BIOIMAGING AND OPTICS PLATFORM EPFL SV PTBIOP FLUORESCENCE MICROSCOPY

Transcription

1 FLUORESCENCE MICROSCOPY Internal course 2014 January 14 th

2 FLUORESCENCE MICROSCOPY Why do we need it? - 2-

3 UNSTAINED SPECIMEN Missing specificity 3

4 DIFFERENT STAINING STRATEGIES Histological stain (Absorption) like e.g H&E staining (Hematoxilin and Eosin staining) Fluorescent dyes: Sensitivity

5 FLUORESCENCE MICROSCOPY Basic principles - 5-

6 ORIGIN OF FLUORESCENCE

7 ABSORPTION 10-3 * / (M -1 cm -1 ) CY3 CY wavelength / nm

8 FLUORESCENCE ENERGY DIAGRAM Jablonski Diagram (very simplified)

9 EXCITATION AND EMISSION SPECTRA Excitation and emission spectra are not discrete.

10 EXCITATION AND EMISSION SPECTRA Scattered excitation light can be efficiently separated from fluorescence

11 EXCITATION AND EMISSION SPECTRA The profile of the emission spectra are independent of the excitation wavelength

12 JABLONSKI DIAGRAM SIMPLIFIED 2 S h ex 4 h emm 6 T 1 S 0 1. Excitation s 2. Internal conversion s 3. Solvent relaxation s 4. Fluorescence 10-9 s 5. Intersystem crossing 10-9 s 6. Phosphorescence 10-3 s

13 BLEACHING Bleaching is irreversible (=fluorophore is destroyed) Bleaching is dependent on the excitation power Bleaching can also cause photodamage bleached bleached

14 FLUORESCENCE MICROSCOPY Detection - 14-

15 FLUORESCENCE DETECTION Excitation light (IE) Sample Most excitation light Fluorescent light (IFL) IE/IFL = 10 4 for strong fluorescence IE/IFL = for weak fluorescence (e.g. in situ hybrid.) In order to detect the fluorescence at 10% background the excitation light must be removed or attenuated by a factor up to 10 11

16 EPIFLUORESCENCE Sample Objective Excitation Light

17 EPIFLUORESCENCE Sample Back-scattered excitation light: IE/100 Objective Fluorescence

18 EPIFLUORESCENCE Sample Objective Excitation Light Dichroic mirror (passes green but reflects blue light)

19 EPIFLUORESCENCE Sample Back-scattered excitation light IE/100 Objective Dichroic mirror (passes green but reflects blue light) Fluorescence Detector

20 EPIFLUORESCENCE REAL WORLD Back-scattered excitation light IE/100 Sample Objective Dichroic mirror (passes green but reflects blue light) Back-scattered excitation light IE/10,000 Fluorescence Detector

21 EPIFLUORESCENCE REAL WORLD Sample Back-scattered excitation light IE/100 Objective Dichroic mirror (passes green but reflects blue light) Back-scattered excitation light IE/10,000 Back-scattered excitation light IE/10 11 Fluorescence Emission filter (passes fluorescence but not back-scattered excitation light) Detector

22 EPIFLUORESCENCE Scattered light Sample Objective HBO 488nm Dichroic mirror Alexa 488 Excitation Filterwheel Detector 520nm Emission Filterwheel

23 DOUBLE FLUORESCENCE Sample Scattered light Objective HBO 488nm Double dichroic mirror (l1 = 505nm +l2 = 560nm) Alexa 488 Excitation Filterwheel (Bandpass) Detector 520nm Emission Filterwheel (Bandpass)

24 DOUBLE FLUORESCENCE Sample Scattered light Objective HBO 550nm Double dichroic mirror Alexa 555 Excitation Filterwheel (Bandpass) Detector 590nm Emission Filterwheel (Bandpass, Longpass)

25 FLUORESCENCE MICROSCOPY Implementation - 25-

26 IMPLEMENTATION OF EPIFLUORESCENCE

27 IMPLEMENTATION OF EPIFLUORESCENCE

28 TYPICAL FILTER PROFILES

29 FILTER CHARACTERISTICS AND NOMENCLATURE 29

30 ANATOMY OF AN INTERFERENCE FILTER 30

31 SINGLE COLOR DETECTION Use longpass filter in the emission!

32 GFP-TRITC DETECTION FILTER CUBES GFP-detection TRITC-detection Bandpass emission filters are necessary in multicolor imaging

