FLUOROMETRI

Lecture 15

Fluorescence spectroscopy and imaging:

Basic principles and sources of contrast

Outline for Fluorescence

I. Princip-Prisip dasar FluoresensiII. Quantum Yield dan LifetimeIII. Spektroskopi Fluoresensi IV. Fluorophores BiologikV. Instrumentasi FluoresensiVI. Pengukuran Fluoresensi

I. Princip-Prinsip Dasar Fluoresensi

1. Luminesensi• Emisi fotons dari keadaan tereksitasi electronik

• Dua tipes luminesensi:

Relakasasi dari keadaan eksitasi singlet excited Relakaksasi dari keadaan eksitasi triplet

I. Princip Dasar Fluoresensi 2. Keadaan singlet dan triplet states

• Keadaan dasar – dua electron per orbital; electron punya spin berlawanan dan berpasangan

• Keadaan eksitasi singlet Electron pada orbital energi lebih tinggi memiliki arah spin berlawanan relative thd electron dalam orbital lebih rendah

• Keadaan eksitasi triplet Elektron valence tereksitasi secara spontan berbailk arah spinnya (spin flip). Proes ini disebut intersystem crossing. Electrons dlm kedua orbital sekarang memiliki arah spin sama

I. Princip Dasar Fluoresensi

3. Jenis emisi• Fluoresensi – kembali dari keadaan eksitasi singlet ke

keadaan dasar; tidak memerlukan perubahan arah spin (relaksasi yang lebih lazim)

• Fosforesensi – Kembali dari keadaan eksitasi triplet ke keadaan dasar; electron perlu perubahan arah spin

• Laji emisi fluoresensi beberapa tingkat lebih cepat daripada fosforesensi (karena perubahan arah spin perlu waktu)

I. Princip Dasar Fluoresensi4. Diagram tingkat energy (Jablonski diagram)

I. Princip Dasar Fluoresensi

5a. Proses Fluoresensi : Population level energi• Pd suhu kamar (300 K), dan level energi electronic dan

vibrational tertentu, dpt dihitung ratio molekule dalam tingkat lebih tinggi dan lebih rendah

kTE

n

n

lower

upper exp

k=1.38*10-23 JK-1 (Boltzmann’s constant)E = perbedaan level energy

I. Prinsip Dasar Fluoresensi5b. Proses Fluoresensi: Eksitasi• Pd suhu kamar, segala sesuatu berawal dari level energi

vibrational terendah pd keadaan dasar.

• Jika sebuah molecule terpapar cahaya pada frekuensi resonan, maka

• Cahaya diserap; utk larutan encer , Hkm Beer-Lambert berlaku koefisien absorpsi molar (extinction coeff) (M-1 cm-1); besarnya menggambarkan probabilitas serapan pada panjang gelombang sesuai dengan pita absorpsinya.

• Eksitasi: setelah penyerapan cahaya, suatu kromofor tereksitasi ke level energi vibrational lbh tinggi, pada level S1 atau S2

• Proses absorpti berlangsung pd skala waktu (10-15 s) jauh lbh cepat d/p vibrasi molekuler → transisi “vertical” (Franck-Condon principle).

So

S1

clA

En

ergy

nuclear configuration

I. Prinsip Dasar Fluoresensi5c. Proses Fluoresensi: relaksasi Non-radiative

• Dlm keadaan tereksitasi, elektron dipromosikan ke orbital anti-bonding → atom dlm ikatan kurang kuat terikat→ bergeser ke kanan kurva energi potential S1 →eletron terpromosikan ke level energi vibrational eksitasi S1 lebih tinggi d/p level vibrational dalam keadaan dasar.

• Deaktivasi vibrational berlangsung lewat tabrakan intermolekuler pd skala waktu10-12 s (lbh cepat d/p proses fluoresensi)

.

