Light Absorption Lambert-Beer Law

Light goes through a number of cuvettes.

I0 I1 I2 I3 I4

I 0 I1 I 2 I 3 = = = =n I1 I 2 I 3 I 4

1 Light Intensity I () 0.8 0.6 0.4 0.2 0 0 1 2 Light pathlength (l ) 3 4

This qualitative relationship is described by the following function for light intensity:

Ii = I o ni

Ii = I o10i logn 10

where i is the number of cuvettes. The last expression may be generalized for any kl cuvette with light path-length l: I = I o 10

where k is a coefficient which depends on concentration, c, and molar absorptivity, : k=c I I I I = I o10 cl = 10 cl 0 = 10 cl log 0 = cl Therefore,
I0 I I

Io Absorbance = A = log = cl I

Transmittance = T =

I = 10 A I0

Lambert-Beer Law Contd.

Dimensions: A dimensionless l cm c M M-1cm-1 (molar absorptivity or extinction coefficient) Connection between absorbance and transmittance:

A 0 1 2 3

T 1 0.1 0.01 0 0

Major application of Beers law - determination of unknown concentration by measuring absorbance:

A c= l

Absorption Spectra
Light absorption depends on wavelength of light Light has dual nature
1. Wave
Absorbance

A = cl

0.4
c

0.3 0.2 0.1 0.0 400

wavelength c speed of light wave propagation = c/ frequency

500

600

700

Wavelength, nm

2. Particle Energy of light particle:

E = h =

hc

Absorption spectrum of red wine (Canadian merlot, 1998)

(or ) determines the color of light

Spectrum of Electromagnetic Waves

Spectrophotometry deals with light within ~2001000 nm

Mechanism of light absorption

Electronic transition is the transition between different electronic states Ground state is the state with lowest electronic energy Excited state is the state with higher electronic energy. Electronic transition to the excited state is caused by absorption of a photon
1) Inter-nuclear distance is greater in the excited state than in the ground state 2) Franck-Condon principle: electronic transition is much faster than rearrangement of nuclei the distance between nuclei is not able to change during electronic transition
Vertical transition results in occupation of higher vibrational energy levels Vibrational energy dissipates producing heat Light absorption is always accompanied by sample heating Diagram of potential energy in diatomic molecule

Vertical transitions are most probable

Small molecules at low temperature

Even at 0K the molecule has vibration. Therefore, a number of vertical transitions can occur to different vibrational levels of the excited state. This transitions will require photons of different energy, resulting in an absorption spectrum. At low temperatures for small molecules transitions to different vibrational levels can be resolved The spectrum consists of very narrow bands.

Multi-atomic molecules at high temperatures

1) Multiatomic molecules have many possible electronic transitions Absorption spectra consist of broad bands 2) At high temperatures excitation can also occur from different vibrational levels of the ground state

Using absorption spectra to study biochemical reactions

Absorption spectrum is sensitive to minor changes in chemical structure of a molecule. Therefore, chemical reactions and changes in biopolymer conformation can be followed by monitoring absorption spectra.
His E7

Example: oxygenation of hemoglobin Hemoglobin is a protein with MW of 70 kDa Contains 4 heme groups Every heme can bind a molecule of oxygen non-covalently (oxygenation) Oxygenation drastically changes the absorption spectrum
N

N N H O N O Fe N N N His F8 N

Heme

Oxygenation of Hemoglobin
1.2
Hemoglobin

1.2 1 Absorption
Oxyhemoglobin

Isosbestic points

1 Absorbance 0.8 0.6 0.4 0.2 0 300

0.8 0.6 0.4 0.2

400

500 Wavelength, nm

600

700

0 300

400 Wavelength, nm

500

- Isosbestic points are wavelengths at which the molar absorptivities of the substances are equal - The presence of isosbestic points is a good indication that only one product forms from a substrate

Analysis of a Mixture
Absorbance is an additive function Absorbance of a mixture is a sum of the absorbances of the components:
A = X [ X ] l + Y [Y ] l + Z [ Z ] l + ...

- It is a linear equation. [X], [Y], [Z] are unknowns. - To find the unknowns the number of unknowns must be equal to the number of equations to determine the unknown concentrations of N substances in a mixture absorbances of the mixture must be measured at N wavelengths The molar absorptivities of all the pure components at N wavelengths must be measured a priory using pure substances.

