BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

FLUORESCENCE MICROSCOPY

PT BIOP COURSE 2012

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

FLUORESCENCE MICROSCOPY

• Why do we need fluorescence microscopy

•Basics about fluorescence

•Fluorescent dyes and staining procedures

•Fluorescent microscopy

•Advanced applications

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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.

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Histological stain (Absorption)

like e.g H&E staining

(Hematoxilin and Eosin staining)

Fluorescent dyes:

Sensitivity / Selectivity

DIFFERENT STAINING STRATEGIES

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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?

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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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OPTICS PLATFORM

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Jablonski Diagram

(very simplified)

FLUORESCENCE ENERGY DIAGRAM

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ABSORBTION/EMISSION SPECTRA

OF FLUOROPHORES

Scattered excitation light can be efficiently separated

from fluorescence

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Excitation and emission spectra

are not discrete.

EXCITATION AND EMISSION SPECTRA

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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

4. Fluorescence 10-9 s Saturation of excited

state possible

5

6

T1

5. Intersystem crossing 10-9 s

6. Phosphorescence 10-3 s

JABLONSKI DIAGRAM (SIMPLIFIED)

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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

”

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

FLUORESCENT MICROSCOPY

• Why do we need fluorescence microscopy

•Basics about fluorescence

•Fluorescent dyes and staining procedures

•Fluorescent microscopy

•Advanced applications

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

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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EPFL–SV–PTBIOP

Sample

Objective

Excitation Light

EPIFLUORESCENCE

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EPFL–SV–PTBIOP

Sample

Objective

Fluorescence

Back-scattered

excitation light:

IE/100

EPIFLUORESCENCE

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OPTICS PLATFORM

EPFL–SV–PTBIOP

Sample

Excitation Light Dichroic mirror

(passes green but reflects blue light)

Objective

EPIFLUORESCENCE

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

Sample

Detector

Fluorescence

Back-scattered

excitation light

IE/100

Dichroic mirror (passes green but reflects blue light)

Objective

EPIFLUORESCENCE

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

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

EPIFLUORESCENCE (REAL WORLD)

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

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

EPIFLUORESCENCE (REAL WORLD)

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

Sample

HBO

Detector

Scattered light

Dichroic mirror

Emission

Filterwheel

Excitation

Filterwheel

Alexa 488

488nm

520nm

Objective

TYPICAL SET-UP FOR 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

SET-UP DOUBLE FLUORESCENCE

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OPTICS PLATFORM

EPFL–SV–PTBIOP

Sample

HBO

Detector

Scattered light

Double dichroic mirror

Alexa 555

550nm

590nm

Objective

Emission

Filterwheel (Bandpass, Longpass)

Excitation

Filterwheel (Bandpass)

SET-UP FOR DOUBLE FLUORESCENCE

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OPTICS PLATFORM

EPFL–SV–PTBIOP

IMPLEMENTATION OF

EPIFLUORESCENCE

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

IMPLEMENTATION OF

EPIFLUORESCENCE

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

IMPLEMENTATION OF

EPIFLUORESCENCE

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

Longpass Bandpass Shortpass

TYPICAL FILTER PROFILES

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DAPI GFP TexasRed

TYPICAL TRIPLE BANDPASS

FILTER

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SINGLE COLOR DETECTION (E.G. GFP)

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Use longpass filter in the emission!

SINGLE COLOR DETECTION

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DOUBLE 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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TRIPLE FILTER CUBE

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Color glass filters (cheap, limited in wavelengths)

Interference filters (high flexibility in wavelengths)

TYPES OF FILTERS TYPICALLY

USED

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Must fit the fluorescent dyes

Must fit the Detectors

LIGHT SOURCES

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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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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 880 GaAlAs/GaA

s

Ultra Red 660 GaAlAs/GaAl

As

Super Red 633 AlGaInP

Super

Orange 612 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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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.

SUMMARY

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Specificity (molecules can be specifically labelled)

Sensitivity (single molecule detection is possible)

Fluorescence can report on the environment

of the labelled molecule

FLUORESCENCE MICROSCOPY

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DAPI

DAPI binds DNA at

AT-rich stretches in

the minor groove

FLUORESCENT STAINS

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Mitotracker LysoSensor

FLUORESCENT STAINS

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IgG labelled IgG

IgG labelled IgG

MOLECULES CAN BE SPECIFICALLY

LABELLED

Fluorescein isothiocyanate

(FITC)

Fluorescein

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(e.g. Immunofluorescence)

MOLECULES CAN BE

SPECIFICALLY LABELLED

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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

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EPFL–SV–PTBIOP

488 nm

Aequorea victoria

(Jellyfish) Chemistry Nobel price 2008

Osamu

Shimomura

Martin

Chalfie Roger Y.

Tsien

GREEN FLUORESCENT PROTEIN

(GFP)

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Two Most common applications of GFP variants

From Chudakov et al, Trends Biotech., 2005

APPLICATIONS OF FLUORESCENT

PROTEINS (FP)

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FLUORESCENT PROTEINS

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10mm

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

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• High Absoption

• High quantum yield

• High stability, little photobleaching

• Compatibility with biological systems

(labeling efficiency)

FEATURES OF A USEFUL FLUOROPHORE

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FLUOROPHORES BRIGHTNESS DEFINITION

•ε : molar decadic

extinction coefficient

E= ε(l)*c*d

E=-lg T

•QE: Quantum efficiency

Brightness: B=QE* ε(l)

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Dye Class Examples Brightnes

s

Environme

nt

Sensitivity

Photo-

stability

Bio-

compability

Coumarin AlexaFluor350 + ++++ + +++

Fluorescein FITC +++ ++++ + +++

BOPIDY BOPIDY FL +++ +++ ++ ++++

AlexaFluor AlexaFluor488 ++++ ++ ++++ ++++

Quantum

Dots

QDot605 +++++ + +++++ +

Cyanines CY3 +++ ++ ++++ +++

Styryl Dyes FM 1-43 ++ +++++ ++ ++

Rhodamines TRITC +++ +++ ++++ ++

Fluorescent

Proteins

EGFP ++ ++ ++ +++++

Table modified from: J. B Pawley, Handbook of Confocal Microscopy, 3rd edition, p. 355

FLOUROPHORES COMPARISON

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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.

SUMMARY

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OPTICS PLATFORM

EPFL–SV–PTBIOP

1. Lecture

Biomicroscopy I + II, Prof. Theo Lasser, EPFL

2. Books

a) Principle of fluorescence spectroscopy,

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

3. Internet

a) http://micro.magnet.fsu.edu

b) b) Web sites of microscope manufactures

Leica

Nikon

Olympus

Zeiss

4. BIOp

EPFL, SV-AI 0241, SV-AI 0140

http://biop.epfl.ch/

MORE ABOUT FLUORESCENCE

MICROSCOPY