Fluorescentna Mikroskopija

Transcript

FLUORESCENTNA MIKROSKOPIJA Dr Milica Markelić Biološki fakultet, Univerzitet u Beogradu [email protected] KONCEPT PREDAVANJA  Fluorescenca i fluorofore  Osnovni principi fluorescentne mikroskopije  Konfokalna VS. Konvencionalna Widefield fluorescentna mikroskopija  Principi konfokalne mikroskopije  Osnovni tipovi konfokalne mikroskopije  Laserska skenirajuća konfokalna mikroskopija (LSCM)  Modalitet dobijanja slike na konfokalnom mikroskopu  Biološke aplikacije konfokalne i fluorescentne mikroskopije  Fluorofore i tehnike fluorescentnog obeležavanja u mikroskopiji ŠTA JE FLUORESCENCA? FOTOLUMINISCENCIJA – apsorpcija i potom reradijacija (emisija) svetlosti • FOSFORESCENCIJA – ukoliko emisija svetlosti traje i do nekoliko sekundi po prekidu ekscitacione energije (svetlosti) • FLUORESCENCIJA – ukoliko emisija svetlosti traje samo tokom apsorpcije ekscitacijskog (svetlosnog) zračenja Sir George Stokes, 1852. Vidljiva svetlost Spektar elektromagnetnog zračenja Što je talasna dužina veća to je energija zračenja niža. Što je talasna dužina manja to je energija zračenja viša Npr. opekotine od sunca izaziva UV zračenje a ne crvena vidljiva svetlost. Jablonsky dijagram excited state - high energy 2. LOSS OF ENERGY ABSORBED LIGHT 1. EXCITATION (ABSORPTION) Excited lifetime 10-15 to 10-9 sec 3. EMISSION ground state - low energy EMITTED LIGHT Single-photon ekscitacija • • KONVENCIONALNA FLUORESCENTNA MIKROSKOPIJA KONVENCIONALNA KONFOKALNA MIKROSKOPIJA Multi-photon ekscitacija • MULTIFOTONSKA MIKROSKOPIJA SVOJSTVA FLUORESCENTNIH MOLEKULA (FLUOROFORA) Emisioni spektar Ekscitacijski spektar Štoksov pomeraj • Kvantni prinos – efikasnost fluorescentnog molekula da konvertuje apsorbovanu svetlost u emitovanu Emisioni spektar NE zavisi od talasne dužine ekscitacijske svetlosti ali intenzitet emisije zavisi. SVOJSTVA FLUOROFORA Photobleaching Fotostabilna fluorofora  Definiše se kao ireverzibilna destrukcija pobuđene fluorofore  Kako izbeći/smanjiti fotoizbeljivanje?      Kraće osvetljivanje/skeniranje Veće uveličanje i objektiv veće NA Primena emisionih filtera sa širim opsegom propuštanja Minimalni intenzitet ekscitacije Primena “antifade” reagensa (ne na živim ćelijama!) – antioksidansi – propil galat, hidrokvinon, p-fenilenediamin. Fotoizbeljena fluorofora OSNOVNI PRINCIPI FLUORESCENTNE MIKROSKOPIJE WIDEFIELD EPI-FLUORESCENTNI MIKROSKOP Kamera EPI-ILUMINACIJA (episkopska iluminacija) Filter cube Objektiv uzorak Lampa TRANS-ILUMINACIJA (dijaskopska iluminacija) OSNOVNI PRINCIPI FLUORESCENTNE MIKROSKOPIJE WIDEFIELD EPI-FLUORESCENTNI MIKROSKOP Objektiv je istovremeno i kondenzor CILJ - razdvajanje ekscitacijske od emitovane svetlosti OSNOVNI PRINCIPI FLUORESCENTNE MIKROSKOPIJE WIDEFIELD EPI-FLUORESCENTNI MIKROSKOP EMISIONI FILTER -razdvajanje pravog signala od background emisije DIHROIČKO OGLEDALO FLUO LAMPA: Hg, Xn, molibdenhalogenska, LED EKSCITACIJSKI FILTER - Selekcija opsega ex. svetlosti FILTERI – „srce“ fluorescentnog mikroskopa • • • • long-pass filteri – propuštaju svu svetlost čija je talasna dužina većaod određene a blokiraju sve ispod iste. short-pass filteri – suprotno od LP filtera bandpass filteri – prpouštaju svetlost u okviru određenog opsega Dihroičko ogledalo (beam splitter) – reflektuje svetlost kraćih a propušta svetlost većih talasnih dužina (od određene) – razdvajanje ex. od em.svetlosti  Excitacijski filteri: X  Emisioni filteri: M  Beamsplitter: bs, dc, FT ("farb teiler“ – color splitter)  480/30 = centralna talasna dužina 450nm /opseg 30 nm [+/- 15]  BP, KP = bandpass, (BP 450-490)  LP = longpass filter- propušta sve iznad prikazane vrednosti (LP 500)  SP = shortpass filter - propušta sve ispod prikazane vrednosti FILTERI – su spakovani u FILTER CUBES Ekscitacioni filter kamera Transmisija (%) Emisioni filter Ekscitacioni filter wave length (nm) Dihroičko ogl. lampa Dihroičko ogledalo Emisioni filter uzorak Ekscitacija / emisija – izbor fluorofora excitation and emission spectra of EGFP (green) and Cy5 (blue) Ex1 Em1 Ex2 