Analysis of two-component mixture (hemoglobin and oxyhemoglobin)

Two components require two wavelengths. The wavelength are chosen to maximize the differential molar absorptivity: H O For wavelength 1: 1 1 = max O For wavelength 2: 2H 2 = max Absorption of the mixture at wavelength 1:
H 1 O 1
1.2
H

1 Ab so rb an ce 0.8 0.6 0.4 0.2 0 300 350 400

450

500

A1 = [ H ]l + [O]l Absorption of the mixture at wavelength 2:

H O [ H ]l + 2 [O]l A2 = 2

Wav e le n g th , n m

To solve this system of equation with regards to [H] and [O] we must: 1) measure the molar absorptivities of pure hemoglobin and H H O O oxyhemoglobin at two wavelengths: 1 , 2 , 1 , and 1

Hemoglobin and Oxyhemoglobin Contd.

Molar A bsorptivity, M cm

2) Measure absorbances, A1 and A2, of the mixture at two wavelengths. 3) Solve the system: From equation 1:

120000
H

-1 -1

100000 80000 60000 40000 20000 0 300 350 400

A1 1O [O]l [H ] = 1H l
Substitute [H] in equation 2:
H A2 = 2

450

500

Wavelength, nm

A1 1O [O]l O l + 2 [O ]l H 1 l

H A21H A1 2 Solve last expression with regards to [O]: [O ] = O H H O ( 2 1 2 1 )l O A1 2 A21O Using expressions 3 and 4: [ H ] = O H H O ( 2 1 2 1 )l

Spectrophotometers
Cuvet
Broad spectrum Narrow line

Monochromator

Detector I0 I

1. Baseline is the measurement of I0 as a function of using a cuvet with only a solvent. 2. Sample reading is the measurement of I as a function of using the cuvet with a sample. 3. Calculation of absorbance: A = log(I0/I) Conventional spectrophotometers Price range CA$ 15-20K

Jablonski Diagram - Fluorescence vs. Phosphorescence

S2
2 1 0

Internal conversion 10-12 s

S1

2 1 0

Inter-system conversion
The rate varies in a large range The influence of heavy metals

Light absorption 10-15 s

Fluorescence 10-8 s

2 1 0

T1
Phosphorescence >10-6 s

hA1

hA2

hF

S0

2 1 0

Fluorescence Lifetime
Fluorescence intensity decays exponentially: I = I0e-kt where k is the monomolecular rate constant of fluorescence decay, which can be determined experimentally from the kinetics of decay Definition: lifetime of fluorescence: = 1/k
do not confuse with half-life time, t1/2= ln2/k

The rate constant of fluorescence decay is in general a function of two components, radiative rate, kr and non-radiative rate, knr: k = kr + knr The lifetime accordingly is = 1/(kr + knr) Natural lifetime, n, is that for knr = 0: n = 1/kr /n = kr/(kr + knr)

Quantum Yield
Quantum yield:
number of emitted photons Q= number of absorbed photons

Substances with the highest Q (~1), such as rhodamines are the brightest emitters Quantum yield relates with the lifetime: Q = kr/(kr+ knr) /n = kr/(kr + knr) = Q

Thus, n can be found as: n = /Q

Note: that both and Q are experimentally measurable parameters

Problem: kr typical for fluorescence is 108 s-1 knr typical is ~ 109 s-1 Calculate Q typical for fluorescence Solution: Q = kr/(kr+ knr) = 108/(108 + 109) 0.1

Quenching of Fluorescence
Two major mechanisms of quenching exist: - Dynamic or collisional (depends on the diffusion of fluorophore and quencher) - Static (the formation of non-radiative complexes with quenchers that does not depend on the diffusion of fluorophore and quencher) Dynamic quenching is characterized by the Stern-Volmer eqn.: I0/I = 1 + kq0[Q] Where I0 and I are fluorescence intensities with and without quencher, kq is a bimolecular rate constant of quenching (M-1s-1), 0 is the lifetime of the fluorophore in the absence of quenchers, [Q] is the concentration of quencher.

Stern-Volmer Equation
* f(t) k = 0-1 Simplified Jablonski diagram: f(t) is the rate of excitation which Q depends on: kq[Q] (i) incident light intensity, Q (ii) concentration of fluorophore, (iii) absorptivity of fluorophore

k = 0-1 is the decay rate of fluorophore in the absence of the quencher 0 is the lifetime of fluorophore in the absence of the quencher [F*] is the concentration of fluorophore in the excited (*) state In the steady state, when f(t) = const, the concentration of the fluorophore in the excited state does not change: [F*] = const Thus: d[F*]/dt = 0 Note: d[F*]/dt = the rate of formation minus the rate of decay

Stern-Volmer Equation contd.

