Fluorescence Microscopy

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1 Fluorescence Microscopy Dr. Arne Seitz Swiss Institute of Technology (EPFL) Faculty of Life Sciences Head of BIOIMAGING AND OPTICS BIOP

2 Fluorescence Microscopy Why do we need fluorescence microscopy Basics about fluorescence Fluorescent dyes and staining procedures Fluorescent microscopy Advanced applications

3 Purpose of fluorescence microscopy Cells are usually transparent and therefore study of dynamic processes is not always easily possible. Thus a staining procedure is needed.

4 Different staining strategies Histological stain (Absorption) like e.g H&E staining (Hematoxilin and Eosin staining) Fluorescent dyes: Sensitivity / Selectivity

5 The term 'fluorescence' was coined Gabriel Stokes in his 1852 paper [1] ; the name was suggested "to denote the general appearance of a solution of sulphate of quinine and similar media". (Phil. Trans. R. Soc. Lond , [quote from page 387). The name itself was derived from the mineral fluorite (calcium difluoride), some examples of which contain traces of divalent europium, which serves as the fluorescent activator to provide a blue fluorescent emission. The fluorite which provoked the observation originally, and which remains one of the most outstanding examples of the phenomenon, originated from the Weardale region, of northern England. (from Wikipedia) What is fluorescence?

6 Absorption 10-3 *ε / (M -1 cm -1 ) CY3 CY wavelength / nm

7 Fluorescence energy diagram Jablonski Diagram (very simplified)

8 Absorbtion and Emission Spectra of Fluorophores => Scattered excitation light can be efficiently separated from fluorescence

9 Excitation and emission spectra of fluorescent dyes Stokes Shifted => Scattered excitation light can be efficiently separated from fluorescence

10 Excitation and Emission Spectra Excitation and emission spectra are not discrete.

11 Excitation and Emission Spectra The profile of the emission spectra are independent of the excitation wavelength

12 Jablonski Diagram (simplified) 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 Saturation of excited state possible 5 T1 6

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

14 Some Features of a Useful Fluorophore High Absoption High quantum yield High stability, little photobleaching Compatibility with biological systems (labeling efficiency)

15 Fluorescence Microscopy Specificity (molecules can be specifically labelled) Sensitivity (single molecule detection is possible) Fluorescence can report on the environment of the labelled molecule

16 Organelle Specific Fluorescent Stains

17 Fluorescent Stains DAPI binds DNA at AT-rich streches in the minor groove DAPI

18 Fluorescent Stains Mitotracker LysoSensor

19 Fluorophore Labeled Proteins/Antibodies

20 Molecules can be specifically labelled Fluorescein Fluorescein isothiocyanate (FITC)

21 Molecules can be specifically labelled IgG labelled IgG IgG labelled IgG

22 Molecules can be specifically labelled (e.g. Immunofluorescence)

23 Protein of interest Production of a specific antibody Proteins can be specifically labelled Fluorescent labbeleing of the antibody Staining of cells, tissue etc. Alternative: Detection via a fluorescently labelled secondary antibody Major limitation: Targeting in live cells.

24 Autofluorescent Proteins

25 Green Fluorescent Protein (GFP) 488 nm Aequorea victoria (Jellyfish) Chemistry Nobel price 2008 Osamu Shimomura Martin Chalfie Roger Y. Tsien

26 Applications of fluorescent proteins (FP) Two Most common applications of GFP variants From Chudakov et al, Trends Biotech., 2005

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28 Protein Localization nucleus nucleolus nuclear envelope cytoplasm nucleus + cytoplasm mitochondria peroxisomes microtubules focal adhesions endoplasmic reticulum Golgi plasma membrane Dr. Arne Seitz 10µm

29 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.

30 Fluorescent Microscopy Why do we need fluorescence microscopy Basics about fluorescence Fluorescent dyes and staining procedures Fluorescent microscopy Advanced applications

31 Fluorescence detection Excitation light (IE) Most excitation light Sample 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

32 Epifluorescence Sample Objective Excitation Light

33 Epifluorescence Sample Back-scattered excitation light: IE/100 Objective Fluorescence

34 Epifluorescence Sample Objective Excitation Light Dichroic mirror (passes green but reflects blue light)

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

36 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 Fluorescence Detector

37 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 Detector Emission filter (passes fluorescence but not back-scattered excitation light)

38 Typical Set-Up for Epifluorescence Sample Scattered light Objective HBO 488nm Dichroic mirror Alexa 488 Excitation Filterwheel Detector 520nm Emission Filterwheel

39 Set-Up for Green-Red Double Fluorescence Sample Scattered light Objective HBO 488nm Double dichroic mirror (λ1 = 505nm +λ2 = 560nm) Alexa 488 Excitation Filterwheel (Bandpass) Detector 520nm Emission Filterwheel (Bandpass)

40 Set-Up for Green-Red Double Fluorescence Sample Scattered light Objective HBO 550nm Double dichroic mirror Alexa 555 Excitation Filterwheel (Bandpass) Detector 590nm Emission Filterwheel (Bandpass, Longpass)

41 Implementation of Epifluorescence Dr. Arne Seitz

42 Implementation of Epifluorescence

43 Implementation of Epifluorescence

44 Typical filter profiles Longpass Bandpass Shortpass

45 Typical Triple Bandpass Filter DAPI GFP TexasRed

46 Single Color Detection (e.g. GFP)

47 Single Color Detection (e.g. GFP) Use longpass filter in the emission!

48 Double Color Detection (e.g. GFP and TRITC)

49 GFP-TRITC Detection Filter Cubes GFP-detection TRITC-detection Bandpass emission filters are necessary in multicolor imaging

50 Triple Filter Cube

51 Types of filters typically used Color glass filters (cheap, limited in wavelengths) Interference filters (high flexibility in wavelengths)

52 Light Sources Must fit the fluorescent dyes Must fit the Detectors

53 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

54 Spectrum of a mercury arc lamp Dr. Arne Seitz Ideal for excitation of GFP2, CFP and DsRed imaging but less convenient for EGFP

55 Spectrum of a Xenon arc lamp

56 Light Emitting Diode Color Variations Light emitting diodes (LED) Pro: long lifetime Fast switching No unwanted excitation Contra: flexibility intensity Color Name Wavelength Semiconductor (Nanometers) Composition Infrared 880 GaAlAs/GaAs Ultra Red 660 GaAlAs/GaAlAs Super Red 633 AlGaInP Super Orange 612 AlGaInP Orange 605 GaAsP/GaP Yellow 585 GaAsP/GaP Incandescent White 4500K (CT) InGaN/SiC 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

57 Summary Epifluorescence microscopy set-up is very sensitive. Bandpass detection filters are necessary for multicolor detection. Ideal excitation light sources should fit the dyes in use.

58 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 Dr. Arne Seitz -