Fluorimeter 0

Fluorolog FL3C-21
User Guide and Tutorial for Using the Fluorolog FL3C-21

in the Polymer Group at Georgia Tech
2nd Edition: April 2012
Georgia Institute of Technology

School of Chemistry & Biochemistry
School of Materials Science and Engineering
Written by: Leandro A. Estrada
1
HJY Fluorimeter Manual Estrada
1. Introduction ................................................................................................................................................... 3
2. Theoretical Aspects of Radiative Decay............................................................................................... 3
3. The Fluorimeter System ............................................................................................................................ 8
3.1. Hardware Configuration ................................................................................................................. 8
3.2. Software................................................................................................................................................. 9
4. Practical Aspects: Fundamentals ........................................................................................................ 11
4.1. Sample Preparation........................................................................................................................ 11
4.1.1 Solutions ................................................................................................................................... 11
4.1.2 Films ........................................................................................................................................... 13
4.1.3 Glasses ....................................................................................................................................... 13
4.2. Turning on the Instrument.......................................................................................................... 14
4.3. Check System Performance......................................................................................................... 14
5. Data Acquisition......................................................................................................................................... 16
5.1. Steady State Fluorescence ........................................................................................................... 16
5.2. Excitation Spectra ........................................................................................................................... 17
5.3. Time Resolved Fluorescence, ..................................................................................................... 17
5.3.1 TCSPC Fluorescence Lifetime ........................................................................................... 19
6. Practical Aspects: Advanced Techniques ......................................................................................... 23
6.1. Phosphorescence Measurements ............................................................................................. 23
6.2. Fluorescence Anisotropy Measurements .............................................................................. 23
6.3. Time Resolved Emission Spectroscopy (TRES) .................................................................. 23
6.4. Advanced TCSPC Measurements: Sequential TCSPC ........................................................ 23
6.5. Multi-Channel Scaling (MCS) Lifetime and Fast MCS ........................................................ 23
6.6. Miscellaneous Measurements: Steady State, Gated Steady State ................................. 23
7. References .................................................................................................................................................... 23
2
1. Introduction
In this manual, we discuss measurement techniques for two optical processes: molecular
fluorescence and phosphorescence. In these methods, the analyte is excited using light to
populate its excited state(s). Depending on the relaxation dynamics, the lowest lying singlet
state, S1, or the lowest-lying triplet state, T1, or both can relax to the ground state S0 via
emission of a photon (viz. photoluminescence). Through the detection of these emitted
photons, important information can be obtained such as the energy, polarity, lifetime, and
radiative and non-radiative rates of the emitting state.
One of the most attractive features of fluorescence spectroscopy is its sensitivity, which can
be 1-3 orders of magnitude higher than that of absorption spectroscopy. For this reason,
quantitative analysis could suffer from serious interference effects from the sample matrix,
if luminescent impurities are present. This implies that the samples to analyze must be
optically pure, in order to avoid any undesired interference; conversely, fluorescence
spectroscopy can be used to test the presence of impurities in materials with known
properties. Finally, fluorescence can also be utilized as a tool to study a variety of physical
and chemical phenomena such as solvatochromism, sensing, and Förster Resonance Energy
Transfer (FRET) to name a few.
This guide is not supposed to act as a replacement of textbooks on Fluorescence

Spectroscopy that have been written with the purpose of teaching the basic theory and
applications of this technique, or the manuals provided by Horiba-Jovin-Yvon (HJY). Rather,
this manual is a support tool to assist in acquiring data from the HJY Fluorolog FL3C-21
spectrofluorimeter and understanding what types of experiments are available through
this instrument, as well as the specifics in terms of sample preparation, setup, and data
acquisition. It is assumed that the user has some beginner-intermediate knowledge of
Origin® which is the software upon which the steady-state HJY software is built, plus basic
understanding of UV-Vis absorption and fluorescence experimental methods.
2. Theoretical Aspects of Radiative Decay1

All absorbers contain bound electrons which are normally distributed in a given quantum
state, where the average distribution is time independent. When a photon is absorbed by a
molecule, a perturbation in the electronic distribution takes place and, if the photon
happens to have the right frequency (often in the UV-Visible region of the electromagnetic
spectrum), a transition to a higher energy quantum state occurs. This higher energy state
possesses a different charge distribution than the previous state, which in turn will
deactivate in order to release that excess of energy in accordance with the principle of
microscopic reversibility.2
However, not all transitions that are energetically allowed actually occur, since the value of
the transition dipole moment (|fi|) can be zero. The set of conditions that govern the
change in quantum numbers of the eigenfunctions of the initial (i) and final (f) states for a
transition are known as the Selection Rules (SRs), which are based on the parity of the
3
quantum numbers for orbital (l), angular momentum (j) and spin (s, ms).3 Considering that
electronic motions are much faster than nuclear motions, the electronic matrix element can
be decomposed into elements that are purely nuclear and electronic. This is the so called
Born-Oppenheimer (BO) approximation.4 Since the electronic spin component is not
position dependent, we finally have:
ˆ fi   f μ i   f μ i   f i  S f Si (1) Comment [W1]: The  integral at the right

