Fluorescence Microscopy

Dr. Arne Seitz PT-BIOP course, Fluorescence Microscopy, EPFL 2010

BioImaging &Optics Platform

Dr. Arne SeitzSwiss Institute of Technology (EPFL)

Faculty of Life Sciences Head of BIOIMAGING AND OPTICS – BIOP

arne.seitz@epfl.ch

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

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.

Histological stain (Absorption) like e.g H&E staining(Hematoxilin and Eosin staining)

Fluorescent dyes:Sensitivity (single molecule detection is possible)

Different staining strategies

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. 1853 143, 385-396 [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?

high energy low energy

The Atomic View

Jablonski Diagram (very simplified)

Fluorescence energy diagram

Absorbtion and Emission Spectra of Fluorophores

Stokes Shifted

=> Scattered excitation light can be efficiently separated from fluorescence

Excitation and emission spectra of fluorescent dyes

Excitation and emission spectra are not discrete.

Excitation and Emission Spectra

The profile of the emission spectra are independentof the excitation wavelength

Excitation and Emission Spectra

1. Excitation 10-15 s2. Internal conversion 10-12 s3. Solvent relaxation 10-11 s4. Fluorescence 10-9 s

Saturation of excited state possible

5. Intersystem crossing 10-9 s6. Phosphorescence 10-3 s

Jablonski Diagram (simplified)

BleachingBleaching is irreversible (=fluorophore is destroyed)Bleaching is dependent on the excitation powerBleaching can also cause photodamage

“bleached”

• High Absoption

• High quantum yield

• High stability, little photobleaching

• Compatibility with biological systems (labeling efficiency)

Some Features of a Useful Fluorophore

Specificity (molecules can be specifically labelled)

Sensitivity (single molecule detection is possible)

Fluorescence can report on the environmentof the labelled molecule

Organelle Specific Fluorescent Stains

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

Fluorescent Stains

Mitotracker LysoSensor

Fluorescent Stains

Fluorophore Labeled Proteins/Antibodies

Fluorescein isothiocyanate(FITC)

Fluorescein

Molecules can be specifically labelled

IgG labelled IgG

Molecules can be specifically labelled

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

Protein of interest

Production of a specific antibody

Fluorescent labbeleing of the antibody

Staining of cells, tissue etc.

Alternative: Detection via a fluorescently labelled

secondary antibody

Major limitation: Targeting in live cells.

Proteins can be specifically labelled

Quantum Dots

band gap

valence band

conduction band

hνWannierexcitonen

band gap

valence band

conduction band

hνWannierexciton

Band gap increasesParticle Size decreases

Picture from: Chan WCW et al. Current Opinion in Biotechnology 2002, 13: 40-46

Size quantization effect

Quantum dotsAdvantages

Quantum yield – Similar, slightly lower as organic dyes

Absorption – Lager cross-section– Reduced photo-bleaching-rate

ZnS-capped CdSe QDs compared with Rhodamine6G– 20 time brighter– 100-200 times more stable

Sensitivity (Single Fluorophores)

Autofluorescent Proteins

488 nm

Aequorea victoria(Jellyfish) Chemistry Nobel price 2008

Osamu Shimomura

Martin Chalfie

Roger Y. Tsien

Green Fluorescent Protein (GFP)

Two Most common applications of GFP variants

From Chudakov et al, Trends Biotech., 2005

Applications of fluorescent proteins (FP)

nucleus nucleolus nuclear envelope cytoplasm

mitochondria peroxisomes microtubules

focal adhesions endoplasmic reticulum Golgi plasma membrane

nucleus + cytoplasm

http://gfp-cdna.embl.de/index.html

Protein Localization

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

Summary

Fluorescent Microscopy

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

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 beremoved or attenuated by a factor up to ≈ 1011

Most excitation light

Fluorescence detection

Sample

Objective

Excitation Light

Epifluorescence

Sample

Objective

Fluorescence

Back-scattered excitation light:

IE/100

Epifluorescence

Sample

Excitation LightDichroic mirror

(passes green but reflects blue light)

Objective

Epifluorescence

Sample

Detector

Fluorescence

Back-scattered excitation light

IE/100

Dichroic mirror(passes green but reflects blue light)