Em2 excitation and emission spectra of EGFP (green) and Cy2 (blue)  Nema filtera koji može da razdvoji ove talasne dužine! KONFOKALNA VS. WIDEFIELD FLUORESCENTNA MIKROSKOPIJA FM ima brojne predosti zbog čega je u širokoj upotrebi ali i nekoliko nedostataka:  Zamućenje  Bleed-through  Photobleaching - KONFOKALNA MIKROSKOPIJA - Upotreba specifičnih talasnih dužina svetlosti (laser= - Eliminacija svetlosti van fokusa (pinhole) Biološke strukture su trodimenzionalne Widefield FM mikrografije debelih uzoraka imaju zamućen izgled usled svetlosti sakupljene sa objekata iznad i ispod fokalne ravni (van fokusa) – smanjenje kvaliteta slike Značajno povećana moć razlučivanja i kolokalizacije malih struktura i molekula uz visoku kontrastnost Kod KM osvetljava se samo mali deo uzorka, znatno manji nego kod konvencionalne FM. Widefield FM KM MONOCHROMATIC LIGHT - Kod KM ekscitacioni pinhole, fokalna ravan u uzorku i detektorski (emisioni) pinhole su u konjugovanim fokalnim ravnima – “konfokalni” - Zahvaljujući pinhole-u osvetljava se samo jedna tačka uzorka (u datom trenutku) umesto celog polja , što omogućava nastanak slika bez zamućenja PRINCIPI KONFOKALNE MIKROSKOPIJE • • • Patent Number: US003013467 • • • Marvin Minsky, 1955 Tipična widefield iluminacija se zamenjuje tačkastom iluminacijom (zahvaljujući ekscitacijskom pinhole-u) U put emitovane svetlosti umeće se apertura emisionog pinhole-a koja omogućava prolazak i detekciju samo one svetlosti koja potiče iz fokalne tačke Svetlost koja se emituje iz osvetljene tačke se putem sočiva objektiva fokusira na malu tačku u ravni slike. Tačkasti izvor svetlosti je u konjugovanom fokusu na uzorku i u ravni slike – konfokalan (“u istom fokusu”) Uzorak se skenira tačkastim osvetljivanjem a informacija slike se sakuplja sekvencionalno – tačku po tačku (orig. pomeranjem postolja, danas – pomeranjem laserskog zraka) Slika uzorka se snima (orig. osciloskopski, danas – kompjuterski) Moderni KM koriste sočivo objektiva i za fokusiranje osvetljenja i za fokusiranje slike – automatski to znači istu fokalnu ravan tj. konfokalnost.  KM su ušli u široku istraživačku upotrebu krajem 80-ih godina XX veka  Tehnološka dostignuća koja su omogućila primenu Minskijevog konfokalnog dizajna: 1. Dovoljno jaki i stabilni laseri 2. Efikasno reflektujuća dihr.ogledala i precizniji filteri 3. Unapređenje metoda skeniranja i elektronike za detekciju slike 4.Fotodetektori visoke kvantne efikasnosti i niskog šuma 5. Unapređenje metoda pripreme uzoraka 6. Brzi računari sa mogućnošću obrade slike 7. Jednostavna software rešenja za analizu slike 8. Visoka rezolucija ekrana računara 9. Bioinformatičke tehnike manipulacije slikom 10. Mogućnost čuvanja velike količine elektronsih podataka 11. Sinteza fluorofora koje su bolje usklađene sa ekscitacijskom linijom lasera Pošto se uzorak osvetljava tačku-po-tačku, a slika takođe dobija tačku-potačku, u datom trenutku se dobija samo slika jedne tačke uzorka. Da bi se detektovala kompletna slika, potrebno je da se uzorak ili snop pomera. Moderni konfokalni mikroskopi – beam scanning mikroskopi  Point scanning CM – LSCM (Laser Scanning Confocal Microscope)  Multipoint (Area) scanning CM – Nipkow (spinning) Disk Confocal Microscope Kako se formira slika? Point Scanning Principle: Scanning mirrors control beam movement in X/Y raster pattern - the image is built up point by point in each frame - RASTER SCAN Beam diameter is limited by a pinhole aperture >> field of illumination and detected signal are pointed Each frame corresponds to a plane in the z-axis – “OPTICAL SECTIONS” – no need to physically slice up the specimen to reconstruct it in three dimensions. Because light is used to image each optical section, these will be in perfect register and so can be reconstructed into a threedimensional model with ease. Optical section Z-series POINT SCANNING MICROSCOPES Laser Scanning Confocal Microscope (LSCM) How is the image formed? 1. The irradiating laser is used to excite a suitable fluorophore. 