In the absence of the quencher: d[F*]/dt = f(t) - k[F*]0 = 0 Where [F*]0 is the concentration of F* in the the absence of quencher In the presence of the quencher: d[F*]/dt = f(t) - k[F*] - kq[F*][Q] = 0 f(t) - k[F*]0 = f(t) - k[F*] - kq[F*][Q] k[F*]0 = (k + kq[Q])[F*] [F*]0 /[F*] = (k + kq[Q]) /k = 1 + kq[Q]/k = 1 + kq0[Q] [F*]0 /[F*] = 1 + kq0[Q] The intensities of irradiation in the absence and the presence of quencher, I0 and I, are proportional to the concentration of fluorophore in the excited state: I0 = k[F*]0, and I = k[F*], then: I0 /I = 1 + kq0[Q] Prove that: 0/ = 1 + kq0[Q]

Stern-Volmer Constant, KSV = kq0

I0/I
Higher temp.

I0 /I = 1 + kq0[Q] = 1 + KSV[Q] KSV can be experimentally measured from the Stern-Volmer plot: - The linearity of the SV plot in general indicates that there is a single class of fluorophore - The increase of the slope with T indicates that quenching is dynamic (collision frequency increases with T) More than 1 fluorophore with at least one of them less accessible to the quencher leads to SV plot deviation toward the [Q]-axis [Q]

Slope = KSV
1 0

[Q]

I0/I

1 0

Efficiency of Quenching
The bimolecular rate constant of quenching, kq, is proportional to the diffusion controlled bimolecular rate constant, kd. The coefficient of proportionality is the efficiency of quenching : kq = kd 1 Determination of : = kq / kd kq can be calculated from kq = KSV/0 KSV can be determined from the SV plots 0 can be determined from the kinetics of fluorescence decay kd can be calculated using the Smoluchovski equation:

4N A kd = ( R f + Rq )( D f + Dq ) 1000
where NA is Avogadro number, Rf and Rq are molecular radii of the fluorophore and quencher, respectively, Df and Dq are diffusion coefficients of the fluorophore and quencher, respectively

Efficiency of Quenching contd.

The radii can be estimated from the molecular structures Diffusion coefficients can be calculated from the Stokes-Einstein eqn.: D = kT/6R where k = 1.38 10-23 JK-1 is the Boltzman constant is the solvent viscosity (~ 9 10-4 kg m-1 s-1 for water)
Example: Find for quenching of tryptophan by oxygen Step 1: finding kq The experimental value of KSV = 32.5 M-1 kq = KSV/0 = 1.2 1010 M-1s-1 The experimental value of 0 = 2.7 ns

Step 2: finding kd At 25C in water: DO2 = 2.5 10-5 cm2/s, Assume that RO2 + Rtryp = 5
2

Dtrypt = 0.66 10-5 cm2/s

4N A kd = ( RO + Rtryp )( DO + Dtryp ) = 1.2 1010 M 1 s 1 1000

2

Step 2: finding

= kq / kd = 1

Fluorescence Polarization or Anisotropy

1. Absorption of Polarized Light
- Polarization is the direction of the electric vector of the electromagnetic wave. G Light is polarized if all photons have the same direction of E (lasers are most convenient sources of polarized light)

E
Photon

- Fluorophores preferably absorb photons whose electric vector is aligned with the G transition G dipole moment, ex , of fluorophore. - The ex has a defined orientation with respect to molecular axes. - Thus, if a fluorophore solution is illuminated by polarized light then only a fraction of randomly oriented molecules will absorb light (those with a favorable orientation). - Thus, the population of molecules excited with polarized light will have the same orientation.
Polarization favorable for absorption of photon

Photon

ex

Polarization unfavorable for absorption of photon

Photon

ex

2. Emission of Polarized Light

- The emission dipole moment, em, is not the same as the excitation dipole moment, em. It is associated with the change of molecule geometry in the excited state. - Therefore, the polarization of emitted light will be different from that of excitation light. - The degree of differences in the polarization of excitation and emission I I I I lights is described by fluorescence anisotropy: r = || = || I|| + 2I I Total where I|| and I are the intensities of fluorescence in the direction parallel and perpendicular to ex respectively.