results in a scalar (number), so there is no need to
put these into vertical bars. The vertical bars at the
left represent the expected value for the transition
Electronic Nuclear Electron dipole moment for the fi transition – no direction
transition overlap spin overlap as denoted by the vector notation.
moment integral integral
One further approximation to that of BO is that the nuclei remain “frozen” at the ground
state equilibrium geometry while an electronic transition occurs. Figure 1 depicts a plot of
the potential energy of the system versus geometrical variations (bold black line). The
electronic transition from ground to excited state, denoted as E1E0, will resemble an
initial change only in energy as depicted by the blue arrow (no change in the geometry of
the system); nuclear reorganization occurs afterwards. The same applies for the case of an
electronic transition from the excited to the ground state, denoted as (E1E0) as depicted
by the green arrow in Figure 1. It is thus said that the electronic transitions are vertical in
nature. Also, the transition between the different vibrational states between both ground
and excited electronic states have different probabilities to occur which are dependent on
their specific vibrational wavefunctions and the population of the initial states. Given that
these electronic transitions also entail vibrational transitions, these are often regarded as
vibronic transitions. This is the spirit of the Franck-Condon (FC) principle which states that
the most intense vibronic transitions will be those corresponding to the maximum overlap
between their wavefunctions.5 Consequently, the absorption probability of a determined
transition will depend on the square of the nuclear overlap integral, f |i2, which value is
regarded as the FC factor.
4
Figure 1. Illustration of the Franck-Condon principle, adapted from Reference 6. Left. Potential energy curves
of the ground and first excited state (bold black lines) with their corresponding vibrational wavefunctions
(orange waves). Right. Absorption and emission spectra corresponding to the energy diagram at the left
(green and blue lines). The colored bands correspond to the probability of the vibronic transitions for the
absorption (green) and emission (blue) processes. The black band corresponds to the 0-0 (adiabatic) vibronic
transition.
Another consequence of the BO approximation is that the spin component is not position
dependent. It can be shown that the spin overlap integral, Sf |Si, is zero when the spin
values are different in the initial and final states (viz. intersystem crossing or ISC).7 Thus,
electronic transitions are allowed between states with the same spin. Since organic
molecules exist in their majority as closed shell singlet (S0), the most favorable absorption
transitions will be of the type SnS0 (usually achieved ~ 10–15 s). Furthermore, the upper
excited states will decay non-radiatively to the lowest excited state of the same multiplicity
with timeframes ~ 10–12–10–6 s (denoted in the case of singlet manifold as SnS1). This
internal conversion (IC) process is the heart of Kasha’s rule,8 the consequence of which is
that the photons emitted from the singlet state (viz. fluorescence) are of lower energy than
those initially absorbed by the chromophore. The energy difference between absorption
and fluorescence maxima, known as the Stokes’ shift, offers qualitative information about
the difference in dipole moments of the chromophore’s ground and first excited singlet Comment [MR2]: Only is the molecule have
non-zero dipole moments. What about the case of
state and the nuclear reorganization after photoexcitation.9 Furthermore, for polyatomic centrosymmetric chromophores?
molecules – and especially for organic molecules – the sharp transitions will be multiplied Comment [W3]: Here, I am talking about the
given the many dimensions conferred by their vibrational freedom. Consequently, it is differences in dipole moments between ground and
often the case where losses of spectral resolution result in a featureless profile. excited states. If the molecule has the same dipole
moment in the ground and excited state, the
Additionally, the spacing of the energy levels in the vibrational manifolds of ground and difference will be zero.
first excited electronic states are usually similar. Thus, ideally, the S1S0 and S1S0 Comment [E4]: This assessment can be
vibronic transitions have mirror image resemblance as presented in Figure 1. complicated for example in polymeric systems
where the Stokes shift is not necessarily dominated
by nuclear relaxation but by charge migration to low
Experimentally, it is known that transitions formally forbidden by the SRs occur to some energy domains
extent. For example the transition T1S0 is known to be possible via perturbations (i.e.
heavy atom effect),10 and the symmetry forbidden n* absorption is known to be present
in many carbonyl containing chromophores. Therefore, we regard this to be a deviation
5
from the BO approximation since the nuclear motion or coupling between states (i.e. spin
and orbital) has to be involved somehow as a perturbation of the initial approximation
(zeroth-order). Many explanations of this phenomenon have been offered within the
borders of Fermi’s Golden Rule (FGR).Error! Bookmark not defined.,10 One of them
suggests that the first-order absorption transition could be seen as having different
“prohibition factors” (f) ranging between (0,1) dependent on electronic (e), vibrational (v)
and spin (s) components which reduce the effectiveness of the transition as follows:10
kobs  k0  k1 f e  f v  f S  (2)
where k0 (zeroth-order
 rate constant) is zero for forbidden transitions according to the
aforementioned SRs. The magnitude of these factors can be approximated to the transition
Hamiltonian (H) via perturbation theory as well (2), leaving us with an extended FGR
expression (3), dependent on electronic and spin-orbit coupling Hamiltonians, and the FC
factor.
H
2
  H  2
 f H SO i  Comment [E5]: ?? This might be my fault; I
 f e i  changed the formatting of the document before
f ~ (5) ; kobs  k0  k1     f | i  (3) checking to see how things should be laid out
E  E 2 E 2 
 