2. Emitted fluorescent light passes back through the objective and is separated from unwanted light by the use of suitable dichroic mirrors. 3. “In-focus” light after passing through the confocal pinhole is detected by a very sensitive light detector – photomultiplier tube (PMT). 4. The analogue signal from PMT is converted to a digital form and displayed on computer screen. The image could be zoomed with no loss of resolution by decreasing the ROI that was scanned by the mirrors – by placing the scanned information into the same number of pixels in the image (without changing the objective) The thickness of the optical section could be adjusted simply by changing the diameter of a pinhole. 0.5 - 1.5 µm POINT SCANNING MICROSCOPES Laser Scanning Confocal Microscope (LSCM) Optical Slicing Optical section Z-series At the heart of confocal microscopy is the ability to take thin "optical slices" through the cell or tissue of interest. - Great improvement of the image quality - Great deal of information on: • 3D structure of the object • Subcellular location of the fluorescence 3D reconstruction http://www.olympusfluoview.com/java/confocalvswidefield/index.html POINT SCANNING MICROSCOPES Laser Scanning Confocal Microscope (LSCM) Components Beam diameter is limited by a pinhole aperture >> field of illumination and detected signal are pointed POINT SCANNING MICROSCOPES Laser Scanning Confocal Microscope (LSCM) Components 1. LASERS – confocal light source Fluo lamps are too weak for point confocal systems. Strong bundled light is generated by LASERs (Light Amplification by Stimulated Emission of Radiation). There are different types of LASERs, gas lasers are predominantly used: • Argon ion • Argon-Krypton • Helium-Neon LASER produces monochromatic light of a discrete wavelength (“laser line”). For the spectral range different LASERs are needed. Depending on the microscope hardware, some of the following lines might be available: LSCM has many components including a way for several different lasers to provide excitation wavelengths and several separate detectors for various emission wavelengths • • • • • • • Argon UV Solid State Argon Krypton-Ar Helium-Cad Helium-Neon Helium-Neon ArUV Violet Ar ArKr HeCd GreNe HeNe 351-364 nm 405 nm 488-514 nm 488-568-648 nm 442 nm 543 nm 633 nm POINT SCANNING MICROSCOPES Laser Scanning Confocal Microscope (LSCM) Components 2. FILTERS – determine the spectral detection CONVENTIONAL: COLORED GLASS FILTERS AND DICHROIC MIRRORS - Inexpensive - Long useful lives - Relatively insensitive to incidence light - Low transmittance - High autofluorescence at longer wavelenghts MODERN: ACOUSTO-OPTICAL DEVICES – the adjustable cristal filters - AOTF (“excitation filter”) - AOBS (“beam splitter”) POINT SCANNING MICROSCOPES Laser Scanning Confocal Microscope (LSCM) Components 3. IMAGE DETECTORS – photomultiplier tubes (PMTs) In confocal microscopy, fluorescence emission is directed through a pinhole aperture positioned near the image plane to exclude light from fluorescent structures located away from the objective focal plane, thus reducing the amount of light available for image formation. As a result, the exceedingly low light levels most often encountered in confocal microscopy necessitate the use of highly sensitive photon detectors that do not require spatial discrimination, but instead respond very quickly with a high level of sensitivity to a continuous flux of varying light intensity. PMTs contain a photosensitive surface that captures incident photons and convert them into electrons which are then multiplied – signal amplification PMTs measure intensity without spectral information resulting in the grayscale images 512x512 1024x1024 2048x2048 More pixels: • smoother looking image - more xy information • more light exposure of specimen • larger file size • slower imaging (less temporal resolution) LUT – Look Up Table PMT1 – Channel 1 PMT2 – Channel 2 PMT3 – Channel 3 Merge The detector signals are adjusted by gain and offset such that maximum number of grey level is included in the