ex

em

Note: 1. (I|| + 2I) is the total intensity of fluorescence, so that fluorescence anisotropy does not depend on fluorescence intensity. 2 in the denominator shows that in a 3-D space there are 2 indistinguishable directions perpendicular to ex 2. Maximum possible anisotropy of 1 (when I = 0) would be observed if: (i) the fluorophore had ex = em , (ii) all fluorophore molecules were oriented favorably and (ii) all fluorophore molecules were immobile 3. Maximum fluorescence anisotropy of randomly oriented solution of fluorophore is 0.4 due to excitation photoselection 4. Light scattering and reflection can produce r = 1

Calculation of Fluorescence Anisotropy

Problem: Calculate fluorescence anisotropy for a solid solution of fluorophore which is oriented so that ex of all molecules are aligned with the direction of polarization of excitation light. The angle between is ex and em Solution: - To calculate the anisotropy we need to know I|| and I. - I|| and I are dependent on the projections of em on the || (em ||) and axes (em 1 and em 2): I1= a em 1 I2= a em 2 I|| = a em || where a is a constant - The projections em 1 and em 2 are identical due to the two axes being indistinguishable from each another, thus: I|| = a em || = a em cos I = I1= I2 = a em sin cos 45 = 0.5 21/2 a em sin
a em cos 0.5 21 / 2 a em sin cos 0.5 21 / 2 sin = = r= I|| + 2I a em cos + 2 0.5 21 / 2 a em sin cos + 21 / 2 sin I|| I

||

ex

em || em 2 2

em

em 1
45

2. Rotational Depolarization of Fluorescence

Reasons for fluorescence anisotropy of a random solution of ex fluorophore to be less than 0.4:

1. ex em

em

2. Rotation of a fluorophore molecule during the lifetime of fluorescence

ex

em

ex em

ex

em

t=0

t = 5 ns

t = 10 ns

Anisotropy Increases with Increasing Size of Fluorophore Molecule

- The rate of rotational depolarization increases with an increasing rate of molecular rotation (rotational diffusion) - The rate of rotational diffusion increases with decreasing the size of fluorophore. - This property of fluorescence anisotropy is used to sense interactions between small fluorophores and large biomolecules: anisotropy increases upon binding of fluorophore to a large molecule. - The equilibrium dissociation constant of the protein-fluorophore complex, Kd = koff/kon, can be calculated based on the anisotropy measurements.
kon Protein + Fluorophore ProteinFluorophore koff

Experimental Design for Anisotropy Measurements

At the interface of two prisms, a two-prism polarization analyzer reflects one direction of polarization (parallel to the plane of interface) and transmits two other directions

B B
E E

Detector 1

E ||

E
Laser

Detector 2

Determination of Kd Using Anisotropy

Assume that fluorophore F forms a dynamic complex with protein P:
kon P + F PF koff

The equilibrium binding constant is defined as:

Kd =

[ P ]eq [ F ]eq [ P F ]eq

where [P]eq, [F]eq, and [PF]eq are equilibrium concentrations of free protein, free fluorophore, and the complex, respectively. We will express [P]eq[F]eq [PF]eq through fluorescence anisotropy and total concentrations of protein and fluorophore, [P]0 and [F]0 using two principles: 1) Anisotropy is an additive function, that is

r = rF
2) Mass balance requires that: and [P]0 = [PF]eq + [P]eq [F]0 = [PF]eq + [F]eq

[F]eq [F]0

+ rPF

[P F]eq [F]0

Determination of Kd Using Anisotropy contd.

[F]eq r = rF + rPF = rF + rPF = rF + rPF 1 [F]0 [F]0 [F]0 [F]0 [F]0 [F]0 [F]eq [F]eq [F]eq [F]eq r rPF rF + rPF rPF = rPF + = =R ( rF rPF ) [F]0 [F]0 [F]0 [F]0 rF rPF [F]eq [F]eq [F]0 -[F]eq [F]eq [P F]eq =

Experimentally measurable parameter Thus: 1. [F]eq = [F]0R 2. [PF]eq = [F]0 [F]eq = [F]0 [F]0R = [F]0 (1 - R) 3. [P]eq = [P]0 [PF]eq = [P]0 [F]0 (1 - R)

By substituting the expressions 1-3 into

Kd [P]0 [F]0 (1 R)}[F]0 R [P]0 (1 R) { = = [F]0 (1 R) (1/R 1)