So

S1

I. Prinsip Dasar Fluoresensi5d. Fluorescence process: Emission

• Molekule relaksasi dari level vibrasional terendah keadaan tereksitasi menuju level energi vibrasional keadaan tereksitasi (10-9 s)

• Relaksasi ke keadaan dasar berlangsung lebih cepat d/p waktu vibrasional molekuler → transisi “vertical”

• Energy foton yg diemisikan lebih kecil

dibandingkan foton datang So

S1

I. Prinsip Dasar Fluoresensi

6a. Pergeseran Stokes • Cahaya fluorescensi adalah “red-shifted” (λ lbh panjang d/p

λ cahaya eksitasi) relatif thdp cahaya terabsorbsi. ("Stokes shift”).

• Konversi internal (see slide 13) dapat mempengaruhi pergeseran Stokes (Stoke’s shift)

• Solvent mempengaruhi reaksi keadaan tereksitasi dan juga mempengaruhi besarnya Stoke’s shift

I. Prinsip Dasar Fluoresensi6b. Invariansi panjang gelombang emission dgn

panjang gelombang eksitasi

• λ emission hanya bergantung pd waktu relaksasi ke level vibrasi

terendah pada level S1

• Utk molekul, λ emisi fluoresensi yg sama muncul berapapun λ eksitasinya

So

S1

I. Prinsip Dasar Fluoresensi6c. Aturan bayangan cermin

• Level vibrational dlm keadaan tereksitasi dan keadaan dasar adalah sama.

• Spektra absorpsi merefleksikan level vibrational keadaan eksitasi electronik

• Spektra emisi merefleksikan level vibrational electron keadaan dasar

• Spektra emisi fluorescensi merupakan bayangan cermin spektra absorpsi

S0

S1

v=0

v=1

v=2v=3v=4v=5

v’=0v’=1v’=2v’=3v’=4v’=5

I. Prinsip Dasar Fluoresensi6d. Konversion Internal vs. Emisi fluoresensi

• Jika energi electronic bertambah, level energi tumbuh more closely spaced

• Sangat boleh jadi akan overlap antara energi vibrasional tinggi level Sn-1 and energi vibrational rendah levels of Sn

• Overlap meyebabkan transisi antar states sangat mungkin

• Konversi nternal mrpk transisi yg terjadi antara states dg multiplisitas sama dan berlangsung pd skala waktu 10-12 dt (lebih cepat d/p proses fluoresensi)

• Perbedaan energi antara S1 and S0 cukup besar dibanding antar states yg berdekatan → lifetime S1 lebih lama → emisi radiative dapat efektif sempurna dengan emisi non-radiatif.

Mirror-image rule typically applies when only S0 → S1 excitation takes place

Deviations from the mirror-image rule are observed when S0 → S2 or transitions to even higher excited states also take place

I. Prinsip Dasar Fluoresensi6e. Intersystem crossing• Intersystem crossing adalah transisi non-radiative antar states (keadaan)yg

berbeda multiplisitasnya

• Ini terjadi via inversi spin electron tereksitasi menghasilkan dua elektron tak berpasangan dengan arah spin sama, memberikan keadaan dg spin = 1 dan multiplisitas 3 (triplet state)

• Transisi antar state berbeda multiplisitas pada dasarna adalah terlarang

• Spin-orbit and vibronic coupling mechanisms decrease the “pure” character of the initial and final states, memungkinkan intersystem crossing terjadi

• Transisi T1 → S0 juga forbidden → lifetime T1 sangat lebih besar d/p lifetime S1 (10-3-102 s)

S0

S1

T1absorption

fluorescencephosphorescence

Intersystemcrossing

I. Prinsip Dasar Fluoresensi

I. Prinsip Dasar FluoresensiIn

ten

sity

Wavelength

Absorbance

DONOR

Absorbance

Fluorescence Fluorescence

ACCEPTOR

Molecule 1 Molecule 2

• Fluorescence energy transfer (FRET)

Inte

nsi

ty

Wavelength

Absorbance

DONOR

Absorbance

Fluorescence Fluorescence

ACCEPTOR

Molecule 1 Molecule 2

Non radiative energy transfer – a quantum mechanical process of resonance between transition dipolesEffective between 10-100 Å onlyEmission and excitation spectrum must significantly overlapDonor transfers non-radiatively to the acceptor

II. Quantum yield and lifetime• Quantum yield of fluorescence, f, is defined as:

• In practice, is measured by comparative measurements with reference compound for which has been determined with high degree of accuracy.

