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Fluorescence Microscopy Ken Jacobson What we will cover What is fluorescence? Fluorescence microscopy: light sources, filters, objectives Special considerations: autofluorescence & photobleaching TIRF Class Exercises

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What we will cover l.jpg
What we will cover

  • What is fluorescence?

  • Fluorescence microscopy: light sources,

    filters, objectives

  • Special considerations: autofluorescence & photobleaching

  • TIRF

  • Class Exercises


Slide3 l.jpg

On line resource: Molecular Expressions, a Microscope Primer at: http://www.microscopy.fsu.edu/primer/index.html

Important reference on fluorescent probes: The Molecular

Probes catalog


Fluorescence fundamentals l.jpg
Fluorescence fundamentals

Fluorescence prop. to [ Light Absorbed]x[quantum yield]

F = I  [c] x Q

Q=[ #photons emitted/#photons absorbed]<1







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GFP Structure: fluorphore formed by cyclization

of Ser65, Tyr66 and Gly 67

M. Ormo et al, Sci. 273:1392, 1996




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Basic design of the epi fluorescence microscope

Objective acts as condenser; excitation light reflected away from eyes


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MICROSCOPE COMPONENTS

Identify

Major

Components

And

Their

Locations

And Functions

Within

Modern

Research Light

Microscope

(See Salmon

And Canman,

2000, Current

Protocols in Cell

Biology, 4.1)

Camera

Binocular

Camera Adapter

Eyepiece

Epi-Condenser

Epi-Lamp Housing

Diaphragm

Epi-Field

Diaphragm

Mirror:

& Centering

Filters

Shutter

Focus and

Beam Switch

Centering

Magnification

Changer

Filter Cube

Changer

Slot for Analyzer

Body Tube

Focus, Centering

Slot for DIC Prism

Objective Nosepiece

Objective

Stage

Trans-Lamp Housing

Condenser:

Diaphragm&Turret

Mirror:

Centering

Focus and

Focus

Centering

Slot for Polarizer

Field Diaphragm

Upright Microscope

Coarse/Fine

Filters

Lamp: Focus, Centering

Stand

Specimen Focus

and Diffuser



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Arc lamps

CAUTION: lamps at hi pressure; do not touch glass envelopes


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Type

Wattage (W)

Luminous density (cd/cm2)

Arc size

h x w (mm)

Lifetime (h)

High-pressure Mercury lamps

HBO 50W/AC

HBO 100W/2

50

100

30 000

170 000

1.0 x 0.3

0.25 x 0.25

100

200

High-pressure Xenon lamps

XBO 75W/2

75

40 000

0.5 x 0.25

400

Tungsten-Halogen lamps

12V 100W

100

4500

4.2 x 2.3

50

TECHNICAL DATA OF THE LIGHT SOURCES FOR INCIDENT-LIGHT FLUORESCENCE MICROSCOPY

From C. Zeiss

Note: small arcs with high luminous density will be brightest


Aligning the light source l.jpg
Aligning the light source

The epi fluorescence microscope is a reflected light microscope with the arc of the lamp imaged at the back focal plane of the objective, ideally just filling the back aperature (Koehler illumination).


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Works because the depth of focus of the collector lens on the lamp housing

is very long: what’s in focus at the back focal plane is ~ in focus at the specimen plane.


Objectives l.jpg
Objectives the lamp housing

High transmittance

Fluorite lenses:  > 350 nm [ok for FURA]

Quartz lenses:  < 350 nm

Employ simple, non plan lenses to minimize

internal elements.

Neglible autofluorescence or solarization [color

change upon prolonged illumination]


Maximizing image brightness b excitation efficiency na 2 b na 4 collection efficiency na 2 l.jpg

1 (NA) the lamp housing4

also B ~ => B ~ , for NA ≤ 1.0

M2 M2

Maximizing image brightness (B)excitation efficiency ~ (NA)2=> B ~ (NA)4collection efficiency ~ (NA)2

at high NA,


Filters the key to successful fluorescence microscopy l.jpg

Filters: the key to successful the lamp housingfluorescence microscopy


Slide27 l.jpg

MICROSCOPE COMPONENTS the lamp housing

Identify

Major

Components

And

Their

Locations

And Functions

Within

Modern

Research Light

Microscope

(See Salmon

And Canman,

2000, Current

Protocols in Cell

Biology, 4.1)

