BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

Internal course 2014

January 14th

FLUORESCENCE MICROSCOPY

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

FLUORESCENCE

MICROSCOPY

Why do we need it?

- 2-

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

3

Missing specificity

UNSTAINED SPECIMEN

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

Histological stain (Absorption)

like e.g H&E staining

(Hematoxilin and Eosin staining)

Fluorescent dyes:

Sensitivity

DIFFERENT STAINING

STRATEGIES

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

FLUORESCENCE

MICROSCOPY

Basic principles

- 5-

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

ORIGIN OF FLUORESCENCE

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

ABSORPTION

400 450 500 550 600 650 700

0

50

100

150

200

250

10

-3*/ (

M-1c

m-1)

wavelength / nm

CY3

CY5

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

Jablonski Diagram

(very simplified)

FLUORESCENCE

ENERGY DIAGRAM

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

Excitation and emission

spectra are not discrete.

EXCITATION AND EMISSION

SPECTRA

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

EXCITATION AND EMISSION

SPECTRA

Scattered excitation light can be efficiently separated

from fluorescence

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

The profile of the emission spectra are independent

of the excitation wavelength

EXCITATION AND EMISSION

SPECTRA

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

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

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

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

FLUORESCENCE

MICROSCOPY

Detection

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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

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

Sample

Objective

Excitation Light

EPIFLUORESCENCE

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

Sample

Objective

Fluorescence

Back-scattered

excitation light:

IE/100

EPIFLUORESCENCE

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

Sample

Excitation LightDichroic 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

EPIFLUORESCENCEREAL 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

EPIFLUORESCENCEREAL WORLD

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

Sample

HBO

Detector

Scattered light

Dichroic mirror

Emission

Filterwheel

Excitation

Filterwheel

Alexa 488

488nm

520nm

Objective

EPIFLUORESCENCE

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

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

BIOIMAGING AND

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)

DOUBLE FLUORESCENCE

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

FLUORESCENCE

MICROSCOPY

Implementation

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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

TYPICAL FILTER PROFILES

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

FILTER CHARACTERISTICS AND

NOMENCLATURE

29

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

ANATOMY OF AN INTERFERENCE

FILTER

30

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

Use longpass filter in the emission!

SINGLE COLOR DETECTION

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

Bandpass emission filters are necessary in multicolor imaging

GFP-detection TRITC-detection

GFP-TRITC DETECTION FILTER

CUBES

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

FLUORESCENCE

MICROSCOPY

Excitation

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BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

FLUORESCENCE MICROSCOPY

• Why do we need fluorescence microscopy?

•Basics about fluorescence

•Fluorescence detection

•Fluorescent excitation

•Fluorescent dyes and staining procedures

•Applications

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

Ideal for excitation of GFP2, CFP and DsRed imaging but

less convenient for EGFP

SPECTRUM OF A MERCURY

ARC LAMP

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

SPECTRUM OF A XENON ARC

LAMP

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

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)

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

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

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

FLUORESCENCE

MICROSCOPY

Staining strategies

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BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

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

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

SMALL MOLECULE STAINING

Mitotracker LysoSensor

DAPI

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

IgG labelled IgG

IgG labelled IgG

ANTIBODIES AS PROBES

Advantages: high-specificity

Disadvantages: availability

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

Major limitation: Targeting in live cells.

ANTIBODY

LABELLING WORKFLOW

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

488 nm

Aequorea victoria

(Jellyfish) Chemistry Nobel price 2008

Osamu

Shimomura

Martin

ChalfieRoger Y.

Tsien

GREEN FLUORESCENT

PROTEIN

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

The fluorescent protein palette: tools for cellular imaging.

Richard N. Day and Michael W. Davidson

Chem. Soc. Rev., 2009, 38, 2887–2921.

GREEN FLUORESCENT

PROTEIN

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

APPLICATIONS OF

FLUORESCENT PROTEINS (FP)

Nathan C. Shaner, Paul A. Steinbach & Roger Y. Tsien

Nat Methods. 2005 Dec;2(12):905-9.

Chudakov DM, Lukyanov S, Lukyanov KA.

Trends Biotechnol. 2005 Dec;23(12):605-13.

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

(A) mOrange2-b-actin-C-7.

(B) mApple-Cx43-N-7.

(C) mTFP1-fibrillarin-C-7.

(D) mWasabi-cytokeratin-N-17.

(E) mRuby-annexin (A4)-C-12.