resulting image (output). •Gain: Amplifies the input signal by multiplication which results in a higher gray level value; bright features are brought closer to saturation, general image brightness is increased. •Offset: sets the gray level of a selected background to zero volts; adjust the darkest features in the image to black. LSCM CONFIGURATION CLSM microscope In a conventional confocal scan head the photons returning from the specimen are separated based on their energies (color) by passing them through a series of filters and collecting each on separate PMTs antivibration table 37 LSCM CONFIGURATION Completely integrated electronic system MULTIPOINT SCANNING MIKROSKOPI Nipkow (spinning) disk konfokalni mikroskop Svetlost iluminatora prolazi kroz seriju pinhole-a uređene distribucije na disku. Rotiranjem diska vrši se rasterizacija i emitovana svetlost koja se vraća kroz otvore na disku će biti konfokalna. Otvori na disku (pinhole-i) služe i kao tačkasti izvori svetlosti i kao konfokalne aperture – tj. i kao ekscitacijski i kao emisioni pinhole. MULTIPOINT SCANNING MIKROSKOPI Nipkow (spinning) disk konfokalni mikroskop PREDNOSTI: 1) Brzina – više tačaka se osvetljuje istovremeno. 2) Fotonska efikasnost CCD kamere 3) Pogodno za žive uzorke – manja snaga lasera 4) Različiti izvori svetlosti mogući 5) Može da se ugradi na već postojeći FM 1) 2) 3) 4) 5) NEDOSTACI: Mala efikasnost – potrebna jaka iluminacija i jak fluorescentni signal Malo vidno polje Crosstalk između susednih pinhola – ograničava debljinu uzorka Nije moguća upotreba visoko-osetljivih detektora poput PMT Kompromis između rezolucije i ukupnog signala CONFOCAL IMAGING MODES • FLUORESCENCE IMAGING MODES o SINGLE OPTICAL SECTIONS o Z-SERIES AND 3-D IMAGING o TIME-LAPSE AND LIVE CELL IMAGING o MULTIDIMENSIONAL IMAGING o X-Z IMAGING o SPECTRAL IMAGING • REFLECTED LIGHT IMAGING MODE • TRANSMITED LIGHT IMAGING MODE CONFOCAL IMAGING MODES – Single Optical Sections optical section - basic image unit in confocal microscopy methods SINGLE-LABEL IMAGING - detection of one fluorophore PMT1 PMT2 lasers PMT3 GRAY-SCALE IMAGES pseudocolor assigned subsequently PMT1 PMT2 MULTI-LABEL (MULTI-CHANNEL) IMAGING - multiple labelled specimens sample Image collecting Simultaneous Sequential Take care to prevent bleed-through (crosstalk) between channels! Channel 1 Channel 2 Channel 3 Merge CONFOCAL IMAGING MODES – Single Optical Sections Crosstalk (Bleed-through) artifacts Green channel FITC Red channel TRITC CONFOCAL IMAGING MODES – Single Optical Sections Crosstalk Crosstalk (Bleed-through) artifacts How to test: • Turn off laser line for the ‘left-handed’ fluorochrome How to reduce: • • • Use better separated fluorochromes Put the weak signal in the ‘left-handed’ channel Sequential imaging rather than simultaneous imaging CONFOCAL IMAGING MODES – Single Optical Sections Crosstalk (Bleed-through) artifacts  Spectral properties of the available dyes limit the experimental freedom.  Often it is even difficult to clearly separate two fluorescence markers.  With more markers, the problem grows increasingly complex. Cross-talk between the FP variants at the excitation and emisson level CONFOCAL IMAGING MODES – Z-series and 3-D reconstruction Z-series (Z-stack) – series of optical sections taken along the z-axis - collected by coordinating step-by-step changes in the fine focus of the microscope with sequential image acquisition at each step - computer-controlled stepping motor that changes focus by predetermined increments Important parameters for Z-series acquisition: • Specimen preparation – 3-D distortions • Image registration – specimen movements, misaligned filter sets • Pixel resolution (“voxel”) – too few or too many optical sections • Image storage – large storage space CONFOCAL IMAGING MODES – Z-series and 3-D reconstruction y x-y y-z x z x-z X-Z and Y-Z imaging CONFOCAL IMAGING MODES – Time-lapse and Live Samples Imaging  Live imaging principal benefit – ability to observe events in cells or tissue as they happen.  