• Ideally, reference compound should have– the same absorbance as the compound of interest at given excitation wavelength– similar excitation-emission characteristics to compound of interest (otherwise, instrument

wavelength response should be taken into account)– Same solvent, because intensity of emitted light is dependent on refractive index

(otherwise, apply correction

– Yields similar fluorescence intensity to ensure measurements are taken within the range of linear instrument response

absorbed photons ofnumber

emitted photons ofnumber f

)(

)(2

2

sn

un

I

Isf

uf

sf

uf

1a. Quantum yield of fluorescence

II. Quantum yield and life time• Another definition for f is

where kr is the radiative rate constant and k is the sum of the rate constants for all processes that depopulate the S1 state.

• In the absence of competing pathways f=1• Radiative lifetime, r, is related to kr

• The observed fluorescence lifetime, is the average time the molecule spends in the excited state, and it is

k

krf

rr k

1

kf

1

1b. Fluorescence lifetime

II. Quantum Yield and Lifetime

2a. Characteristics of quantum yield• Quantum yield (hasil Kuantum) fluoresensi bergantung

pd lingkungan biologis

• Contoh: spektrum eksitasi Fura 2 dan spektrum emisi Indo-1 dan hasil quantum berubah ketika terikat pada Ca2+

Fura-2 changes in response to varying [Ca2+]

Indo-1 changes in response to varying [Ca2+]

II. Quantum yield and lifetime

2b. Characteristics of life-time• Provide an additional dimension of information

missing in time-integrated steady-state spectral measurements

• Sensitive thd mikrolingkungan biochemis, termasuk pH lokal, oksigenasi dan ikatan

• Lifetimes tak terpengaruh oleh variasi in intensitas eksitasi, konsentrasi atau sumber hilangnya keoptikan

• Kompatible dg pengukuran klinik in vivo

Courtesy of M.-A. Mycek, U Michigan


3a. Fluorescence emission distribution• Utk λ eksitasi tertentu,

transisi emisi terdistribusi di antara level energi vibrational yg berbeda

• Utk λ eksitasi tunggal dpt mengukur suatu spectrumemisi fluoresensi

Inte

nsit

y

Emission Wavelength (nm)

ExcEmm


3b. Heisenberg’s uncertainty principle• Values of particular pairs of observables cannot be

determined simultaneously with high precision in quantum mechanics

• Example of pairs of observables that are restricted in this way are:

• Momentum and position

• Energy and time


3c. Heisenberg’s uncertainty principle

• Momentum and position :

• Energy and time:

2

hxpx

2

htE


3d. Effect on fluorescence emission• Suppose an excited molecule emits fluorescence in

relaxing back to the ground state

• If the excited state lifetime, is long, then emission will be monochromatic (single line)

• If the excited state lifetime, is short, then emission will have a wider range of frequencies (multiple lines)

Inte

nsit

yEmission Wavelength (nm)

Exc Emm

Inte

nsit

y


Exc Emm

Large – small Small – large

III. Fluorescence Intensity

1. Fluorescence intensity expression

2. Fluorescence spectra

III. Fluorescence Intensities

1a. Fluorescence intensityThe fluorescence intensity (F) at a particular excitation (x) and emission wavelength (m) will depend on the absorption and the quantum yield:

where,

IA – light absorbed to promote electronic transition – quantum yield

mxAmx IF ,


1b. From the Beer-Lambert law, the absorbed intensity for a dilute solution (very small absorbance)

where,

Io – Initial intensity – molar extinction coefficient

C – concentrationL – path length

1 <<CL for

CLI303.2I

x

xoxA


1c. Fluorescence intensity expressionThe fluorescence intensity (F) at a particular excitation (x) and emission wavelength (m) for a dilute solution containing a fluorophore is:

where,

Io – incident light intensity – quantum yieldC – concentration – molar extinction L – path length coefficient

mxomx CLIF 303.2,


1d. Measured fluorescence intensityIf we include instrument collection angle:

where,Z – instrumental factor

Io – incident light intensity – molar extinction coefficientC = concentrationL – path length