(F) mEGFP-H2B-N-6.

(G) EBFP2-b-actin-C-7.

(H) mTagRFP-T-mitochondria-N-7.

(I) mCherry-C-Src-N-7.

(J) mCerulean-paxillin-N-22.

(K) mKate-clathrin (light chain)-C-15.

(L) mCitrine-VE-cadherin-N-10.

(M) TagCFPlysosomes-C-20.

(N) TagRFP-zyxin-N-7.

(O) superfolderGFP-lamin B1-C-10.

(P) EGFP-a-v-integrin-N-9.

(Q) tdTomato-Golgi-N-7.

(R) mStrawberry-vimentin-N-7.

(S) TagBFP-Rab-11a-C-7.

(T) mKO2-LC-myosin-N-7.

(U) DsRed2-endoplasmic reticulum-N-5.

(V) ECFP-atubulin-C-6.

(W) tdTurboRFP-farnesyl-C-5.

(X) mEmerald-EB3-N-7.

(Y) mPlum-CENP-B-N-22.

FLUORESCENT PROTEINS

The fluorescent protein palette: tools for cellular imaging.

Richard N. Day and Michael W. Davidson, Chem. Soc. Rev., 2009, 38, 2887–2921.

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

Re

ym

on

dL

, L

ukin

avič

ius

G,

Um

eza

wa

K,

Ma

ure

lD

, B

run

MA

, M

ash

arin

aA

, B

ojk

ow

ska

K, M

ollw

itz

B,

Sch

en

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, G

riss

R,

Jo

hn

sso

n K

.

Ch

imia

(Aa

rau

). 2

011;6

5(1

1):

868

-71.

FlAsH;ReAsH Fluoresceine, Oxazine

TMP-tag Fluorescein, Atto655

SNAP-tag Tetramethylrhodamine (TMR), Atto633

HaloTag TMR

Coumarine ligase Coumarin

TAG TECHNOLOGY

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

LABELLING STRATEGIES

SUMMARY

Fixed specimens

• Small, organelle specific molecules

• Pro: availability, stability

• Con: low specificity

• Antibody labeled with fluorophore

• Pro: flexibility, specificity

• Con: complex production, stability

Living specimens

• Small, organelle specific molecules

• Pro: ease of use,

• Con: Toxicity, permeability, specificity

• Fluorescent Proteins

• Pro: intrinsic labeling, known stoichiometry, flexibility

• Con: slow, genetic tools

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

FLUOROPHORESBRIGHTNESS DEFINITION

•ε : molar decadic

extinction coefficient

E= ε(l)*c*d

E=-lg T

•QE: Quantum efficiency

Brightness: B=QE* ε(l)

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

FLUOROPHORESBRIGHTNESS DEFINITION

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

Dye Class Examples Brightness Environment

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

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

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

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

FLUORESCENCE

MICROSCOPY

Resolution

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BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

RESOLUTION

Lateral resolution

Axial resolution

2NA2

nR

z

l

NA61.0

l R

l=540 nm, NA=1.4, n=1.52: 235 nm - lateral, 838 nm - axial

Shortest distance between two points on a

specimen that can still be distinguished by the

observer or camera as separate entities.

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

POINT SPREAD FUNCTION

1E-6

1E-5

1E-4

1E-3

0.01

0.1

1

1E-6

1E-5

1E-4

1E-3

0.01

0.1

1

NA=0.8 NA=1.4

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

LOW PASS FILTERING

Object

Imag

e

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

1 nm

10-9m

1 µm

10-6m

1 mm

10-3m

1 cm

10-2m

1 m1 Å

10-10m

Cells Worm HouseflyOrganelles

light microscopy

resolution limit

Homo sapiens

Light

MicroscopyProteins

RNA

Atom

Molecule

LIGHT MICROSCOPY IN LIFE

SCIENCE

Lateral resolution

obj

AiryNA

r 061.0l

Lord Rayleigh

sin2

0min

nd

l

Ernst Abbe

sinnNA58

BIOIMAGING AND

OPTICS PLATFORM

EPFL–SV–PTBIOP

• Epifluorescence microscopy set-up is very sensitive.

• Ideal excitation light sources should fit the dyes in use.

• Specificity (molecules can be specifically labelled)

• Sensitivity (single molecule detection is possible)

• Fluorescence can report on the environment

of the labelled molecule

SUMMARY

BIOIMAGING AND

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