33 FLUORESCENCE MICROSCOPY Excitation - 33-

34 FLUORESCENCE MICROSCOPY Why do we need fluorescence microscopy? Basics about fluorescence Fluorescence detection Fluorescent excitation Fluorescent dyes and staining procedures Applications

35 SPECTRUM OF A MERCURY ARC LAMP Ideal for excitation of GFP2, CFP and DsRed imaging but less convenient for EGFP

36 SPECTRUM OF A XENON ARC LAMP

37 LIGHT EMITTING DIODES (LED) Pro: long lifetime Fast switching No unwanted excitation Contra: flexibility intensity Color Name Wavelength (Nanometers ) Infrared 880 Ultra Red 660 Semiconduct or Composition GaAlAs/GaA s GaAlAs/GaAl As Super Red 633 AlGaInP Super Orange 612 AlGaInP Orange 605 GaAsP/GaP Yellow 585 GaAsP/GaP Incandescen t 4500K (CT) InGaN/SiC White Pale White 6500K (CT) InGaN/SiC Cool White 8000K (CT) InGaN/SiC Pure Green 555 GaP/GaP Super Blue 470 GaN/SiC Blue Violet 430 GaN/SiC Ultraviolet 395 InGaN/SiC

38 LIGHT SOURCES Halogen lamp Continuous spectrum: depends on temperature For 3400K maximum at 900 nm Lower intensity at shorter wavelengths Very strong in IR Mercury Lamp (HBO) Most of intensity in near UV Spectrum has a line structure Lines at 313, 334, 365, 406, 435, 546, and 578 nm Xenon lamp (XBO) Even intensity across the visible spectrum Has relatively low intensity in UV Strong in IR Metal halide lamp (Hg, I, Br) Stronger intensity between lines Stable output over short period of time Lifetime up to 5 times longer

39 FLUORESCENCE MICROSCOPY Staining strategies - 39-

40 LABELLING CELLULAR COMPONENTS Organelle specific dyes/molecules: Nucleus: DAPI, Hoechst. Mitochondria: DiOC 6, MitotrackerRed/Green Endoplasmic Reticulum: DiOC 6, ER tracker Golgi Aparatus: BODIPY FL Advantage: easy to use, availability Disadvantage: limited specificity

41 SMALL MOLECULE STAINING Mitotracker LysoSensor DAPI

42 ANTIBODIES AS PROBES IgG labelled IgG IgG labelled IgG Advantages: high-specificity Disadvantages: availability

43 ANTIBODY LABELLING WORKFLOW Major limitation: Targeting in live cells.

44 GREEN FLUORESCENT PROTEIN 488 nm Aequorea victoria (Jellyfish) Chemistry Nobel price 2008 Osamu Shimomura Martin Chalfie Roger Y. Tsien

45 GREEN FLUORESCENT PROTEIN The fluorescent protein palette: tools for cellular imaging. Richard N. Day and Michael W. Davidson Chem. Soc. Rev., 2009, 38,

46 APPLICATIONS OF FLUORESCENT PROTEINS (FP) Nathan C. Shaner, Paul A. Steinbach & Roger Y. Tsien Nat Methods Dec;2(12): Chudakov DM, Lukyanov S, Lukyanov KA. Trends Biotechnol Dec;23(12):

47 FLUORESCENT PROTEINS The fluorescent protein palette: tools for cellular imaging. Richard N. Day and Michael W. Davidson, Chem. Soc. Rev., 2009, 38, (A) morange2-b-actin-c-7. (B) mapple-cx43-n-7. (C) mtfp1-fibrillarin-c-7. (D) mwasabi-cytokeratin-n-17. (E) mruby-annexin (A4)-C-12. (F) megfp-h2b-n-6. (G) EBFP2-b-actin-C-7. (H) mtagrfp-t-mitochondria-n-7. (I) mcherry-c-src-n-7. (J) mcerulean-paxillin-n-22. (K) mkate-clathrin (light chain)-c-15. (L) mcitrine-ve-cadherin-n-10. (M) TagCFPlysosomes-C-20. (N) TagRFP-zyxin-N-7. (O) superfoldergfp-lamin B1-C-10. (P) EGFP-a-v-integrin-N-9. (Q) tdtomato-golgi-n-7. (R) mstrawberry-vimentin-n-7. (S) TagBFP-Rab-11a-C-7. (T) mko2-lc-myosin-n-7. (U) DsRed2-endoplasmic reticulum-n-5. (V) ECFP-atubulin-C-6. (W) tdturborfp-farnesyl-c-5. (X) memerald-eb3-n-7. (Y) mplum-cenp-b-n-22.