Orbital Spin-Orbit FC factor

Interactions Interactions
The possible events that occur after accessing S1 via photoexcitation are outlined in the
Jablonski diagram displayed in Figure 2. The average time during which the fluorophore
remains in S1, t, is equal to the fluorescence lifetime, , the period after which the excited
state population is reduced to 1/e of the initial value, if the decay follows a single
exponential model ( I   I 0  e1 ). These quantities (I(t), ) permit the calculation of
important parameters such as the fluorescence quantum yield (F), defined as the ratio of
emitted photons with respect of absorbed photons (4), which in turn is related to the rates
of fluorescent (kF) and non-radiative (kNR) decay as follows (5−6):
IF kF F 1 F
F   (4) kF  (5) k NR  (6)
Iabs kF  kNR  
 The evaluation of the fluorescence rate constants are also indicative of the type of excited
state that is carrying out the S1S0 deactivation. Therefore, the rate of fluorescence allows
6
for a calibration of the maximum time that the competitive processes have for them to
actually occur. For example, for an allowed * transition the largest kF is ~ 109 s–1 (e.g. p-
terphenyl) whereas for a forbidden n* transition the kF value is expected to be ~ 105 s–1
(e.g. Acetone).
The non-radiative deactivation of S1 can occur through many potential processes such as
ISC to the triplet manifold and IC from the singlet ground state. The rate of ISC can be
quantified once the measurement of the triplet formation quantum yield (ISC) has been
carried out, following the expression:
ISC
kISC 
 (10)
Experimental methods that  allow its measurement range from quenching methods,11 to
triplet sensitization of processes with known quantum yield of formation such as stilbene
isomerization.12
Figure 2. Modified Jablonski diagram, adapted from reference Error! Bookmark not defined.. See
that Kasha’s rule implies vibronic deactivations via IC for states higher in energy than the lowest vibrational
state for S1.
7
3. The Fluorimeter System

3.1. Hardware Configuration
A schematic diagram and a photograph of the HJY FL3C-21 are presented in Figure 3. The
spectrofluorimeter contains a 450 W xenon CW lamp as a steady state excitation source
and a pulsed xenon lamp for phosphorimetry, both contained in the lamp housing box.
Solid-state NanoLEDs are also available as ns-pulsed excitation sources (370 nm, 455 nm,
570 nm, and 625 nm with pulse duration of < 1.4 ns), although these are not housed in the
lamp housing box (not shown). The light path is directed to a double-grating
monochromator where the slit sizes and wavelength calibration are automated ensuring
reproducibility at all times. The sample compartment is bifurcated to accommodate two
monochromator/detector sets: one consists of a single grating monochromator attached to
a fully automated iHR spectrometer with dual detection mode (250-1050 nm) and a single
monochromator attached to a thermoelectrically cooled InGaAs NIR PMT module (950-
1400 nm). The iHR spectrometer possesses two detectors: a multialkali PMT detector (250-
850 nm) and a thermoelectrically cooled NIR PMT (250-1050 nm), which is attached to a
water cooling device to dissipate heat. This NIR detector is less sensitive than the
multialkali PMT, thus it is intended for use as a crossover between the multialkali PMT and
the InGaAs NIR PMT module (or for experiments for which it is critical to have low dark
counts). All detectors are operated in the single photon counting mode; the signals from the
detector are then amplified and sent to a data acquisition system and a computer. Fully
automated polarizers are available. The sample holder contains a cuvette holder that can
be changed for other accessories such as a film holder, an integrating sphere or a liquid N2
dewar assembly. The former accessory contains a fiber optic that permits the integration of
the in/out photons and the calculation of universal fluorescence quantum yields. On the
other hand, the latter accessory permits the measurement of fluorophores in a glass matrix
at low temperatures. A detailed description of these accessories will be offered in the
appropriate sections of this manual.
8
Figure 3. Top. Schematics of the HJY Fluorolog FL3C-21 Spectrofluorimeter. Bottom. Picture of the assembled
spectrophotometer.
3.2. Software
FluorEssenceTM is a software package developed by the Horiba Sci Labs in which a full
integration between the steady state acquisition modules and Origin® has been achieved.
All aspects of the spectrofluorimeter control are available with minimum overlapping of
windows and screens. The data can be previewed while it is being recorded and the output
is immediately exported to an Origin data sheet. This permits an easy data workup prior to
exporting the whole acquired set for further workup and ultimate analysis. Furthermore,
the software permits the use of scripts for data workup in areas such as graphical
templates, mathematical operations and much more.
9
The way Origin works is by projects which hold a collection of workbooks of data.* These
may hold:
Graphs. These may contain multiple information including separate layers describing the Formatted: Bullets and Numbering
data, axes, background colors, etc.
Worksheets. These contain the raw numerical data ready for processing. Also, it contains
processed data through selected mathematical operations.
Notes. These contain any type of string you may like for you to remember what type of
processing was done to the raw data and how this processing was performed.
The Origin software is available for all types of steady state measurements. This includes
measurements of fluorescence and phosphorescence in samples ranging from solutions,
films, and low temperature matrices. It also processes universal quantum yield data as
acquired through the integrating sphere.
On the other hand, all time correlated data acquisition (e.g. lifetimes measurements) and
processing are not integrated with either Origin or with each other. Therefore, two
different software packages are needed: HJY DataStation and the DAS6 software packages.
The DataStation is required for the control of the FluoroHub-B, which is the component in
charge of counting photons and the time in which these arrive. The data analysis is
accomplished through the DAS6 software package. The presentation screens for both data
acquisition software packages used for the fluorimeter are presented in Figure 4, whose
icons can be easily found at the computer’s Desktop.
Figure 4. Presentation screen of the HJY FluorEssence and DataStation software packages when loaded.
*
The reader is referred to the manual of Origin 8.5 to get more information about the software organization and
further features.
10
An illustration of the basic elements

