t

15、o a protein of interestfluorescent probes and dyes - 3dyes that bind cellular structures or organellesdapicrystal structure of dapi bound to dnasporulating bacillus subtilisfm4-64 and dapidyes that bind organelles - 1chemical conjugation of fluorescent dyes to chemicals that bind cellular structures

16、rhodamine-coupled phalloidin(phalloidin is a mushroom toxin that binds to f-actin)dyes that bind organelles -2immunofluorescence microscopyfluorophoresecondary antibodyprimary antibodyuse antibodies raised against your protein of interestorchemical dyes -1mouseanti-mouserabbitanti-rabbitepitope tags

17、 in fluorescence microscopycommon epitopes = myc, hagene x6xhax fuse protein of interest to an epitope “tag”x buy commercially-available antibodies to the epitope and use as primary antibody for ifadvantage: fast (do not need to raise antibodies)disadvantages: protein fusion may not be fully functio

18、nalproblems of specificity of antibodies to tagchemical dyes - 2fluorophores for microscopyfluorescein (igg-coupled)(fitc)520nm - greentexas red (igg-coupled)601 nm - redtetramethylrhodamine (dextran coupled) (tritc)573 nm - redfluorescein and rhodamine derivativescoupled with isothiocyanates - allo

19、ws attachment via amino groups in proteinschemical dyes -3improved dyescydyes (cyanine dye-based) amersham-pharmacia incalexafluor (molecular probes/invitrogen)(brighter, more stable)chemical dyes -4qdot nanocrystals extremely photostable(molecular probes/ invitrogen)different wavelengths achieved b

20、y varying size of crystalsmall semi-conductorschemical dyes -5cadmium/seleniumzinc sulphidemulticolour labelingx can simultaneously image multiple fluorophores e.g to localize multiple proteins in the same cellx need to isolate the signal from each fluorophore individually1) choose fluorophores with

21、 minimum emission overlap 2) choose filter sets that minimize “bleed through” into another channelsuitablenot suitablechemical dyes -6fluorescent proteinsgreen fluorescent protein (gfp) isolated from the jellyfish aequorea victoriamy proteingfpshort flexible linkerfusion proteinadvantages:can use in

22、 live cellsfixing artefacts avoideddynamicsdisadvantages: photobleachingfolding environment dependentfunctionality of fusion proteinfluorescent proteins -1mutagenisation of gfp- more stable - spectrally shifted variantsother fluorescent proteins from other organismse.g. dsred from discosoma (26% hom

23、ology with gfp)gfp variantsshaner nc, steinbach pa, tsien ry (2005) a guide to choosing fluorescent proteins. nat methods. 2(12):905-9. gfp (wt)395/475509green fluorescent proteinsegfp484507acgfp480505turbogfp482502emerald487509azami green 492505zsgreen493505blue fluorescent proteinsebfp383445sapphi

24、re399511t-sapphire399511cyan fluorescent proteinsecfp439476mcfp433475cerulean433475cypet435477amcyan1458489midori-ishi cyan472mtfp1 (teal) 462492orange and red fluorescent proteinskusabira orange548morange548562dtomato554581dtomato-tandem554dsred558583dsred2563582dsred-express (t1)555dsred-monomer55

25、6mtangerine568585mstrawberry574596asred2576592mrfp1584607jred584610mcherry587610hcred1588618mraspberry598625hcred-tandem590mplum590649yellow fluorescent proteinseyfp514527topaz514527venus515528mcitrine516529ypet517530phiyfp525537zsyellow1529539mbanana540553fluorescent proteins -2photobleachingphotob

26、leaching: a fluorophore permanently loses the ability to fluoresce due to photon-induced chemical damage and covalent modification.largely due to the generation of free oxygen radicals that attack and permanently destroy the light-emitting properties of the fluorochrome.absorption(10-15 sec)fluoresc

27、ence(10-9 - 10-12 sec)(nsec-psec)internalconversion(heat)phosphorescence(102 - 10-2 sec)(100sec-0.01sec)*triplet state*triplet state - very reactive may interact with another molecule to produce irreversible covalent modifications (photobleaching)ground state excited state photobleaching and quenchi

28、ng - 1how to reduce photobleachingx chemical reactivity of the fluorophorex intensity and wavelength of the excitation light x intracellular chemical environmentphotobleaching influenced by:reduce photobleaching by:x choice of fluorophorex limit exposure time (but will reduce emission)x use of antif

29、ade reagentsphotobleaching and quenching - 2antifade reagentsact by scavenging reaction oxygen species common antifade reagentsdiy (buy from sigma)p-phenylenediaminen-propyl gallatedabcoproprietyslowfademolecular probes (invitrogen)prolong antifade kitmolecular probes (invitrogen)vectashieldvector l

30、aboratoriesphotobleaching and quenching - 3frap (fluoresence recovery after photobleaching)photobleaching and quenching - 4x phenomenon of photobleaching is exploited in frapx frap- learn how dynamic a protein is by monitoring recovery of fluoresence after photobleachingbleachtime taken to recoverqu

31、enchingphotobleaching and quenching - 5x quenching - reduced fluoresence intensity as a result of the presence of oxidizing agents or the presence of salts of heavy metals or halogen compoundsx quenching reduces emissionx quenching sometimes results from the transfer of energy to other “acceptor molecules” close to the excited fluorophore = resonance energy transfer x resonance energy transfer has been exploited to measure the proximity of tw