Partha Roy

Transcription

1 Fluorescence microscopy Partha Roy 1

2 Lecture Outline Definition of fluorescence Common fluorescent reagents Construction ti of a fluorescence microscope Optical considerations Biological applications of fluorescence microscopy - Immunofluorescence - FRAP (Fluorescence Recovery After Photobleaching) - FRET (Fluorescence Resonance Energy Transfer) 2

3 Definition of fluorescence: Property of a molecule to absorb light (photons) at one wavelength and emit light at a different (higher) wavelength. Molecules capable of fluorescing fluorescent molecule fluorescent dye fluorochrome Fluorescent dye Protein/lipid/DNA Dye conjugated to macromolecule 3

4 λex max λem max - Stoke s shift t / i /i d 4

5 Properties of fluorescent molecule 1) Stoke s shift ( λ emission >λλ excitation ) - useful flfor microscopy 2) Extinction coefficient (ε) measure of efficiency to absorb photons 3) Quantum efficiency or yield (Q): Efficiency to emit photons = photons emitted /photons absorbed 5

6 4) Quenching : Loss of fluorescence Dynamic quenching: collision with a second non-fluorescent molecule results in transfer of energy (Reduces Q) Non-excited surrounding fluorophore or another molecule Excited fuorophore Increases with proximity(concentration) of fluorescent molecules (can reduce fluorescence protein protein by quenching) Static quenching : conjugation with a second molecule (quencher) reduces the absorption o of photons (protein can act as a quencher) e 6

7 5) Photobleaching: Fading of fluorescence as a result of repeated excitation of fluorochrome Example of photobleaching due to prolonged excitation A Compare B vs A B Requires interaction of the fluorophore with light and oxygen in the environment (fluorophore undergoes irreversible chemical modification) Reduction of photobleaching : 1) by reducing environment (oxyrase); antifade agents (n-propyl-galate) OK for fixed cells 2) by cutting down the excitation light 7 (slows down the rate) - Live cells

8 Common fluorophores and their ex/em characteristics DAPI (diamidino-2-phenylindole) 358 (UV) /461 (purplish blue) Binds to DNA (nuclear probe) Fluorescein isothiocynate FITC 494 (blue) /520 (green) High QE, photobleaches quickly Aromatic molecules Rhodamine 543 (yellow) /567 (red) Good photostability Texas Red ~ (red) /~615 (far red) 8

9 Problem with labeling cellular proteins with fluorophores: fixing the cell Fluorescent proteins GFP (green fluorescent protein): ajell jellyfish fishaequorea protein that absorbs blue light and emit green light Variants a : B(blue)FP; C(cyan)FP, Y(yellow)FP, R(red)F or DsRed RFP source: Discosoma coral E - enhanced Advantage: GFP protein Genetically engineered fluorescent protein (GFP-fusion) DNA into cell GFP is expressed when your protein expressed 9

10 Principle of fluorescence microscopy object Emission light Excitation light 1) Excite the specimen at the right wavelength 2) Collect ONLY the fluorescence emission and block the excitation light (very important) Possible because of stoke s shift (important) 10

11 Has peaks (365, 400, 440, 546, 580 nm) Good for UV excitation, excitation of DAPI (358), FITC (494), Rhodamine (543) Uniform spectral response in the visible range (useful for ion imaging) 11

12 excitation emission FITC: blue/green Principles of a fluorescence microscope 1) Excite the specimen with blue light White light P B Dichroic mirror (45 o ) Reflects most of blue (<500), Transmits higher wavelengths Excitation filter Filter passes blue (say upto 495) White light P B specimen 12

13 2) Collect the emitted light ONLY by the specimen in the green wavelength Dichroic mirror fluorescence G Y/G/B G Emission/barrier filter (allows green and higher wavelength) White light excitation emission Y/G/B G fluorescence Excitation filter specimen B G greenish blue FITC: blue/green Note : stoke s shift becomes handy 13

14 Construction of a fluorescence microscope (reflection type) eye eyepiece Objective also acts as a condenser No condenser needed 14

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16 Same principle: field diaphragm controls the field of view aperture diaphragm controls the NA of illumination i Same principle on conjugate planes 16

17 What determines the brightness of fluorescence images n α Remember light gathering power of objective NA 2 objective acts as both exciter and collector of fluorescence Excitation efficiency NA 2 Efficiency to gather emission NA 2 Brightness NA 4 Image Brightness NA 4 /M 2 Brightness 1/M 2 (M magnification) 17

18 RIGHT CHOICE of filters and dichroic is key to proper fluorescence microscopy EX DM EM Biht Brighter image, but not good dfor imaging i more than one fluorophores at a time because of wide spectral characteristics (can excite multiple fluorophores simultaneously; can collect emission from multiple fluorophores simultaneously) Rhodamine SP ex For FITC LP em For FITC FITC Rhodamine FITC Rhodamine FITC excitation emission Also loss of contrast from autofluorescence (from flavins, fatty acid etc) 18

19 Alternative: bandpass filters Bandpass ex Rhodamine For FITC Bandpass em for FITC FITC FITC Rhodamine excitation FITC Rhodamine emission Adv: specificity (good for imaging multi-fluorophore, more contrast Disadv: weaker signal Widening the bandwidth causes Excitation and Emission bleedthrough 19