Live samples are usually examined in a time-lapse mode: image selection at pre-selected time intervals, images are placed into a single file, usually viewed as a movie.  Imaging living samples with the LSCM is substantially more difficult than imaging fixed specimens, and is not always a practical option because the specimen may not tolerate the conditions involved.  Extreme care must be taken to keep your sample alive and healthy Schmitz M. et al. Nature Cell Biology 12, 886–93 (2010) CONFOCAL IMAGING MODES – Multidimensional Imaging  Z-series of the same living sample are taken at periodic intervals over time and reconstructed 3-dimensionally - 4-D imaging (3-D stereo movies or projections over time)  Collection of multichannel images as Z-series over time – 5-D imaging  Applications: - Analysis of embryonic development - Tracing cell lineages - Imaging rapid cellular processes (signal pathways, ion fluctuations…) t CONFOCAL IMAGING MODES – Spectral Imaging (spectral fingerprinting, lambda stack, lambda scan) - a region of interest in the x-y dimension (optical section) is examined along the wavelength axis to determine how pixel intensity and/or color changes due to signal level variations at different emission bands (λ planes) - A series of images within a user-defined wavelength range is recorded – each image will be recorded at a specific emission wavelength - Applications: - Measurement of the emission spectrum of new fluorophores, - Determination of the emission maximum of a fluorophore in a specific sample to optimize detection, - Detection of autofluorescence(s) where the spectrum can be unknown http://www.microscopyu.com/tutorials/flash/spectralimaging/lambdastack/index.html CONFOCAL IMAGING MODES – Reflected and Transmitted Light Imaging Reflected (Backscattered) Light Imaging - Probes that reflect light (nanogold, silver particles) collagen Transmitted Light Imaging - Bright field Phase contrast, Differential interference contrast (DIC), Dark field A transmitted light detector is used to collect light passing through the specimen, and a fiber optic light guide transmits the signal to one of the PMTs in the microscope system's scan head RL imaging BF imaging DF imaging Bright field Phase contrast DIC Dark field The transmitted light images and confocal epifluorescence images can be acquired simultaneously using the same illumination beam, ensuring that all of the images are in registration. When the images are combined or merged using image processing software, the precise location of labeled cells within the tissues can be mapped. LSCM MEASURING TECHNIQUES • DEPTH AND THICKNESS MEASUREMENTS • FLUORESCENCE INTENSITY MEASUREMENTS - CLSM software, Image J, FIJI… - calibration is necessary! - controls are necessary! • CO-LOCALIZATION MEASUREMENTS • SPECIAL MEASURING TECHNIQUES • FRET • FLIM • FRAP • … CLSM MEASURING TECHNIQUES – Co-localization Co-localization does not mean interaction! Co-localization - presence of two or more different molecules residing at the same physical location in a specimen. - Widely used to determine the relationships between various macromolecules and subcellular structures - In the context of digital imaging, the term refers to colors emitted by fluorescent molecules sharing the same pixel (voxel) in the image. - Accurate c-olocalization analysis is only possible if the fluorescence emission spectra are sufficiently well separated between fluorophores and the correct filter sets (or spectral slit widths) are using during the acquisition sequence. The ability to determine co-localization in a confocal microscope is limited by the resolution of the optical system and the wavelength of light used to illuminate the specimen. - theoretical resolution of approximately 200 nm, - in practice, this number drops to a value between 400 and 600 nm for a variety of reasons (misalignment of the microscope, refractive index fluctuations, optical aberrations, and improper specimen preparation). - Co-localization is difficult to interpret In almost