Kd =

[ P ]eq [ F ]eq [ P F ]eq

we get:

r rPF where R = rF rPF

[P]0 (1 R) Kd = (1/R 1)

where R =

r rPF , 0 < R <1 rF rPF

Determination of r, rF and rPF

- rF is anisotropy of free fluorophore. It is measured in the absence of the protein - r is the anisotropy of fluorophore at a finite concentration of protein so that only part of fluorophore is bound to a protein:
r = rF [F]eq [F]0 + rPF [P F]eq [F]0 and 0< [F]eq [F]0 < 1, preferably [F]eq [F]0 0.5

- rPF is the anisotropy of the PF complex, that can be experimentally found by using high enough concentration of protein to ensure that [PF]eq/[F]0 1 and [F]eq/[F]0 0 and thus
r = rF [F]eq [F]0 + rPF [P F]eq [F]0 = rF 0 + rPF 1 = rPF

- Practically, r is measured as a function of [P]0 (Scotchard plot) to find the saturating value of r which is assumed to be equal to rPF

Other Parameters that Can be Used for the Determination of Kd

- Anisotropy can be used for the determination of Kd because it is an additive parameter (anisotropy of a mixture is equal to the sum of anisotropies of its components) - Any additive parameter that has a different value for F and PF can be used to find Kd - Can fluorescence lifetime or intensity be used? Home - Can fluorescence resonance energy transfer (FRET) be used? assignment - Mobility in capillary electrophoresis can be used for calculation of Kd, because:
= F
[F]eq [F]0 + PF [P F]eq [F]0 ,

v L = E tE

where t is migration time

1 1 [F]eq 1 [P F]eq = + t tF [F]0 tPF [F]0

Fluorescence Energy Transfer

Reaction involved in ET between donor (D) and acceptor (A): D + hv D* kET D* + A D + A* + heat Two types of processes are distinguished: Non-Resonance Energy Transfer requires collision of D and A: d[A*]/dt = kET[D*][A] where kET = kd, is the efficiency of ET and kd is a diffusion-controlled rate constant. The process is similar to quenching! It is rare (e.g. spin-forbidden TS or ST) and is not widely used as an analytical tool. 2. Fluorescence Resonance Energy Transfer (FRET) by the Frster mechanism is widely used to study the dynamics of molecular interactions. The rate constant of FRET is conventionally expressed as a monomolecular one:

1 R0 k FRET ( r ) = D 0 r
where 0D is the lifetime of D in the absence of A, R0 is the Frster radius, and r is the distance between the D and A.

FRET the Meaning of R0

k FRET ( r ) =
What is the physical meaning of R0?
R0 is the distance between D and A at which the efficiency of energy transfer is equal to 1/2 (half of energy is transferred from D to A). Indeed, when r = R0, then:

1 R0 D 0 r

Typical R0 are 20 90

1 1 1 k FRET ( r = R0 ) = D = D 0 1 0

Q: What is the efficiency of ET for r = R0? Recall that the rate constant, k, of radiative+non-radiative transitions in D is the same as that of FRET: k = kFRET = 1/0D

k FRET ( r = R0 ) =

0D

hv

k=

0D

heat

The efficiency of ET: = kFRET/(kFRET + k) (=1/2 for r = R0)

Calculation of R0
9000(ln 10) QD 4 R0 = 6 F ( ) ( ) d D 5 4 128 N A n 0
2

where - QD is the quantum yield of D in the absence of A - n is the refractive index of the medium (1.4 for biomolecules in aqueous solns.) - NA is Avogadros number - 2 is the orientation factor (2/3 for randomly oriented molecules) - FD( ) is the normalized spectrum of fluorescence of D and () is the extinction coefficient of A
4 F ( ) ( ) d D 0

is called the integral of spectral overlap

What is the reason of 6 in (R0/r)6?

Dipole-dipole coupling mechanism makes the distance dependence so strong

Types of intermolecular interactions in solution

FRET The Bioanalytical Use

1. Calculation of the distance between two fluorophores: - The distance between tyrosine and tryptophan in a protein - R0 is calculated using the formula R = 9000(ln10) Q F ( ) ( ) d 128 N n and, then, r is calculated as follows:
2

R06 r=6 = D 6 k FRET ( r ) 0

R06 R06 = = 6 (1 / 1) R06 k 1 6 0D 1/ 1 1/ 1

k = 1/0D

= kFRET/(kFRET + k)

The efficiency of FT, is determined experimentally

2. FRET-based reporters such as molecular beacons

Light Sources. Lamps

Never touch optical surfaces with bare fingers
Characteristics of lamps as light sources: Tunable (requires monochromator), Low cost, Low power at any fixed , No polarization

Widely used in fluorescence microscopes

Mercury and Xenon arc lamps require caution during operation because of the danger of explosion due to very high internal gas pressures and extreme heat generated during use.