of the user interface of DataStation is
presented in Figure 5. The
Measurement Chart is the largest
element and displays the data listed
on the measurement tree in graphical
form. It consists of three separate
areas, the display chart, the InfoBar
and the RateBar. The InfoBar is
above the chart and shows
information about the currently
selected item. The RateBar is below
the chart and shows the live
ratemeter values (see later sections).
Figure 5. The DataStation workspace showing prompt and
The Menus and Toolbars are used
decay data.
to access many of the DataStation’s
features, whereas the Measurement Tree displays the various measurements in progress.
Selecting an item from the tree will normally display the associated data in the
Measurement Chart, right-clicking on an item will usually pop-up a menu of further
options. Right-clicking on the empty background of the tree can also show a menu
depending on the measurement template currently in use. The Hardware Tree allows
quick access to the various hardware parameters, and the controls are grouped depending
on function. Again, the number of icons displayed depends on the options installed and on
the measurement template selected. For example, the NanoLED group will not be displayed
if the Hub-NL card is not detected when the Hub is switched on. Similarly, the TAC Range
control will not appear in the Hub group if the measurement template does not use TCSPC
(e.g. steady-state template). The controls are described in more detail in the DataStation
Manual. Finally, the Status Bar is where information (not displayed elsewhere) relating to
the status of the Hub or the measurement progress is displayed. Note that as the mouse
cursor is moved over certain interface elements a message is displayed in the Status Bar
describing the function of that element. This is in addition to the brief “ToolTips” displayed
next to the mouse cursor.
4. Practical Aspects: Fundamentals

4.1. Sample Preparation
4.1.1 Solutions13
The shape and intensity of the fluorescence spectrum is highly dependent on the nature of
the sample, its concentration, and the solvent of choice. Given that small particles within
the solution induce light scattering, the scattered photons may find their way to the
detector introducing measurement errors. On the other hand, the fluorescence intensity is
linearly dependent on the sample concentration only over a limited range of optical
densities. This is illustrated in Figure 6A using quinine sulfate as example.Error!
Bookmark not defined. The threshold concentration above which deviations from the
11
linear behavior are observed depends on the chromophore; for the quinine sulfate example
the threshold O.D is about 0.05 over a 1 cm pathlength. Fluorophores with particularly
small Stokes shifts are sensitive to inner filter effects (or reabsorption), to the point in
which distortions of the emission spectrum can be observed. As example, the fluorescence
spectrum of anthracene can be visibly affected by the increasing fluorophore concentration
as shown in Figure 6B.
A B
Figure 6. (A) Plot of fluorescence intensity at 450 nm versus optical density of quinine sulfate at 346 nm for
solutions of various analyte concentrations. FOBS corresponds to the experimentally observed signal intensity
at the corresponding O.D. FCORR corresponds to the corrected signal intensity by using an empirical correction
factor14 (B) Fluorescence spectra of anthracene in a 1cm 2 cuvette using
right angle observation.
Additionally, Figure 7 summarizes common errors related to

sample preparation or choice of instrumental conditions. If the
concentration is too high, the photons will be absorbed at the
surface facing the light source which limits the fluorescence
intensity as the population of excited chromophores is
unevenly distributed within the cell.† On the other hand, the
purity of the solvent is very important as well as the
cleanliness of the cell. Contaminants in the solvent/cell could
affect the fluorescence signal. Other problems present are
when the sample contains excess of Rayleigh (elastic) or
Raman (inelastic) scattering which translates into unstable or
too noisy signals.‡ This symptom is likely of scattering coming
from floating particles in solutions (dust, silica gel, etc.). Comment [E6]: This is really good information,
but to keep the “practical aspects” section
Figure 7. Common errors in streamlined, let’s move the majority of this to a
Due to all these phenomena, it is recommended to prepare a different section (maybe the theory section) and here
sample preparation
stock solution with a concentration approaching 1 M (for just have one or two summarizing sentences to lead
into the next paragraphs
†
This is usually the case, unless one is using front face collection which is possible in this instrument. For further
information about this type of experiment, please refer to Dr. M. Rumi (Marder’s Group).
‡
Rayleigh and Raman scatterings are ALWAYS present. There is no material that has zero scattering cross section
and zero Raman cross section for all the modes so it is expected that the signal contains some degree of “noise”.
Care should be exercised, however, to avoid extra noise than necessary.
12
polymers the approach is a little different), and then prepare the diluted solutions
accordingly. It is best to have these solutions freshly filtered before adding to the cell. Comment [MR7]: Depends what measurement
you are doing. If you want to determine the quantum
yield and measured the absorbance of the stock
The cleanliness of the cell/cuvette is very important. Usually, immersing these in solution (as the absrobance of the diluted one is
concentrated nitric acid overnight the day before use is a good practice. Afterwards, a good typically too low for accurate measurement (unless a
very long path lenght is used), then filtering should
wash with deionized water, followed by ethanol and/or acetone will suffice. Be sure to be aviouded, as it could change the concetration.
rinse the cell with pure solvent or solution twice before measuring, as this prevents any Comment [W8]: I think I am confused by this
concentration gradients at the time of measurement. point you have made. The sensitivity of
spectrophotometers could go as low as 0.03 mOD.
Usually for quantum yields, I get to use ODs of 10,
It is a good practice to measure the solvent background in order to identify either solvent 30, 50 mOD, and so on as long as I am within the
impurities or scattering coming from inelastic or elastic collisions of the photons with the linear regime (the maximum relative error for this
would be 0.3%). I have never had a problem
solvent. This will also provide a signal that can be subtracted from the spectrum of the measuring this kind of data before as recommended
analyte. by one of the Horiba Engineers.
Comment [E9]: I’ve personally never filtered my
Finally, it is strongly recommended that, once an excitation wavelength has been chosen, a solutions. Is the noise negligible? Is it sufficient to
filter the solvent but not the analyte, to prevent
plot of optical density versus fluorescence intensity is generated in order to determine if changes in concentration? Also, on an unrelated
the measurement is done in the linear regime and that there is no reabsorption. This is note, don’t you bubble the solution with inert gas for
phosphorescence measurements?
important at the time of measuring fluorescence quantum yields using the comparative
method as such relies on the linearity of the optical densities with respect of the
fluorescent photon flux. Comment [E10]: This can be elaborated in more
detail later in the manual
4.1.2 Films
To be added later. A figure will be added as deemed appropriate (Figure 8). Comment [MR11]: We need to tack about this.
There are no “general” rules with films as there is
usually no or little flexibility in concentration and
4.1.3 Glasses thickness. We can say something about the sample
configuration accessible with this instrument, but the
In the setup we will use, glasses can be made using glass-forming solvents such as other choice of the experimental parameters really
depends on what information people want to obtain.
methylcyclohexane (MeCH), methyltetrahydrofuran (MeTHF) and 1:1 ethanol/methanol So, what details should we included here and for
mixture. The sample is then placed into what cases?
an EPR-like tube that will be housed in a Comment [E12]: Since this is the “sample
preparation” section, maybe a comment about which
custom-made Dewar containing liquid- substrates and sizes could be used (?)
N2. Given the reduced optical path of Recommended techniques (spin-coating, etc.)
General rules of thumb on how concentrated a
this tube (3-5 mm diameter), the solution should be? Probably no more than a few
concentration of the sample can be sentences necessary.
increased 2-3 fold. Usually, samples