Camera

Binocular

Camera Adapter

Eyepiece

Epi-Condenser

Epi-Lamp Housing

Diaphragm

Epi-Field

Diaphragm

Mirror:

& Centering

Filters

Shutter

Focus and

Beam Switch

Centering

Magnification

Changer

Filter Cube

Changer

Slot for Analyzer

Body Tube

Focus, Centering

Slot for DIC Prism

Objective Nosepiece

Objective

Stage

Trans-Lamp Housing

Condenser:

Diaphragm&Turret

Mirror:

Centering

Focus and

Focus

Centering

Slot for Polarizer

Field Diaphragm

Upright Microscope

Coarse/Fine

Filters

Lamp: Focus, Centering

Stand

Specimen Focus

and Diffuser


Slide30 l.jpg

Filter cube must provide excitation, reflect the excitation onto sample while

transmitting emission, and pass the fluorescence


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Cut off filters onto sample while


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Bandpass Filters onto sample while

Most bandpass filters are interference type with multilayer

dielectric coatings that pass or reject certain wavelengths

with great selectivity




Filter selection l.jpg
Filter selection onto sample while

  • Broadband filters: more excitation, less contrast [more autofluorescence may be excited].

  • Narrowband filters:less signal, more contrast.

  • Note: eye responds to contrast while detectors respond to signal.


Multiple band pass filters l.jpg
Multiple Band-Pass Filters onto sample while

From E.D. Salmon


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Multi-Wavelength Immunofluorescence onto sample while

Microscopy


Special issues autofluorescence which causes unwanted background obscuring weak signals l.jpg

Special issues: autofluorescence onto sample whilewhich causes unwanted background obscuring weak signals


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COMMON SOURCES OF AUTOFLUORESCENCE onto sample while

Autofluorescent SourceTypical Emission Wavelength (nm)Typical Excitation Wavelength (nm)

Flavins 520 to 560 380 to 490

NADH and NADPH 440 to 470 360 to 390

Lipofuscins 430 to 670 360 to 490

Elastin and collagen 470 to 520 440 to 480

Lignin 530 488

Chlorophyll 685 (740) 488

From Biophotonics International



Photobleaching l.jpg
Photobleaching onto sample while

  • Photochemical lifetime: fluorescein will

    undergo 30-40,000 emissions before bleaching. (QYbleaching ~ 3x10-5)

  • At low excitation intensities, pb occurs but at lower rate.

  • Bleaching is often photodynamic--involves light and oxygen.


Slide42 l.jpg

Photochemistry often begins from the long-lived onto sample while

triplet state


Slide43 l.jpg

1 onto sample whileD + photon 1D*3D*

isc


Slide44 l.jpg

  • 1 onto sample whileD + photon 1D*3D*

  • isc

  • (i) 3D*+ [O] oxidized dye


Slide45 l.jpg

  • 1 onto sample whileD + photon 1D*3D*

  • isc

  • (i) 3D*+ [O] oxidized dye

  • (ii) 3D*+3O21O2+1D


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  • 1 onto sample whileD + photon 1D*3D*

  • isc

  • (i) 3D*+ [O] oxidized dye

  • (ii) 3D*+3O21O2+1D

  • +1D photobleached dye

  • 1O2

  • + other substrates ox. substrate


Slide47 l.jpg

Singlet oxygen has a lifetime of ~ 1 onto sample whiles and

a diffusion coefficient ~ 10 E-5 cm2/s.

Therefore, potential photodamage radius from fluor is ~ 50nm.


Reducing photobleaching live cells l.jpg
Reducing Photobleaching (live cells) onto sample while

Deoxygenate: Oxyrase (Ashland, OH)--bacterial

membrane fragments that reduce oxygen to water

if glucose present

OR

Catalase + glucose + glucose-oxidase to use mol

oxygen


Reducing photobleaching fixed cells anti fades l.jpg
Reducing photobleaching (fixed cells:anti-fades) onto sample while

  • Increase viscosity of medium (e.g. 95% glycerol)

  • Add singlet oxygen quenchers and free radical traps (e.g. histidine, water soluble

    caratenoids)