ZCLIF mxomx 303.2,

III. Fluorescence Intensities2a. Fluorescence spectra

• Emission spectrum– Hold excitation wavelength fixed, scan emission

– Reports on the fluorescence spectral profile

» reflects fluorescence quantum yield, k(m)

ZCLIF mxomx 303.2,

III. Fluorescence Intensities2b. Fluorescence spectra

• Excitation spectrum– Hold emission wavelength fixed, scan excitation

– Reports on absorption structure

» reflects molar extinction coefficient, (x)

ZCLIF mxomx 303.2,

Fluo

resc

ence

Int

ensi

ty


Fixed Excitation Wavelength

(b)

Fluo

resc

ence

Int

ensi

ty

Excitation Wavelength (nm)

Fixed Emission Wavelength

(a)


III. Fluorescence Intensities2c. Fluorescence spectra

• Composite: Excitation-Emission Matrix» Good representation of multi-fluorophore solution

Flu

ores

cen

ce I

nte

nsit

y


Fixed Excitation Wavelength

Exc

itat

ion

Wav

elen

gth

(n

m)


Emission spectrum Excitation-emission matrix


IV. Biological Fluorophores

1. Table

2. EEMs of Epithelial cell suspension

3. EEMs of Collagen

IV. Biological Fluorophores–Endogenous Fluorophores

amino acids

structural proteins

enzymes and co-enzymes

vitamins

lipids

porphyrins

–Exogenous Fluorophores

Cyanine dyes

Photosensitizers

Molecular markers – GFP, etc.


TCL-1Emission(nm)

Exc

itatio

n(nm

)

300 350 400 450 500 550 600 650 700250

270

290

310

330

350

370

390

410

430

450

470

490

510

530

550

9.767e-001

6.646e+004

1.306e+005

1.947e+005

4.785e+005

1.120e+006

1.761e+006

2.923e+006

9.335e+006

1.575e+007

2.216e+007

Epithelial Cell SuspensionFluorescence intensity excitation-emission

matrix

FAD

NADH

Tryp.

Courtesy of N. Ramanujam

CarbohydratesFatty Acids and Glycerol Amino Acids

Acetyl CoA

CITRIC ACID CYCLE

CoA

CO2

FADH2

NADH

NADH-QReductase

Cytochrome Reductase

Cytochrome Oxidase

Q

Cytochrome C

O2

FAD NAD+

Oxidation of NADH and FADH2 by O2 drives synthesis of ATP

ELECTRON TRANSPORT

Metabolic Indicators

Metabolism

Redox Ratio: FAD / (FAD+NADH)

Redox ratio ~ Metabolic Rate

Emission(nm)

Exc

itatio

n(nm

)

Highest value: 13223388.1 270/260Scaled @: 4916595.85 305/270

M

S

UT Austin Mar-2000 UU

300 350 400 450 500 550 600 650 700250

270

290

310

330

350

370

390

410

430

450

470

490

510

530

550

7.551e+003

2.210e+004

3.613e+004

5.016e+004

1.123e+005

2.526e+005

3.929e+005

6.473e+005

2.051e+006

3.454e+006

4.857e+006

0 days

Emission [nm]

Exc

itatio

n [n

m]

Emission(nm)

Exc

itatio

n(nm

)


M

S


300 350 400 450 500 550 600 650 700250

270

290

310

330

350

370

390

410

430

450

470

490

510

530

550

5.021e+003

1.514e+004

2.490e+004

3.467e+004

7.788e+004

1.755e+005

2.731e+005

4.501e+005

1.426e+006

2.403e+006

3.379e+006

7 days

Collagen I (gel)

K. Sokolov

Emission(nm)

Exc

itatio

n(nm

)


M

S


300 350 400 450 500 550 600 650 700250

270

290

310

330

350

370

390

410

430

450

470

490

510

530

550

1.097e+004

2.559e+004

3.969e+004

5.379e+004

1.162e+005

2.572e+005

3.982e+005

6.538e+005

2.064e+006

3.474e+006

4.883e+006

39 days


Collagen• It is the major extracellular matrix component, which is

present to some extent in nearly all organs and serves to hold cells together in discrete units

• Collagen fluorescence in load-bearing tissues is associated with cross-links, hydroxylysyl pyridoline (HP) and lysyl pyridinoline (LP).