all cases, however, the optical resolution limit of a perfectly tuned confocal microscope is not sufficient to determine whether two fluorescent molecules are attached to a single target, or whether they even reside within the same organelle. Software Analysis of Colocalization The degree of fluorophore colocalization in a specimen is measured by comparing color values for the equivalent pixel position in each of the acquired images – scatterplot (fluorogram) ROI - indicates threshold levels of signal to be included in the analysis microtubules/mitochondria/nuclei scatterplot co-localization mask merge SPECIAL MEASURING TECHNIQUES – FRET Förster (Fluorescence) Resonance Energy Transfer - Measurements of protein-protein interactions inside cells - Two fluorophores: emission of the first one (the donor) serves as the excitation source for the second one (the acceptor) – resonance energy transfer - FRET only occurs when the donor and the acceptor molecules are extremely close to one another, at a distance less than 100 Å or (preferable 20-50 Å) - In this way, sub-resolution molecular measurements are made SPECIAL MEASURING TECHNIQUES – FLIM, FRAP, FLIP FRAP: Fluorescence Recovery After Photobleaching - This technique uses the high light flux from a laser to locally destroy fluorophores labeling specific macromolecules to create a photobleached zone. - The observation and recording of the subsequent movement of undamaged fluorophores into the bleached zone using confocal microscopy gives a measure of molecular mobility. Confocal Microscopy Applications CONFOCAL MICROSCOPE IS (NOT) JUST A MICROSCOPE ! - CONFOCAL MICROSCOPY – EXCITING, BUT EASILY MISSLEADING TECHNOLOGY REQUIRES A WIDE RANGE OF SKILLS: IMAGE ANALYSIS LIGHT MICROSCOPY DIGITAL IMAGING SYSTEM BIOLOGY SOFTWARE LIGHT PHYSICS FLUORESCENCE LASERS BIOCHEMISTRY IMMUNOLOGY As you gain a better understanding of the various technologies that are involved in confocal microscopy you will not only produce more reliable data, but you will find that the confocal microscope is capable of gathering a great deal more information from your sample than just a "pretty picture" . FLUOROFORE (FLUOROHROMI) Fluorofore su molekuli koji imaju sposobnost fluoresciranja Generalno, poseduju aromatični prsten Npr. kod proteina veći deo fluorescence potiče od indolskog prstena triptofana Klasifikacija:  Fluorofore prisutne u uzorku  Fluorofore dodate u uzorak – fluorescentne probe http://www.lifetechnologies.com/rs/en/home/references/molecular-probes-the-handbook.html FLUOROFORE PRISUTNE U UZORKU AUTOFLUORESCENCA          B vitamins, Fatty acids Lipofuscin Serotonin Cateholamins Macrophages Neurons Sperms Aldehyde fixation (glutaraldehyde) PARAFORMALDEHID Fiksativ izbora za FM!!! FLUORESCENTNE PROBE - Dodavanje sintetskih boja ili modifikovanih jedinjenja u uzorak sa ciljem produkcije fluorescence specifičnih spektralnih karakteristika. FLUORESCENTNE PROBE – tehnike ugradnje fluorescence u specifične molekulske strukture unutar ćelija i tkiva. - Direktno bojenje specifičnih struktura - Fluorescentno obeležavanje nefluorescirajućih proba “Idealna fluorofora”: • Adekvatna ekscitacijska talasna dužina – u skladu sa lampama/laserima FM/KM • Visok kvantni prinos • Uzak emisioni spektar (pri upotrebi više fluorofora) • Emisioni spektar u skladu sa dostupnim optičkim filterima • Minimalna podložnost fotoizbeljivanju • Minimalan uticaj na ćelijske procese (kod live cell imaging-a) • Visoka specifičnost obeležavanja KLASIFIKACIJA FLUORESCENTNIH PROBA: • Fluorescentne boje • Quantum dots i nanopartikule (Ag, Au) • Fluorescentni proteini METODE APLIKACIJE FLUORESCENTNIH PROBA: • Direktno bojenje ćelijskih struktura • Fluorescentno obeležavanje nefluorescentnih proba • Obeležavanje antitela - Imunofluorescenca • Obeležavanje nukleinskih kiselina - Fluorescentna In Situ Hibridizacija (FISH) • Praćenje ćelijskih linija - Cell tracing • Obeležavanje receptora • Citohemijske aplikacije • Detekcija bioloških struktura, procesa i interakcija • Ekspresija