48 Reymond L, Lukinavičius G, Umezawa K, Maurel D, Brun MA, Masharina A, Bojkowska K, Mollwitz B, Schena A, Griss R, Johnsson K. Chimia (Aarau). 2011;65(11): TAG TECHNOLOGY FlAsH;ReAsH TMP-tag SNAP-tag HaloTag Coumarine ligase Fluoresceine, Oxazine Fluorescein, Atto655 Tetramethylrhodamine (TMR), Atto633 TMR Coumarin

49 LABELLING STRATEGIES SUMMARY Fixed specimens Small, organelle specific molecules Pro: availability, stability Con: low specificity Antibody labeled with fluorophore Pro: flexibility, specificity Con: complex production, stability Living specimens Small, organelle specific molecules Pro: ease of use, Con: Toxicity, permeability, specificity Fluorescent Proteins Pro: intrinsic labeling, known stoichiometry, flexibility Con: slow, genetic tools

50 FLUOROPHORES BRIGHTNESS DEFINITION ε : molar decadic extinction coefficient E= ε(l)*c*d E=-lg T QE: Quantum efficiency Brightness: B=QE* ε(l)

51 FLUOROPHORES BRIGHTNESS DEFINITION

52 FLOUROPHORES COMPARISON Dye Class Examples Brightness Environment Sensitivity Photostability Biocompability Coumarin AlexaFluor Fluorescein FITC BOPIDY BOPIDY FL AlexaFluor AlexaFluor Quantum Dots QDot Cyanines CY Styryl Dyes FM Rhodamines TRITC Fluorescent Proteins EGFP Table modified from: J. B Pawley, Handbook of Confocal Microscopy, 3 rd edition, p. 355

53 SUMMARY Organelles and molecules can be labeled by: Organelle and protein specific fluorescent stains (e.g. DAPI). Labeling of antibodies/proteins with fluorophores. Autofluorescent proteins (e.g. GFPs). Live cell imaging: FP (Fluorescent proteins, e.g. GFP) are the method of choice to label proteins or organelles. Injection of labeled antibodies is possible. Organelle specific stains like e.g. DAPI can be toxic for the cell.

54 FLUORESCENCE MICROSCOPY Resolution - 54-

55 RESOLUTION Shortest distance between two points on a specimen that can still be distinguished by the observer or camera as separate entities. Lateral resolution R Axial resolution l 0.61 NA R z 2 ln 2 NA l=540 nm, NA=1.4, n=1.52: 235 nm - lateral, 838 nm - axial

56 POINT SPREAD FUNCTION NA=0.8 NA= E-3 1E-4 1E-5 1E E-3 1E-4 1E-5 1E-6

57 Image LOW PASS FILTERING Object

58 LIGHT MICROSCOPY IN LIFE SCIENCE Atom Molecule Proteins RNA Light Microscopy Organelles Cells Worm Housefly Homo sapiens 1 Å m 1 nm 10-9 m 1 µm 10-6 m 1 mm 10-3 m 1 cm 10-2 m 1 m light microscopy resolution limit Lateral resolution l0 dmin 2 n sin NA n sin Ernst Abbe l 0 rairy 0.61 NAobj Lord Rayleigh 58

59 SUMMARY Epifluorescence microscopy set-up is very sensitive. Ideal excitation light sources should fit the dyes in use. Specificity (molecules can be specifically labelled) Sensitivity (single molecule detection is possible) Fluorescence can report on the environment of the labelled molecule

60 MORE ABOUT FLUORESCENCE MICROSCOPY 1. Lecture Biomicroscopy I + II, Prof. Theo Lasser, EPFL 2. Books a) Principle of fluorescence spectroscopy, Joseph R. Lackowicz, Springer 2 nd edition (1999) 3. Internet a) b) b) Web sites of microscope manufactures Leica Nikon Olympus Zeiss 4. BIOp EPFL, SV-AI 0241, SV-AI