with 0.2-0.3 O.D as measured in a 1 cm
cuvette are used for this purpose.
Figure 9. Immersion of the sample tube into the liquid N2
dewar. Once the tube is filled with enough
sample or solvent (3-5 cm high) the
tube is immersed into the liquid-N2 bath slowly. A schematic illustration of these steps is
presented in Figure 9. A distinct sound originates after the sample attains cryogenic
conditions at the liquid N2/air interface, which alerts the user to immerse the tube a little
more. Once the total sample height has been immersed and equilibrated with the liquid-N2
bath, the rest of the tube can be immersed at once.
13
It is a good practice to measure the solvent background not only to identify either solvent
impurities or scattering coming from inelastic or elastic collisions of the photons with the
solvent, but also reflections from the Dewar. The residual signal should be subtracted from
the measured signal from the analyte in question.
4.2. Turning on the Instrument15 Comment [E13]: Pictures will be useful here.
Maybe use Figure 3 with arrows and labels pointing
to the various on/off switches
1.Firstly turn on the power (fan) switch and then the lamp switch from the lamp housing.
Formatted: Bullets and Numbering
This is important as to avoid any damages to the computing equipment given that a voltage
spike can feed back down into the electrical line. The lamp takes 15-20 minutes to warm up
to an equilibrated temperature and give steady emission.
2.Start any fluorimeter accessories (e.g., temperature bath) used with the Fluorolog.
3.Turn on the SpectrAcq controller

4.Start any peripheral controllers:
a.Turn on the Hub Station

b.Turn on the iHR spectrophotometer
c.Turn on the chiller and piezo cooler if the R2659 PMT (crossover NIR detector) will be
used
d.If needed, turn on the Hamamatsu InGaAs NIR controller (key).

5.Start host computer.
a.Switch on the host computer

b.Double-click on the appropriate software to be used. For steady state measurements use
FluorEssence and for time-resolved experiments use Data Station.
4.3. Check System Performance15
1.In FluorEssence, click the experiment menu button ( ), or select Experiment Setup from Formatted: Bullets and Numbering
the collect menu, and select FL3-2(iHR-320)1 as the Hardware Configuration. Wait until
the system initialization process completes. Write down your name, the starting lamp turn
on time, and the research group to which you belong in the logbook.
2.Perform an excitation calibration check using the following steps:
a.Once the system initialization process completes, the Main Experiment menu should
appear (this can be also brought up to the screen using the experiment menu button).
Select spectra.
b.Choose excitation and click next.

c.Use the default parameters for monochromator and detector (File:
DfltSpectralExcitation.xml – It will be already pre-loaded). The spectral acquisition
14
[excitation] menu should appear at this point. The excitation scan will be performed from
200-600 nm in 1 nm steps with the emission monochromator parked at 350 nm. Both
excitation and emission slits are set at 1 nm. Activate the iHR-320 detector enabling only
the R1 signal, then click run.
d.In the scanned spectrum (200-600 nm), you
0.10 467
will find a broad band accompanied by a 0.09
structured signal from 400-500 nm. In these, the
Intensity (MicroAmps)
0.08
maximum peak should be located at 4671 nm. 0.07
If this is the case, the excitation monochromator 0.06 Lamp Spectrum