fluorescentnih proteina FLUORESCENTNE BOJE - Direktno bojenje ćelijskih struktura (DNK, organele…) - Obeležavanje nefluorescentnih proba (antitela, proteini, lipidi, ugljeni hidrati, nukleinske kiseline…) • • Boje koje ne prodiru u žive ćelije Boje koje prodiru u žive ćelije (vitalne boje) FLUORESCENTNE BOJE Bojenje nukleinskih kiselina Brojne DNK-specifične probe: PROBA Ex. (nm) Em. (nm) NOTES DAPI Boje koje ne prodiru u žive ćelije DAPI 358 461 (blue) UV laser Propidium iodide 536 617 (red) Ethidium bromide 518 605 (red) TOTO dyes (cyanine dimers) range Blue to red YOYO, BOBO, POPO, LOLO… Cyanine monomers range Blue to red TO-PRO, LO-PRO, BO-PRO SYTOX range blue- to orange Propidium iodide Vitalne boje Hoechst 33258 352 461 (blue) Acridine orange 500 (DNA) 460 (RNA) 526 (DNA) 650 (RNA) Dihydroethidium 518 605 SYTO dyes range Blue to red UV laser Hoechst Ethidium bromide /Acridine orange Acridine orange FLUORESCENTNE BOJE Detekcija organela - Raznovrsne fluorescentne probe specifične za određene organele - Nakon obeležavanja mogu se fiksirati in situ nakon čega se može izvršiti dodatno fluorescentno obeležavanje, npr. IF PROBA Ex. (nm) Em. (nm) NOTES Mitochondrial probes MitoTrackers: - MT green - MT red - MT orange 490 578 551 516 599 576 BODIPY FL C5-ceramide 505 511 (green) 620 (red) BODIPY TR C5-ceramide 589 617 (red) 374 430-640 - Aldehyde fixable dyes - MT Green accumulates in mitochondria regardless of membrane potential, - MT Red and Orange in active mitochondria BODIPY FL C5-ceramide Golgi specific probes ER tracker Lysosomes – red ER specific probes ER-Tracker UV laser Broad emission spectrum (influenced by the polarity of the environment) DiOC7 482 504 ER of plants DilC6 549 565 (red) General membrane stain LysoTracker dyes various various LysoSensor dyes various various Lysosomal probes pH indicator, specific for lysosomes Lyso tracker Hoechst YFP FLUORESCENTNE BOJE Ostale ćelijske probe PROBA Ex. (nm) Em. (nm) LIPOtox+DAPI +CellTracker NOTES Membrane probes – hydrophobic molecules DiOC7 482 504 DilC6 549 565 (red) BODIPY FL C5-ceramide 505 511 (green) BODIPY TR C5-ceramide 589 617 (red) BODIPY C9 505 515 CellTracker dyes various various Fluorescin diacetate 488 520 (green) Lucifer Yellow 488 500-560 Bound to phospholipids Calcein Cell tracers Aldehyde fixable Neuronal tracer, aldehyde fixable Cell integrity probes – determining the permeability of cellular membranes Calcein 494 517 (green) ActinGreen ActinRed 495 555 518 565 Phalloidins various various AF-conjugates with phalloidin TubulinTracker 494 522 Taxol conjugates Cytoskeletal probes Lipid probes LIPOtox BODIPY 505/515 TubulinTracker™ FLUORESCENTNE BOJE Jonski indikatori Brojne fluorescentne probe koje menjaju spektralni odgovor prilikom vezivanja specifičnog liganda. - NEDOSTATAK - naelektrisani – ne mogu da prođu kroz membrane Rešenja: - Elektroporacija - Mikroinjektiranje - Transfekcija - Lipid-rastvorne boje - ...     Kalcijumski indikatori Indikatori drugih metalnih jona pH indikatori Probe membranskog potencijala QUANTUM DOTS fluorescentni semikonduktorski nanokristali QUANTUM DOTS Prednosti:  Visok kvantni prinos – veoma jaka fluorescenca  Izuzetna fotostabilnost – malo fotoizbeljivanja  Promena veličine istog kristala – ražličit emisioni spektar  Apsorbuju širok spektar talasnih dužina – mogućnost ekscitacije većeg broja fluorofora istom laserskom linijom  Uzak emisioni spektar – pogodne za multikolorno obeležavanje Nedostaci:  Krupne – usled dodatnih slojeva koji ih čine vodorastvornim – preko 10 nm  Citotoksičnost– ekscitacija može dovesti do oslobađanja toksičnog Cd Veličina i hidrofilnost fluorescentnih proba otežava njihov transport kroz membrane ćelije. Rešenje – sinteza fluorofora u ćeliji FLUORESCENTNI PROTEINI GFP - Prirodni fluorescentni protein prvi put izolovan iz meduze Aquorea victoria Gen za GFP lako se može zakačiti za bilo koji gen od interesa putem genske fuzije Marker unutarćelijske lokacije i kretanja proteina Kompaktna struktura – zaštita od fotoizbeljivanja i od promena