Objective

Epifluorescence

Sample

Detector

IE/100

IE/10,000

Objective

Fluorescence

Epifluorescence (real world)

Sample

Detector

IE/100

Emission filter(passes fluorescence but notback-scattered excitation light)

IE/10,000

IE/1011

Objective

Fluorescence

Epifluorescence (real world)

Sample

Detector

Scattered light

Dichroic mirror

Emission Filterwheel

Excitation Filterwheel

Alexa 488

Objective

Typical Set-Up for Epifluorescence

Sample

Detector

Scattered light

Double dichroic mirror(λ1 = 505nm +λ2 = 560nm)

Emission Filterwheel(Bandpass)

Excitation Filterwheel(Bandpass)

Alexa 488

Objective

Set-Up for Green-Red Double Fluorescence

Sample

Detector

Scattered light

Double dichroic mirror

Alexa 555

Objective

Emission Filterwheel

(Bandpass, Longpass)

Excitation Filterwheel(Bandpass)

Set-Up for Green-Red Double Fluorescence

Implementation of Epifluorescence

Transmission• Focus on the specimen• Close field diaphragm• Focus condenser until field diaphragm is seen sharp• Center field diaphragm• Close field diaphragm up to 80 – 90 %• Remove eyepiece, look down to the aperture diaphragm• Center (if possible) aperture diaphragm• Open/Close aperture diaphragm up to 80 – 90 %

Fluorescence• Focus on the specimen• Swing in focusing aid (if available) • Focus image of arc sharply• Swing out focusing aid• Close field diaphragm• Center field diaphragm

Köhler illumination in Epifluorescence

Typical filter profiles

Longpass Bandpass Shortpass

Typical Triple Bandpass Filter

DAPI GFP TexasRed

Single Color Detection (e.g. GFP)

Use longpass filter in the emission!

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

GFP-TRITC Detection Filter Cubes

Bandpass emission filters are necessary in multicolor imaging

GFP-detection TRITC-detection

Triple Filter Cube

Color glass filters (cheap, limited in wavelengths)

Interference filters(high flexibility in wavelengths)

Types of filters typically used

Light Sources

Must fit the fluorescent dyes

Must fit the Detectors

Light sourcesHalogen 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

Spectrum of a mercury arc lamp

⇒Ideal for excitation of GFP2, CFP and DsRed imaging butless convenient for EGFP

Spectrum of a Xenon arc lamp

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.

What is special aboutfluorescence microscopy?

Specificity (molecules can be specifically labelled)

Sensitivity (single molecule detection is possible)

Fluorescence can report on the environmentof the labelled molecule

1 nm10-9m

1 µm10-6m

1 mm10-3m

1 cm10-2m

1 m1 Å10-10m

Cells Worm HouseflyOrganelles

LMlimit

FRAPFCSFRETPALM,STORM

Molecular dynamics,molecular interactions

FRAP: Fluorescence recovery after photobleaching

FCS: Fluorescence correlation spectroscopy

FRET: Fluorescence resonance energy transfer

Light microscopyElectron microscopy

Quantum Yield ”Q” = number of emitted versus absorbed photons

kf+ knr

Lifetime “ τ ” = average time moleculespends in the excited state

k= events/sec

τ 0knr = ∑ ki

Nonfluorescent relaxation can be due to:

Donor Acceptor

Fluorescence Resonance Energy Transfer (FRET)

itatio

1−= DD kτDonor Fluorescence

itatio

( ) 1−+= TDD kkτ

QuenchedDonor Fluorescence

SensitizedEmission

Excitation

Summary

Fluorescence is dependant on the environment of the molecule.Parameters which can change due to the environment are:• Intensity• Fluorescence lifetime•Frequency (=spectral shift)

Fluorescence can be used as a reporter of the environment.

More about fluorescence microscopy1. Lecture

Biomicroscopy I + II, Prof. Theo Lasser, EPFL

2. Booksa) Principle of fluorescence spectroscopy,

Joseph R. Lackowicz, Springer 2nd edition (1999)

3. Interneta) http://micro.magnet.fsu.edub) b) Web sites of microscope manufactures

LeicaNikonOlympusZeiss

4. BIOpEPFL, SV-AI 0241, SV-AI 0140http://biop.epfl.ch/

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