• Collagen crosslinks are altered with age and with invasion of cancer into the extracellular matrix

V. Fluorescence Instrumentation

1. Introduction

2. Components of a spectrofluorometer

3. Description of key components


1. Introduction• Fluorescence is a highly sensitive method (can measure

analyte concentration of 10-8 M)

• Important to minimize interference from:Background fluorescence from solventsLight leaks in the instrumentStray light scattered by turbid solutions

• Instruments do not yield ideal spectra:Non-uniform spectral output of light sourceWavelength dependent efficiency of detector and

optical elemens

V. Fluorescence Instrumentation• Illumination source

– Broadband (Xe lamp)– Monochromatic (LED, laser)

• Light delivery to sample– Lenses/mirrors– Optical fibers

• Wavelength separation (potentially for both excitation and emission)– Monochromator– Spectrograph

• Detector– PMT– CCD camera

2. Major components for fluorescence instrument


Components of the spectrofluorometer (standard fluorescence lab instrument for in vitro samples)• Xenon lamp (> 250 nm)

• Excitation and emission monochromatorEach contains two gratings to increase purity of the lightAutomatic scanning of wavelength through motorized gratings

• Sample compartment

• Photo multiplier tube

PMT

Xenon Source

ExcitationMonochromator Emission

Monochromator

Sample compartment

V. Fluorescence Instrumentation2. Spectrofluorometer schematic


3a. Xenon light source• Continuous output from Xenon: 270-1100 nm

• Power – typically 200-450 W

• Lifetime of ~2000 hours

• Strong dependence on wavelength


3a. Xenon light source: broad illumination in the near UV-visible range

PMT

Xenon Source


Monochromator

Sample compartment


V. Fluorescence Instrumentation3b. Monochromator: only a small range of wavelengths are focused

at the exit slit determined by angle of light incident on the diffraction grating

constant

wavelength

mmper lines of #n

ordern diffractioK where

,10sinsin 6

D

Kn

Principle of diffraction grating operation


3b. Monochromator – Spectral ResolutionInversely proportional to product of dispersion

(nm/mm) of grating and the slit width (mm)

~ 5 nm sufficient for fluorescence measurements of

biological media

Signal increases with the slit width


3b. Monochromator – Stray light• Light which passes through monochromator besides

that of desired wavelength

• Double grating monochromator (stray light rejection is 10 -8 – 10-12) but signal is decreased


3b. Monochromator – Signal efficiency• Grating has a wavelength dependent efficiency

• Can choose the wavelength at which grating is blazed (maximal efficiency)

• Excitation monochromator should have high efficiency in the UV; emission monochromator should have high efficiency in the visible

PMT

Xenon Source


Monochromator

Sample compartment



3c. Photomultiplier tube• Contains a photocathode:

light sensitive material, which yields electrons upon interaction with photons based on photoelectric effect.

• Electrons are multiplied by a series of dynodes

• Provides current output proportional to light intensity


3c. PMT – Linearity response• Current from PMT is proportional to light intensity

• Under high intensity illumination, PMT will saturate (dynamic range); at low intensity, limited by dark noise

• Excessive light can damage photocathode, resulting in loss of gain and increased dark noise (thermal noise)


3c. PMT – Quantum efficiency• Quantum efficiency gives the photon to electron

conversion efficiency

• Highly dependent on wavelength


3c. Key components – Noise• Dark current – Noise due to thermal generation;

increases with temperature and high voltage

• Shot noise – proportional to the square root of the signal

VI. Fluorescence Measurements

VI. Fluorescence measurements1. Instrument non-uniformities2. Excitation wavelength calibration3. Emission wavelength calibration4. Setup parameters for emission spectrum5. Routine experimental procedure6. Collection geometry 7. Blank scans8. Typical fluorescence spectrum


