FLUORESCENTNA MIKROSKOPIJA · digital form and displayed on computer screen. The image could be zoomed with no loss of resolution by ... Nipkow (spinning) disk konfokalni mikroskop

FLUORESCENTNA MIKROSKOPIJA

Dr Milica MarkelićBiološki 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

Sir George Stokes, 1852.

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

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.

ground state- low energy

excited state- high energy

1. EXCITATION(ABSORPTION)

2. LOSS OF ENERGY

3. EMISSION

Excited lifetime10-15 to 10-9 sec

ABSORBED LIGHT

EMITTED LIGHT

Jablonsky dijagram

Single-photon ekscitacija Multi-photon ekscitacija

• KONVENCIONALNA FLUORESCENTNA

MIKROSKOPIJA

• KONVENCIONALNA KONFOKALNA

MIKROSKOPIJA

• MULTIFOTONSKA

MIKROSKOPIJA

SVOJSTVA FLUORESCENTNIH MOLEKULA (FLUOROFORA)

• Kvantni prinos – efikasnost fluorescentnog molekula da konvertuje apsorbovanu svetlost u emitovanu

Emisioni spektar NE zavisi od talasne dužine ekscitacijske svetlosti ali intenzitet emisije zavisi.

Štoksov pomeraj

Ekscitacijski spektarEmisioni spektar

Fotostabilna fluorofora Fotoizbeljena 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.

SVOJSTVA FLUOROFORA

Photobleaching

Lampa

Filter cube

Objektiv

uzorak

Kamera

OSNOVNI PRINCIPI FLUORESCENTNE MIKROSKOPIJE

WIDEFIELD EPI-FLUORESCENTNI MIKROSKOP

TRANS-ILUMINACIJA (dijaskopska iluminacija)

EPI-ILUMINACIJA(episkopska iluminacija)

CILJ - razdvajanje ekscitacijske od emitovane svetlosti

Objektiv je istovremeno i kondenzor



FLUO LAMPA:

Hg, Xn, molibden-

halogenska, LED

EKSCITACIJSKI FILTER

EMISIONI FILTER

DIHROIČKO OGLEDALO

- Selekcija opsega ex. svetlosti

-razdvajanje pravog

signala od

background emisije



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

Transmisija

(%)

wave length (nm)

Ekscitacioni filter

Emisioni filter

Dihroičko ogl.

lampa

uzorak

kamera

Emisioni filter

Dihroičko ogledalo

Ekscitacioni filter

FILTERI – su spakovani u FILTER CUBES

Ekscitacija / emisija – izbor fluorofora

excitation and emission spectra of EGFP (green) and Cy5 (blue)

excitation and emission spectra of EGFP (green) and Cy2 (blue)

Nema filtera koji

može da razdvoji ove

talasne dužine!

Ex1 Ex2Em1 Em2

KONFOKALNA VS. WIDEFIELD FLUORESCENTNA MIKROSKOPIJA

KONFOKALNA MIKROSKOPIJA- Upotreba specifičnih talasnih dužina svetlosti (laser=- Eliminacija svetlosti van fokusa (pinhole)

Značajno povećana moć razlučivanja i kolokalizacije malih struktura i molekula uz visoku kontrastnost

FM ima brojne predosti zbog čega je u širokoj upotrebi ali i

nekoliko nedostataka:

Zamućenje

Bleed-through Photobleaching

- 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

Widefield FM KM

MONOCHROMATIC

LIGHT

Kod KM osvetljava se samo mali deo uzorka, znatno manji nego kod konvencionalne FM.

- 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

Patent Number: US003013467

PRINCIPI KONFOKALNE MIKROSKOPIJE

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-po-tačku, u datom trenutku se dobija samo slika jedne tačke uzorka. Da bi sedetektovala 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

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 three-

dimensional model with ease.

Optical section Z-series

Beam diameter is limited by a pinhole aperture

>> field of illumination and detected signal are pointed

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



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

http://www.olympusfluoview.com/java/confocalvswidefield/index.html



Components

Beam diameter is limited by a pinhole aperture

>> field of illumination and detected signal are pointed



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 ArUV 351-364 nm

• Solid State Violet 405 nm

• Argon Ar 488-514 nm

• Krypton-Ar ArKr 488-568-648 nm

• Helium-Cad HeCd 442 nm

• Helium-Neon GreNe 543 nm

• Helium-Neon HeNe 633 nm



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”)



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.

37

CLSM microscope

antivibration table

LSCM CONFIGURATION

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

LSCM CONFIGURATION

Completely integrated electronic system

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 kamere3) Pogodno za žive uzorke – manja snaga

lasera4) Različiti izvori svetlosti mogući5) Može da se ugradi na već postojeći FM

NEDOSTACI:1) Mala efikasnost – potrebna jaka

iluminacija i jak fluorescentni signal2) Malo vidno polje3) Crosstalk između susednih pinhola –

ograničava debljinu uzorka4) Nije moguća upotreba visoko-osetljivih

detektora poput PMT5) Kompromis između rezolucije i

ukupnog signala

MULTIPOINT SCANNING MIKROSKOPI

Nipkow (spinning) disk konfokalni mikroskop

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

MULTI-LABEL (MULTI-CHANNEL) IMAGING - multiple labelled specimens

Channel 1 Channel 2 Channel 3 Merge

Image collecting

- Simultaneous

- Sequential

GRAY-SCALE IMAGES

pseudocolor assigned subsequently

PMT1 PMT2 PMT3

Take care to prevent bleed-through (crosstalk) between channels!