20 Use of mutipass dichroic and emission filters dichroic emission DAPI (ex) Single filter cube has the Dichroic and emission filter Change exciters on the filterwheel. FITC (ex) cy5 (ex) Cy3/ Rhodamine/ Tx red (ex) 1- DAPI (em) 2-FITC (em) 3- Rh/cy3 (em) 4- Tx-red (em) 5-Cy5 (em) Chroma corp. website 20

21 Applications of fluorescence microscopy 1) To visualize molecules (structures) in cells 2) To study dynamics (mobility) of molecules in cells 3) To study protein-protein interactions in cells in real-time 21

22 1) To visualize molecules (structures) in cells a) Using a fluorescent dye that t directly binds to the molecule l of interest t Chromosome staining by DAPI (a common dye for nuclei) b) Coupling a fluorescent dye to a probe that directly binds to the molecule of interest FITC-conjugated phalloidin (binds to actin filaments) Nuclear staining by DAPI fluorophore phalloidin actin filaments 22

23 C) Immunofluorescence (using an antibody that binds to a specific molecule) 1 o antibody Molecule of interest cell X Fluorochrome attached to antibody Ex: 1 o - rabbit-anti X 2 o - goat-anti rabbit 1 o antibody Molecule of interest Fluorochrome cell 2 o antibody Direct Indirect Rhodaminephalloidin labeled actin filaments Actin filaments Cell-substrate adhesion (integrin) Integrin immunostaining showing cell-substrate adhesion (FITC staining) 23

24 2) To study molecular dynamics Time-lapse imaging of fluorescent proteins in living cell SEE the movie: microtubule dynamics (cells injected with fluorescent tbli tubulin- building block of microtubule) n[tubulin] = microtubule Shrinking microtubule Growing microtubule Inject fluor. tubulin Endogenous tubulin Fluor. tubulin is incorporated in microtubule 24

25 Measuring mobility (diffusion) of a molecule cell (high intensity) Protein conjugated with fluorochrome Exchange between bleached and Unbleached region Near Immobile (restricted movement) Diffusion constant =f (τ 1/2, area of bleached region) Freely mobile: F =F i ; Immobile molecule: F << F i 25

26 Example: FRAP of FITC-conjugated thymosinβ4 (cytoplasmic) in cell Time: -60 ms ms 1000 ms Pre-bleach Bleach Recovery Cytoplasmic proteins more diffusible Membrane proteins - often restricted in diffusion 26

27 3) To study molecular interaction in real-time FRET (Fluoroscence Resonance Energy Transfer) Principle: Transfer of energy from one fluorophore (donor) to a second fluorphore (acceptor) when two fluorophores come close in that case excitation of donor leads to emission from acceptor. (prereq: spectral overlap between donor emission and acceptor excitation) FRET efficiency 1/R 6 Donor emission (decreases) D- donor fluorophore A- acceptor fluorophore 12 1,2 interacting protein pair R o (forster distance) typically < 100 nm Donor emission Acceptor excitation Spectral overlap 27 (causing FRET)

28 Common FRET pairs Donor Acceptor BFP GFP BFP YFP BFP RFP CFP YFP CFP RFP GFP Rhodamine FITC Rhodamine FITC Cy3 Cy3 Cy5 Alexa-488 Rhodamine 28

29 Microscopic imaging: Excite Donor fluorophore (donor excitation filter) Collect emission from acceptor fluorophore (acceptor emission filter) If 1 and 2 bind: FRET between D and A fluorescence in A channel If 1 and 2 do not bind: No FRET between D and A No fluorescence in A channel Filter comb (Ex/Em): Donor/Donor: Donor distribution Acceptor/Acceptor : Acceptor distribution Donor/Acceptor: Interaction between donor and acceptor 29

30 FRET design Intramolecular FRET (donor and acceptor on the same molecule) Bimolecular FRET (donor and acceptor on different molecules) 30

31 Example: Using intermolecular FRET as a biosensor Rac a molecular switch (binds to either GTP or GDP) Active when it is bound to GTP; Inactive when it is bound to GDP Use FRET to determine when and where Rac is active When active it can bind to PAK (p21 activated kinase): No binding if it is inactive. Rac PAK Ex/Em: GFP/Alexa PBD (PAK binding domain) GFP/GFP Higher activity of Rac Higher conc. Of Rac GFP donor; A- alexa 546 (acceptor) Blue: low; red - high 31 Science 2000:290 ( )

32 Intramolecular l FRET as a biosensor (ex: monitor protease activity) i excitation I D Protease recognition sequence I A donor acceptor Protease activity: loss of FRET excitation I D I A=0 X donor acceptor 32

33 Intramolecular FRET as a biosensor (ex: receptor activation) EGFR EGF (epidermal (p growth factor) (EGF receptor) EGFR activation phosphorylation (py) of EGFR py py by EGFR itself py- phosphotyrosine Phosphatase converts pegfr to dpegfr (receptor inactivation) shc No binding between dp-egfr and shc binding between p-egfr and shc FRET construct Use FRET to determine the activation status of EGFR linker CFP YFP donor acceptor Shc EGFR Phosphorylation (binding region) Substrate (EEAEYMNMAPQ) 33

34 Inactive EGFR Shc can t bind EGFR phosphorylation substrate Longer distance between CFP and YFP: Low FRET CFP Shc (binding region) YFP EGFR Phosphorylation Substrate (EEAEYMNMAPQ) (No phosphorylation of Y) Active EGFR Shc binds to (p)egfr substrate Smaller distance between CFP and YFP: high FRET CFP YFP shc py by EGFR YFP/CFP ratio Fibroblast with EGFR construct and EGF stimulation Ting et al.,