FLUORESCENCE MICROSCOPY - EPFL BIOP .FLUORESCENCE MICROSCOPY •Why do we need fluorescence microscopy?

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  • BIOIMAGING AND

    OPTICS PLATFORM

    EPFLSVPTBIOP

    Internal course 2014

    January 14th

    FLUORESCENCE MICROSCOPY

  • BIOIMAGING AND

    OPTICS PLATFORM

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    FLUORESCENCE

    MICROSCOPY

    Why do we need it?

    - 2-

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    3

    Missing specificity

    UNSTAINED SPECIMEN

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    Histological stain (Absorption)

    like e.g H&E staining

    (Hematoxilin and Eosin staining)

    Fluorescent dyes:

    Sensitivity

    DIFFERENT STAINING

    STRATEGIES

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    FLUORESCENCE

    MICROSCOPY

    Basic principles

    - 5-

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    ORIGIN OF FLUORESCENCE

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    ABSORPTION

    400 450 500 550 600 650 700

    0

    50

    100

    150

    200

    250

    10

    -3*/ (

    M-1c

    m-1)

    wavelength / nm

    CY3

    CY5

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    Jablonski Diagram

    (very simplified)

    FLUORESCENCE

    ENERGY DIAGRAM

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    Excitation and emission

    spectra are not discrete.

    EXCITATION AND EMISSION

    SPECTRA

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    EXCITATION AND EMISSION

    SPECTRA

    Scattered excitation light can be efficiently separated

    from fluorescence

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    The profile of the emission spectra are independent

    of the excitation wavelength

    EXCITATION AND EMISSION

    SPECTRA

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    1. Excitation 10-15 s

    2. Internal conversion 10-12 s

    3. Solvent relaxation 10-11 s

    JABLONSKI DIAGRAM SIMPLIFIED

    1

    2

    3

    4h ex h emm

    5

    6

    T1

    S0

    S1

    4. Fluorescence 10-9 s

    5. Intersystem crossing 10-9 s

    6. Phosphorescence 10-3 s

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    BLEACHING

    Bleaching is irreversible (=fluorophore is destroyed)

    Bleaching is dependent on the excitation power

    Bleaching can also cause photodamage

    bleached

    bleached

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    FLUORESCENCE

    MICROSCOPY

    Detection

    - 14-

  • BIOIMAGING AND

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    Sample

    Excitation

    light (IE)

    Fluorescent light (IFL)

    IE/IFL = 104 for strong fluorescence

    IE/IFL = 1010 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 1011

    Most excitation light

    FLUORESCENCE DETECTION

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    Sample

    Objective

    Excitation Light

    EPIFLUORESCENCE

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    Sample

    Objective

    Fluorescence

    Back-scattered

    excitation light:

    IE/100

    EPIFLUORESCENCE

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    Sample

    Excitation LightDichroic mirror

    (passes green but reflects blue light)

    Objective

    EPIFLUORESCENCE

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    Sample

    Detector

    Fluorescence

    Back-scattered

    excitation light

    IE/100

    Dichroic mirror(passes green but reflects blue light)

    Objective

    EPIFLUORESCENCE

  • BIOIMAGING AND

    OPTICS PLATFORM

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    Sample

    Detector

    Back-scattered

    excitation light

    IE/100

    Dichroic mirror(passes green but reflects blue light)

    Back-scattered

    excitation light

    IE/10,000

    Objective

    Fluorescence

    EPIFLUORESCENCEREAL WORLD

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    Sample

    Detector

    Back-scattered

    excitation light

    IE/100

    Dichroic mirror(passes green but reflects blue light)

    Emission filter(passes fluorescence but not

    back-scattered excitation light)

    Back-scattered

    excitation light

    IE/10,000

    Back-scattered

    excitation light

    IE/1011

    Objective

    Fluorescence

    EPIFLUORESCENCEREAL WORLD

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    Sample

    HBO

    Detector

    Scattered light

    Dichroic mirror

    Emission

    Filterwheel

    Excitation

    Filterwheel

    Alexa 488

    488nm

    520nm

    Objective

    EPIFLUORESCENCE

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    Sample

    HBO

    Detector

    Scattered light

    Double dichroic mirror

    (l1 = 505nm +l2 = 560nm)

    Emission

    Filterwheel(Bandpass)

    Excitation

    Filterwheel(Bandpass)

    Alexa 488

    488nm

    520nm

    Objective

    DOUBLE FLUORESCENCE

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    Sample

    HBO

    Detector

    Scattered light

    Double dichroic mirror

    Alexa 555

    550nm

    590nm

    Objective

    Emission

    Filterwheel(Bandpass, Longpass)

    Excitation

    Filterwheel(Bandpass)

    DOUBLE FLUORESCENCE

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    FLUORESCENCE

    MICROSCOPY

    Implementation

    - 25-

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    IMPLEMENTATION OF

    EPIFLUORESCENCE

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    IMPLEMENTATION OF

    EPIFLUORESCENCE

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    TYPICAL FILTER PROFILES

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    FILTER CHARACTERISTICS AND

    NOMENCLATURE

    29

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    ANATOMY OF AN INTERFERENCE

    FILTER

    30

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    Use longpass filter in the emission!

    SINGLE COLOR DETECTION

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    Bandpass emission filters are necessary in multicolor imaging

    GFP-detection TRITC-detection

    GFP-TRITC DETECTION FILTER

    CUBES

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    FLUORESCENCE

    MICROSCOPY

    Excitation

    - 33-

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    FLUORESCENCE MICROSCOPY

    Why do we need fluorescence microscopy?

    Basics about fluorescence

    Fluorescence detection

    Fluorescent excitation

    Fluorescent dyes and staining procedures

    Applications

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    Ideal for excitation of GFP2, CFP and DsRed imaging but

    less convenient for EGFP

    SPECTRUM OF A MERCURY

    ARC LAMP

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    SPECTRUM OF A XENON ARC

    LAMP

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    Color Name

    Wavelength

    (Nanometers

    )

    Semiconduct

    or

    Composition

    Infrared 880GaAlAs/GaA

    s

    Ultra Red 660GaAlAs/GaAl

    As

    Super Red 633 AlGaInP

    Super

    Orange612 AlGaInP

    Orange 605 GaAsP/GaP

    Yellow 585 GaAsP/GaP

    Incandescen

    t

    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

    Pro:long lifetime

    Fast switching

    No unwanted excitation

    Contra:

    flexibility

    intensity

    LIGHT EMITTING DIODES (LED)

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    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

    LIGHT SOURCES

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    FLUORESCENCE

    MICROSCOPY

    Staining strategies

    - 39-

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    OPTICS PLATFORM

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    LABELLING CELLULAR

    COMPONENTS

    Organelle specific dyes/molecules:

    Nucleus: DAPI, Hoechst.

    Mitochondria: DiOC6 , MitotrackerRed/Green

    Endoplasmic Reticulum: DiOC6 , ER tracker

    Golgi Aparatus: BODIPY FL

    Advantage: easy to use, availability

    Disadvantage: limited specificity

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    SMALL MOLECULE STAINING

    Mitotracker LysoSensor

    DAPI

  • BIOIMAGING AND