PMT2PMT1

lasers

sample


Crosstalk (Bleed-through) artifacts

Green channel

FITC

Red channel

TRITC

Crosstalk

How to reduce:

• Use better separated fluorochromes

• Put the weak signal in the ‘left-handed’ channel

• Sequential imaging rather than simultaneous imaging

How to test:

• Turn off laser line for the ‘left-handed’ fluorochrome



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

z

x-y

x-z

y-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)

http://www.microscopyu.com/tutorials/flash/spectralimaging/lambdastack/index.html

- 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 Transmitted Light Imaging

RL imaging DF imaging BF imaging

- Probes that reflect light (nanogold, silver particles)

- collagen

- 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

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.

Bright field

Dark field

Phase contrast

DIC

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 - 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)

microtubules/mitochondria/nuclei co-localization maskscatterplot merge

ROI - indicates threshold levels of signal to be included

in the analysis

Co-localization does not mean interaction!

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 tolocally destroy fluorophores labeling specificmacromolecules to create a photobleached zone.- The observation and recording of the subsequentmovement of undamaged fluorophores into the bleachedzone using confocal microscopy gives a measure ofmolecular mobility.

Confocal Microscopy Applications

CONFOCAL MICROSCOPE IS (NOT) JUST A MICROSCOPE !

- CONFOCAL MICROSCOPY – EXCITING, BUT EASILY MISSLEADING TECHNOLOGY

- REQUIRES A WIDE RANGE OF SKILLS:

SYSTEM BIOLOGY

LIGHT MICROSCOPY

IMAGE ANALYSIS

DIGITAL IMAGING

SOFTWARE

FLUORESCENCE

BIOCHEMISTRY

IMMUNOLOGY

LASERS

LIGHT PHYSICS

As you gain a better understanding of the various technologies that are involved in confocal microscopy you will not onlyproduce more reliable data, but you will find that the confocal microscope is capable of gathering a great deal moreinformation from your sample than just a "pretty picture" .

FLUOROFORE(FLUOROHROMI)

Fluorofore su molekuli koji imaju sposobnost fluoresciranjaGeneralno, poseduju aromatični prstenNpr. 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)

PARAFORMALDEHIDFiksativ 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

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

Vitalne boje

Hoechst 33258 352 461 (blue) UV laser

Acridine orange 500 (DNA)460 (RNA)

526 (DNA)650 (RNA)

Dihydroethidium 518 605

SYTO dyes range Blue to red

DAPI

Hoechst

Propidium iodide

Acridine orangeEthidium bromide

/Acridine orange

FLUORESCENTNE BOJE

Detekcija organela


Mitochondrial probes

MitoTrackers:- MT green- MT red- MT orange

490578551

516599576

- Aldehyde fixable dyes- MT Green accumulates in mitochondria regardless of membrane potential,- MT Red and Orange in active mitochondria

Golgi specific probes

BODIPY FL C5-ceramide 505 511 (green)620 (red)

Lysosomes – red

BODIPY TR C5-ceramide 589 617 (red)

ER specific probes

ER-Tracker 374 430-640 UV laserBroad emission spectrum (influenced by the polarity of the environment)

DiOC7 482 504 ER of plants

DilC6 549 565 (red) General membrane stain

Lysosomal probes

LysoTracker dyes various various

LysoSensor dyes various various pH indicator, specific for lysosomes

- Raznovrsne fluorescentne probe specifične za određene organele

- Nakon obeležavanja mogu se fiksirati in situ nakon čega se može izvršiti dodatno fluorescentno

obeležavanje, npr. IF

BODIPY FL C5-ceramide

ER tracker

Lyso trackerHoechstYFP

TubulinTracker™

FLUORESCENTNE BOJE

Ostale ćelijske probe


Membrane probes – hydrophobic molecules

DiOC7 482 504

DilC6 549 565 (red)

BODIPY FL C5-ceramide 505 511 (green) Bound to phospholipids

BODIPY TR C5-ceramide 589 617 (red)

BODIPY C9 505 515

Cell tracers

CellTracker dyes various various Aldehyde fixable

Fluorescin diacetate 488 520 (green)

Lucifer Yellow 488 500-560 Neuronal tracer, aldehyde fixable

Cell integrity probes – determining the permeability of cellular membranes

Calcein 494 517 (green)

Cytoskeletal probes

ActinGreenActinRed

495555

518565

Phalloidins various various AF-conjugates with phalloidin

TubulinTracker 494 522 Taxol conjugates

Lipid probes

LIPOtox

BODIPY 505/515

LIPOtox+DAPI+CellTracker

Calcein

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

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

QUANTUM DOTS

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

2008-Nobel price in chemistry

Shimomura O., Chaltfie M., Tsien R.Y.

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

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

(+) Potrebna niža koncentracija antitela

(-) Mogućnost nespecifične reakcije

(+) Visoka specifičnost

(-) Veliki značaj otkrivanja epitopa

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

• Mapiranje gena – identifikacija položaja gena • Karyotyping – dijagnostika hromozomskih

aberacija• Spektralni karyotyping – mFISH• Analiza interfaznih hromozoma• Komparativna genomska hibridizacija

DNK FISH probe

RNK FISH proba

• 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-probes-the-handbook.html

Fluorescence SpectraViewer: https://www.lifetechnologies.com/rs/en/home/life-science/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.html

http://www.microscopyu.com/

http://www.olympusmicro.com/

http://micro.magnet.fsu.edu/

https://www.lifetechnologies.com/rs/en/home/references/molecular-probes-the-handbook.html

https://www.lifetechnologies.com/rs/en/home/life-science/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.html

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FLUORESCENTNA MIKROSKOPIJA · digital form and displayed on computer screen. The image could be zoomed with no loss of resolution by ... Nipkow (spinning) disk konfokalni mikroskop