Ex 250-600nm
is calibrated and no further measures need to be

0.05 Slits 1.0nm
Inc 1.0nm Int 0.5sec
0.04 Mode R
taken. Otherwise, contact the person(s) in 0.03
450W XE OFR
S/O 199514, S/N 1505C-0812-FL
charge of the instrument. A representative 0.02
excitation spectrum is presented in Figure 10. 0.01
0.00
250 300 350 400 450 500 550 600
3.Perform an emission calibration check. For Wavelength (nm)
this, place into a quartz cuvette a sample of Figure 10. Xe-lamp excitation spectra. The
research-quality, triple-distilled or deionized main peak ought to be at 467 nm.
water. HPLC grade (18M spec.) or equivalent
is recommended for this. Then, follow these
steps:
a.Insert the cuvette into the sample compartment, and close the lid.
b.On the main FluorEssence toolbar select the experiment button and choose spectra.
c.Choose emission and click next. The spectral acquisition [emission] menu should
appear at this point.
d.Use the default parameters for monochromator and detector (multialkali PMT) (File:
DfltSpectralEmission.xml – It will be already pre-loaded). Make sure that the manual shutter
at the detector is open, then click run. 100
397
e.A new system should display a water Raman 80 Water Raman

S2-Side R2658(ANC1588)
Intensity (kCPS)
signal with a peak intensity of at least 450,000

Em 365-450nm Slits 5nm
Ex 350nm Slits 5nm
60 Inc 0.5nm Int 0.5sec
counts/s. This peak should be centered at 3971 450W XE OFR

S/O 199514, S/N 1505C-0812-FL
nm. If this is the case, the excitation 40
monochromator is calibrated and no further

20
measures need to be taken. Otherwise, contact
the person(s) in charge of the instrument. A 0
representative water Raman emission spectrum 370 380 390 400 410 420 430 440 450
is presented in Figure 11. Wavelength (nm)
Figure 11. Water Raman emission spectra. The

f.Record the water-Raman intensity in the log- main peak ought to be at 397 nm.
book.
15
5. Data Acquisition
5.1. Steady State Fluorescence15
After verifying that the performance of the system is OK, the signal acquisition is now
trustworthy. To acquire an emission spectrum, insert your sample in the compartment,§ Comment [MR14]: Depending on which
detector is going to be used, there may be some cable
close the lid, and perform the following steps: swapping to be done. Is this described somewhere
else?
1.Get to the emission spectral acquisition menu. Click on Real Time Control (RTC). Formatted: Bullets and Numbering
a.Position the desired excitation and emission wavelengths and slit widths. Also, set the Comment [MR15]: At which point is the choice
of detector made in the RTC mode?
desired integration time for the S1 signal.
b.Make sure of the following:

i.Status is Intermediate slit: 5nm set at Critical
ii.Shutter mode is set in auto.

c.After making sure that the Continuous box is unchecked, click run. See what number of
counts-per-second (CPS) you get. Perform a skim of many emission wavelengths until you
get a feel for where the maximum might be. Make sure to aAdjust your excitation/emission
slits so that the number of counts is below 2 million per second (this is important as we do
not want to saturate the detector – About 1 million counts per second is optimum).
d.After the conditions have been setfound with acceptable signal intensity at the emission
maximum for the desired excitation wavelength, click Transfer. The adjusted parameters
should now appear at the spectral acquisition [emission] menu.
2.Once the right settings have been transferred, make sure of the following:
a.In the monochromator menu ( ), the
Excitation 1 should be activated with wavelength parked at the desired number. Also, the
iHR-320 detector is activated (S1), the mirrors are set in axial at the entrance and exit of Comment [MR16]: If that is the detector of
interest. I think we need to give a generic procedure
the detector, and the range in set to the desired wavelengths. for all emission spectra acquisition. So, we should
say how to select the right detector.
b.Click the detector icon ( ) to confirm that the S1 channel detector is active. If desired,
enter a description of the experiment in the comment field and a data identifier. If needed,
signal correction can be applied including dark offset, blank subtraction, and correction
factors for dispersion of the instrument or detector response. A brief description of these
will be given in the following sections. Finally, select the integration time (0.1 s is the Comment [MR17]: Where will this be? I don’t
see a section title or space-holder for this.
preset) and the signal algebra (usually S1 and a corrected signal such as S1c/R1c). A
number of accumulations and scan cycles can be also accommodated at will, depending on
the actual experimental needs (the standard is that 1 accumulation and 1 cycle are used).
3.Open the manual shutter for the detector of choice. Click run. You will watch the
incoming data in real time on the Intermediate Display screen along with the variations in
§
The sample is assumed to be prepared using the specifications of Section IVa.
16
the positioning of the accessories. The scan may be paused, continued, or aborted as
deemed necessary. After all data is recorded, the Intermediate Display screen vanishes
and the Select Project File window opens. Close the detector shutter.
4.Enter a name for the project, or browse for an existing project with the Browse button.
Click the OK button, and do post-processing as needed.
Figure 12. Intermediate display screen (left), select project file (center), and plotted data in Origin after
acquisition (right).
5.2. Excitation Spectra15
To acquire an excitation spectrum perform the following steps:
1.Click the experiment menu button ( ), and choose excitation as the experiment type. Comment [MR18]: Why not use the RTC in this
case too?
2.In the monochromator menu ( ), Formatted: Bullets and Numbering

select the appropriate wavelength range for the excitation scan along with the wavelength
at which the emission monochromator will be parked. Select the appropriate slits sizes for
the excitation and emission monochromators.
3.Click on the detector icon ( ) and enable both, the reference and signal detectors.
Remove the R1 signal from the Formulas window and include S1 and S1/R1 signals, where Comment [MR19]: Why do this. You are
measuring the excitation scan, so it not a bad idea to
the latter includes the correction for the lamp response (use further correction factors if record the raw data from the lamp too (I would
deemed necessary, as described in the subsequent sections). recommend doing it).
4.Enter a description of the experiment in the comments field and then add a data
identifier.
5.Click run to perform the experiment. Notice that both signals will be acquired at the same
time. See that the uncorrected signal should be under the 2 million cps limit at all times.
Once the data is in the Origin window, it will be immediately ready for post-processing and
analysis. See that the data to analyze is the one with the correction for the lamp response.
5.3. Time Resolved Fluorescence16,17