  • Exotic: build triplet state quenchers into flour


Reducing photobleaching anti fade reagents for fixed specimens l.jpg
Reducing Photobleaching: Anti-Fade Reagents for Fixed Specimens

  • p-phenylenediamine: The most effective reagent for FITC. Also effective for Rhodamine. Should be adjusted to 0.1% p-phenylenediamine in glycerol/PBS for use. Reagent blackens when subjected to light exposure so it should be stored in a dark place. Skin contact is extremely dangerous.G. D. Johnson & G. M. Araujo (1981) J. Immunol. Methods, 43: 349-350

  • DABCO (1,4-diazabi-cyclo-2,2,2-octane): Highly effective for FITC. Although its effect is slightly lower than p-phenylenediamine, it is more resistant to light and features a higher level of safety.G. D. Johnson et. al., (1982) J. Immunol. Methods, 55: 231-242.

  • n-propylgallate: The most effective reagent for Rhodamine, also effective for FITC. Should be adjusted to 1% propylgallate in glycerol/PBS for use. H. Giloh & J. W. Sedat (1982), Science, 217: 1252-12552.

  • mercapto-ethylamine: Used to observe chromosome and DNA specimens stained with propidium iodide, acridine orange, or Chromomysin A3. Should be adjusted to 0.1mM 2-mercaptotheylamine in Tris-EDTAS. Fujita & T. Minamikawa (1990), Experimental Medicine, 8: 75-82



Parameters for maximizing sensitivity l.jpg
Parameters for Maximizing Sensitivity Specimens

  • Use High Objective NA and Lowest Magnification:

    Ifl ~ IilNAobj4/Mtot2

    -Buy the newest objective: select for best efficiency

  • Close Field Diaphragm down as far as possible

  • Use high efficiency filters

  • Use as few optical components as possible

  • Reduce Photobleaching

  • Use High Quantum Efficiency Detector in Camera

Adapted from E.D.Salmon


Live cell considerations l.jpg
Live Cell Considerations Specimens

  • Minimize photobleaching and photodamage (shutters)

  • Use heat reflection filters for live cell imaging

  • Image quality: Maximize sensitivity and signal to noise (high transmission efficiency optics and high quantum efficiency detector)

  • Phase Contrast is Convenient to Use with Epi-Fluorescence

    • Use shutters to switch between fluorescence and phase

    • Phase ring absorbs ~ 15% of emission and slightly reduces resolution by enlarging the PSF

Adapted from E.D. Salmon



Slide55 l.jpg

EPI Specimens

TIRF


Slide56 l.jpg

Refraction Specimens

Snell’s Law:

n1sin(1)=n2sin(2)

n2

q2

n1

q1

when n1 > n2 (dense to less dense), light is bent away from the normal upon entering the less dense medium

i.e. q2>q1


Slide57 l.jpg

Total internal reflection and the critical angle Specimens

n1 sin(qc) = n2 sin(90)

n2

qr= 90

n1

qc

The critical angle for the glass-water interface  67.5


Slide58 l.jpg

d Specimens

Evanescent Wave

Z

n2

n1

qc

Intensity in Z direction

I(z)=I°e-z/d

d < l[30-300nm]

qi



Slide60 l.jpg

TIRF excitation using the objective Specimens

Iino and Kusumi, J. Fluorescence (2001)



Slide62 l.jpg

EPI Specimens

TIRF


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FRAP: Specimensfluorescence recovery after photobleaching


Slide65 l.jpg

CURRENT USES Specimens

  • Membrane and cytoplasmic diffusion

  • Exchange rates with static structures

  • Directed motion



Slide67 l.jpg

FLIP--fluorescence loss in photobleaching Specimens

Repeated photobleaching of a GFP-ER membrane protein causes

Loss of fluorescence from entire ER-->membrane continuity

Lippincott-Schwartz et al, Nat. Cell Biol. [2001]


Set magnification so that the psf corresponds to 2 3 pixels on camera l.jpg
Set magnification so that the PSF Specimenscorresponds to 2-3 pixels on camera

Example: MMax = 3*Pixel Size of Detector/Optical Res.

pixel size = 7 mm

NA = 1.4;  = 520 nm

measure of PSF dimension: 0.61 /NA

MMax = 3*7 mm/[0.6 *520nm/1.4] = 91X


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