Light Sources. Lasers

Advantages: monochromatic light, high power, high level of polarization Disadvantages: high cost, impossible to tune Examples - Helium-cadmium laser, 325nm, 442 nm - Helium-neon laser, 543 nm, 594 nm, 632.8 nm - Diode-pumped solid-state lasers, 457 nm, 640-685 nm (temp-dependant), 1064 nm. - Argon-ion, 488 nm, 514 nm Lasers deliver 1-200 mW in light. Eye damage is possible during direct exposure

Objectives
Magnification: The ratio between the sizes of the image and object when object is at the working distance. Working distance: Distance from the objective to the object at which the image will be in focus Angular Aperture: AA = sin Numerical Aperture: NA = n(sin ) where n is the refractive index of the imaging medium between the front lens of the objective and the specimen cover glass, a value that ranges from 1.00 for air to 1.51 for specialized immersion oils.

Most objectives in the magnification range between 60x and 100x (and higher) are designed for use with immersion oils.

Manufacturers: Melles Griot, Newport, Leica, Olympus, etc.

How Much of Isotropic Fluorescence Light Does the Air Lens of Known Angular Aperture Collect?
specimen l
d dS d

Objective lens R

Working distance

The part of light collected by a lens is the ratio between the solid body S that defines the aperture and the solid body Ssph of the sphere: S/Ssph = S/4l2 dS = l d Rd = l d (l sin )d = l 2 sin d d
S = l 2 (sin d )d = l 2 (cos | 0 )d =
0 0 0 2 2

l 2 (cos 1)d = l 2 (1 cos )d = 2 l 2 (1 cos )

S/Ssph = S/4l2 = 2 l2 (1- cos)/4l2 = (1- cos)/2

How Much of Isotropic Fluorescence Light Does an Air Lens of Known AA Collect? Contd.
AA = sin = arcsin (AA) S/Ssph = (1- cos)/2 = = {1 cos(arcsin(AA))}/2

Angular aperture

Raman Scattering on H2O

Cut-off filters:

Transmittance, % Transmittance, %

Filters

Wavelength
Absorption spectra are important for estimation of quality of cutting light

Band-pass filters:

Wavelength

Nomenclature: 530DF30 means that the transmittance band is centered at 530 nm and has a width of 30 nm (530 15 nm).
Manufacturers: Omega Opticals (www.omegafilters.com), Chroma

Mirrors
- In household mirrors, the reflecting surface is protected by glass, we look through glass. - In optical mirrors the reflective surface is exposed; therefore it should be protected from oxidation by a layer of dielectric coating

Domestic mirror

Optical mirror

Glass Reflective coating

Manufacturers: Omega Opticals (www.omegafilters.com), Chroma

Dichroic Mirrors (Beamsplitters)

Dichroic beamsplitters typically operate at 45 to incident light. They are designed to separate light of short and long wavelengths into two separate channels. Dichroic mirrors reflect low-wavelength light and transmit high-wavelength light
Nomenclature: for example 490DCLP means DiChroic Long Pass. 490 nm corresponds to 80%/20% transmittance/refraction

Manufacturers: Omega Opticals (www.omegafilters.com), Chroma

Filter Cubes
Filter cubes are used in fluorescence microscopes and fluorescence detection systems to allow excitation of fluorescence and collection of fluorescence light with the same objective lens
specimen

exciter

dichroic mirror

EYE or detector

emitter

Manufacturers: Omega Opticals (www.omegafilters.com), Chroma

Light Detectors - PMTs

+1 kV

Modes of operation: 1) Current (higher noise, higher dynamic range) 2) Photon counting (noise reduction due to the discrimination of pulses that correspond to single electrons born on the photocathode. Lower dynamic range due to the need to resolve between single photons. The pulse width is 50 ns (5 10-8 s). The dynamic range is 0 105 counts/s.
Manufacturers: Hamamatsu (www.hamamatsu.com)