Make sure to close the FluorEssence software after saving all experiment files of interest
and closing the experiment setup window. Additionally, make sure all the cables from the
pulse converter/discriminator (PCD) belonging to the desired detector are connected to
17
the FluoroHub. If they are not, turn off all the peripheral controllers (e.g. FluoroHub), and
the ACQ Controller. If the Xe-lamp is not needed at this time, also turn it off (Do not turn
off the Power button of the lamp housing yet, until its fully cooled). Then, proceed with
the pertinent connection(s) (contact the person in charge of the instrument for
training on this if you are not familiar with the system). An illustration of the rear panel
of the FluoroHub is presented in Figure 13. If NanoLEDs are to be utilized as light source, Comment [MR20]: Should we mention here how
to mount the NanoLED on the side or the sample
connect the corresponding cable from the FluoroHub (nanoLED Power, red dot) to the LED. compartment. Or maybe just give the page number
Likewise, the cables from the PCD should be connected to the TAC start/stop (blue dots) of the manual where this step is described.
and detector power (green dot) ports as well. Finally, turn on the ACQ Controller, and the
peripheral controllers.
Figure 13. Signal connections on

the rear panel of the FluoroHub
(main power inlet, switch and fan
not shown). The blue, green, and
red spheres mark the connections
for the detector signal output
(TAC start/stop), detector power,
and NanoLED power (Drive),
respectively.
In the time-correlated single photon counting (TCSPC) technique, the TAC** range is a
hardware parameter that defines the time window in which photons will be collected.
When a photon is collected, the TAC determines the time of arrival of the photon relative to
the excitation event, and one channel of the histogram is subsequently incremented by one
count according to the time of arrival. It is possible to run a TCSPC experiment in forward
TAC or reverse TAC mode. In forward mode the TAC start signal is taken from the
excitation source’s synchronization NIM†† output and the single-photon detector’s NIM
output provides the TAC Stop signal (Figure 14). Forward mode is used when the source
operates at a repetition rate lower than or equal to 100 kHz. Reverse mode is used when
the source operates at repetition rate above 100 kHz.
In reverse mode the connections to the TAC Start and TAC

Stop are reversed at the rear of the Hub in order to maximize
TAC conversion efficiency, so that the source synchronization
signal arrives after the detector signal. This is achieved by
increasing the amount of delay applied to the TAC Stop signal.
The amount of delay required in reverse mode depends on
the lifetime to be measured, and is typically just less than the
TAC range. The total amount of delay available is 95 ns
(coaxial) plus 200 ns (NanoLED sync) so the 500 ns TAC
range is longest possible in reverse mode operation. This is
not a significant limitation because the repetition rate must
**
TAC: Time-to-Amplitude Converter
††
Nuclear Instrumentation Module
18
Figure 14. TCSPC Schematic