uslova sredine 2008-Nobel price in chemistry Shimomura O., Chaltfie M., Tsien R.Y. FLUORESCENTNI PROTEINI The Fluorescent Protein Palette APPLICATIONS OF FLUORESCENT PROTEINS • • • • • • • • • • • • • • Reporter gene Fusion Tag - determination of subcellular location and dynamics of protein Gene Transfer - efficacy of gene transfer for the development of human gene therapy Cell lineage tracer - to trace cell lineage pH indicator - the fluorescence of particular mutants is pH sensitive Molecular proximity - FRET pairs can be created by using GFP and a longer wavelength derivative or related FRET based protease assay - FRET pair that will change in fluorescence emission on being cleaved (the FRET pair separated) by intracellular proteases. FRAP - to study the dynamics of protein of interest Calcium concentration - calcium sensitive GFP-calcium chelator fusions have been developed Embryogenesis - cell lineage during embryogenesis can be followed using GFP fusion proteins Whole animal studies - whole animals can be grown with a GFP fusion present Protein degradation in vivo - fusion proteins eontaining GFP as areporter protein and the protein under study. Organelle tagging Cellular dynamics – following the dynamics of cellular processes in real time.  Relativno krupni  Mogućnost uticaja na funkciju proteina za koji su vezani  Vreme maturacije  Detekcioni limit – nije moguća amplifikacija signala  pH zavisnost  Moguće loše savijanje pri fuziji sa drugim proteinom IMMUNOFLUORESCENCA - Upotreba fluorescentno obeleženih antitela u svrhu detekcije specifičnih ciljnih proteina (antigena) - Imunohemijska tehnika - Fluorescentna imunohistohemija - Fluorescentna imunocitohemija (+) Visoka specifičnost (-) Veliki značaj otkrivanja epitopa (+) Potrebna niža koncentracija antitela (-) Mogućnost nespecifične reakcije FLUORESCENTNA IMUNOHISTO/CITOHEMIJA (IMUNOFLUORESCENCA) Opšti IF protokol Priprema uzoraka:   ĆELIJE: fiksacija/permeabilizacija TKIVA: fiksacija/kalupljenje/sečenje 1. TKIVA: Deparafinizacija i rehidracija 2. Otkrivanje epitopa (antigen retrieval) 3. Ispiranje (PBS, TBS, PB) 4. Blokiranje nespecifičnog vezivanja antitela 5. Inkubacija - primarno anititelo 6. Ispiranje 7. Inkubacija – fluorescentno obeleženo sekundarno antitelo 8. Ispiranje 9. Montiranje (u antifading medijum – medijum koji umanjuje fotoizbeljivanje) 10. Mikroskopska analiza Od ovog koraka u MRAKU! FLUORESCENTNA IN SITU HIBRIDIZACIJA (FISH) - Primena fluorescentno obeležene DNK/RNK sekvence u cilju identifikovanja položaja DNK/RNK sekvence in situ - DNK FISH proba - RNK FISH proba RNK FISH proba DNK FISH probe • Mapiranje gena – identifikacija položaja gena • Karyotyping – dijagnostika hromozomskih aberacija • Spektralni karyotyping – mFISH • Analiza interfaznih hromozoma • Komparativna genomska hibridizacija • • • • Determinacija tipa ćelije Determinacija stadijuma u diferencijaciji ćelije Detekcija abnormalne/izmenjene ekspresije iRNA Tkivna lokalizacija ekspresije određene iRNA expression Combination of different fluorescent techniques We just scratched the surface… REFERENCES  http://www.microscopyu.com  http://www.olympusmicro.com  http://micro.magnet.fsu.edu  Pawley J, Ed. “Handbook of Biological Confocal Microscopy”, 3rd ed.  White J, Paddock SW, Eds. “Confocal Microscopy Methods and Protocols”, 2nd ed.  Robert L, Price W, Gray (Jay) J, “Basic Confocal Microscopy”  The Molecular Probes® Handbook—A Guide to Fluorescent Probes and Labeling Technologies: https://www.lifetechnologies.com/rs/en/home/references/molecular-probesthe-handbook.html  Fluorescence SpectraViewer: https://www.lifetechnologies.com/rs/en/home/life-science/cellanalysis/labeling-chemistry/fluorescence-spectraviewer.html “Prilikom mikroskopiranja, zaboravite očekivanja, ne gajite predubeđenja, jer u suprotnom lako ćete napraviti grešku i videti ono što želite da vidite“. “Ne zaboravite da tragate za istinom, i ukoliko napravite grešku, ne dozvolite da vas sujeta zavede da u njoj i istrajete.“ Henry Baker, The Microscope Made Easy, 1742.