1a. Ideal spectrofluorometer• Light source must yield constant photon output at all

wavelengths

• Monochromator must pass photons of all wavelengths with equal efficiency

• The PMT must detect photons of all wavelengths with equal efficiency

VI. Fluorescence MeasurementsL

ight

Int

ensi

ty

Wavelength

LIGHT SOURCE

Eff

icie

ncy

Wavelength

MONOCHROMATOR

Eff

icie

ncy

Wavelength

PMT


1b. Distortions in excitation and emission spectra• Light intensity from light source is a function of

wavelength

• Monochromator efficiency is a function of wavelength

• The PMT does not have equal efficiency at all wavelengths


1c. Calibration• Correction of variations in wavelength of Xenon lamp

and excitation monochromatorNeed to do when measuring excitation spectra or emission spectra at multiple excitation wavelengths

• Correction of emission monochromator and PMTNeed to do when measuring emission spectra

VI. Fluorescence Measurements2a. Excitation wavelength calibration

• Excitation spectra are distorted primarily by the wavelength dependent intensity of the light source

• Can use reference photodetector (calibrated) next to sample compartment to measure fraction of excitation light

• The measured intensity of the reference channel is proportional to the intensity of the exciting light

Io

F

SampleCompartment

To PMT

To Reference Photodiode

VI. Fluorescence Measurements2b. Effect of excitation wavelength calibration

VI. Fluorescence Measurements3a. Emission wavelength calibration

• Need correction factors• Measure wavelength dependent output from a

calibrated light source• Standard lamps of known and calibrated

spectral outputs are available from the National Institute of Standards and Testing (NIST)

• This measurement is typically done by factory; it is difficult to perform properly with commercial fluorimeter

To PMT

Sample Compartment

Calibrated Lamp


3b. Emission wavelength calibration procedure• Measure intensity versus wavelength (I()) of standard

lamp with spectrofluorometer

• Obtain the spectral output data (L()) provided for the lamp

• Correction factor: S() = L()/ I()

• Multiply emission spectrum with correction factor

VI. Fluorescence Measurements3c. Emission wavelength calibration curve

0.0E+00

2.0E+00

4.0E+00

6.0E+00

8.0E+00

1.0E+01

1.2E+01

300 350 400 450 500 550 600 650 700

Wavelength (nm)

Inte

nsi

ty (

c.u

.)


5. Routine experimental procedures• Check wavelength calibration of excitation

monochromator

• Check wavelength calibration of emission monochromator

• Check throughput of spectrofluorometer

0.0E+00

1.0E-02

2.0E-02

3.0E-02

4.0E-02

5.0E-02

6.0E-02

260 310 360 410 460 510 560

Wavelength (nm)

Inte

nsity

(cp

s)

467 nm

Xe lamp scan

Hg lamp spectrum scan

0.0E+00

2.0E+06

4.0E+06

6.0E+06

8.0E+06

1.0E+07

1.2E+07

475 525 575 625 675

Wavelength (nm)

Inte

nsity

(c.

u.)

575 nm

Rhodamine standardscan

VI. Fluorescence Measurements6a. Collection geometry in sample compartment

• Front face – collection is at a 22 degree angle relative to the incident beam; appropriate for an optically absorbing / scattering sample; more stray light

• Right angle – collection is at a right angle to the incident light; appropriate for optically transparent sample; less stray light

Io F Io

F

Front Face Right Angle

VI. Fluorescence Measurements6c. Features of right angle illumination

• Appropriate for optically transparent sample

• At high optical densities, signal reaching detector will be significantly diminished


6d. Features of front face illumination• Appropriate for an optically absorbing / scattering

sample

• At high optical densities, light is absorbed near the surface of the cuvette containing the absorber; therefore fluorescence is detectable

• Fluorescence independent of concentration at high optical densities


7. Blank scan• Blank is identical to sample except it does not contain

fluorophore

• Measuring the fluorescence of these samples allows the scattering (Rayleigh and Raman) to be assessed

• In addition, such samples can reveal the presence of fluorescence impurities, which can be subtracted

VI. Fluorescence Measurements8. Typical fluorescence emission spectrum at 340 nm

excitation (the different components)

0

500000

1000000

1500000

2000000

2500000

3000000

300 350 400 450 500 550 600

Wavelength (nm)

Flu

ore

sc

en

ce

In

ten

sit

y (

a.u

.)

Raman

Rayleigh (exc = emm)

Fluorescence

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FLUOROMETRI