using forward TAC mode.
be decreased below 1 MHz as the TAC range is increased above 500 ns, and so it is
appropriate to revert to forward mode. The range of coaxial delay settings is sufficient for
all TAC ranges in forward TAC mode.
The nanosecond flashlamp is always run in forward mode, but the NanoLEDs should be
connected according to the repetition rate. Please ensure that the correct connections have
been made prior to commencing. Also, refer to the Hub Manual to verify that all other
connections have been made correctly. Comment [E21]: Nice information, but relocate
to a theoretical section in order to keep the practical
section more streamlined and focused on the “how
Open the DataStation software by double clicking the DS shortcut icon on the Windows to” rather than the “why”.
Desktop ( ). At this point the splash start-up window should appear on the screen. Once
the software has been loaded and all the preliminary checks have been performed, the New
Measurement window appears. Each icon displayed therein corresponds to an available
type of measurement. In our setup, we should be able to measure TCSPC lifetime, TCSPC
anisotropy, TCSPC TRES. For a detailed description of each of these experiments, please
refer to the manufacturer’s manual. Select the appropriate experiment and click New. For
this section, we will only focus on the routine TCSPC lifetime measurements.
5.3.1 TCSPC Fluorescence Lifetime
To measure the TCSPC decay signal, place a sample of your analyte solution into a cuvette,
place the cuvette into the sample holder and close the lid. Follow the next steps:
1.Select Decay in the Measurement Tree window. Formatted: Bullets and Numbering
2.In the Hardware Tree:
a.Go to the System Optics menu and:
i.Select the Ex Mono and input the excitation wavelength and the size of the slits (only if a
Flash Lamp is being used).
ii.Feed the wavelength number and the size of the slit in the Em Mono 1.
b.Go to the TCSPC Mode Controls and place the desired settings for the measurement (e.g.
Peak Preset at 10,000 counts).
i.The TAC range can be set between 50 ns and 2 ms. As a first approximation, the TAC range
should be set to be about 20 times longer than the expected lifetime of the sample.
ii.A coaxial cable delay module is provided inside the Hub module and this is used to offset
all other delays so that the decay trace can be positioned correctly within the TAC Range.
The coaxial delays do not drift and do not introduce any error into the decay measurement.
The range of delay settings available using the Hub is 0-95 ns (variable in 1 ns steps) and
this is applied to the signal arriving at the TAC Stop input. Additional delay can be provided
externally if desired (e.g. using a gate-and-delay generator) and additional electronic
synchronization delay is available on the NanoLED controller. For example, this is set at 65
ns for a TAC range of 50 ns.
19
iii.The run time (RT) preset causes the decay measurement to stop after a preset time.
Setting the RT preset to zero disables it. The RT preset is typically set to zero.
iv.The Peak preset causes the measurement to stop when a certain peak counts is reached.
Setting the Peak preset to zero disables it. The Peak preset is typically set to 10,000.
v.The Hub-TE is a biased amplifier stage connected at the output of the TAC (internally in
the Hub). When the Time Expander is off, the decay is acquired in the normal way over the
full TAC range, and the yellow markers are visible. When the Time Expander is on, the time
region between the yellow markers is electronically amplified to fit the full ADC window
giving an increase factor of ten in time resolution.
vi.Ensure that the Reverse button is depressed on the toolbar if you are using NanoLED
excitation in reverse TAC mode. Select logarithmic display using the button on the toolbar.
c.Go to the NanoLED Controls and place the desired parameters for the measurement (e.g.
Repetition Rate = 1 MHz). For high repetition rates, the synchronization delay should be 0
ns and the Sync mode should be set to internal. Always ensure that the NanoLED Output
control is in the OFF state while swapping sources.
3.Click Start. The decay data should now accumulate in the Measurement Chart and the RT
indicator on the InfoBar should display the current run time. The icon associated with the
Decay item in the Measurement Tree will animate while the decay trace acquires.
Accumulation should stop when the peak count reaches 10,000 (the final peak count will
be slightly over 10,000) as shown in Figure 15.
The full decay, including the rising edge and tail, should fit within the TAC range. If
necessary the decay position can be shifted by adding or subtracting some delay (you will
need to erase the existing data and reacquire a trace at the new delay setting). If the decay
is too long to fit within the TAC range, then select the next longest TAC range and reacquire
the decay. Similarly, if the decay fills less than half the TAC range it may be preferable to
select the next shortest TAC range. When the TAC range and delay settings have been
selected it is time to optimize the count rate.
20
Figure 15. Data points accumulation after reaching the preset peak number.
It is important in TCSPC to limit the count rate () to about 2% of the excitation source
repetition rate to avoid photon pile-up. In this example, the TAC Out rate should be limited
to 20 kcps in the case of NanoLED excitation at 1 MHz repetition rate, or to 800 cps in the
case of nanosecond flashlamp excitation at 40 kHz. The alpha indicator on the Status Bar
illuminates green when the TAC Out rate is below 2% and red when the TAC Out rate is
above 2%.
If you need to reduce the count rate to satisfy the pile-up condition then you can:
Decrease the monochromator slit widths (if applicable) but always use the same slit Formatted: Bullets and Numbering
widths for the sample and prompt measurements.
Close the iris on the flashlamp (if applicable).

Insert neutral density filters into the filter holders inside the sample chamber.
Select a detection wavelength which is not at the peak of the emission.

Dilute the sample.
21
It is desirable, but not essential, to collect counts at a rate close to the 2% pile-up limit. If
you need to increase the count rate then you can:
Increase the monochromator slit widths (if applicable) but always use the same slit Formatted: Bullets and Numbering
widths for the sample and prompt measurements.
Open the iris on the flashlamp (if applicable).

Adjust the beam steering mirror on the flashlamp while viewing the Foreground
Ratemeter window (this procedure should always be carried out after cleaning the
flashlamp electrodes).
Check that inner filter effects are not present in the sample. To avoid inner filter effects,
ensure that the optical density of the sample is less than 0.1 at the excitation wavelength. If
the sample is too concentrated then diluting it can give more counts.
Optimize the position of the cuvette using the x-y-z stage (if installed). View the
Foreground Ratemeter window while adjusting the position.
Check that the correct excitation and emission wavelengths and/or filters are being used
One or both of the monochromators (if applicable) can be set to zero order (wavelength
setting 0nm) to pass all wavelengths, and filters used instead.
Adjust the lenses in the NanoLED sources, or check that the output lens on the flashlamp
is located back towards the lamp body and away from the excitation monochromator.
Remove neutral density filters from the filter holders inside the sample chamber.
Increase the concentration of the sample.

In the case of FluoroCube or 5000-Series systems, optimize lens positions within the
sample compartment while viewing the count rate shown on the Foreground Ratemeter
window. If your system is equipped with motorized lenses then try running a Focus Scan Comment [MR22]: Where can the general user
find out if this is the case?
(select ToolsFocus Scan).
Once the sample count rate has been optimized, erase the data in the current Decay item
and start the decay measurement again. Remove the sample from the cuvette holder and
insert the scatter sample for the prompt measurement. Replace the sample compartment Comment [MR23]: Do you think we should
make a suggestion as to what material could be a
lid and click on the Prompt item in the Measurement Tree to highlight it. good scatterer for this purpose?
If you are using the emission monochromator, set its wavelength to the excitation
wavelength, but do not adjust its slit width. If you are using spectral filters, remove the
filter in the emission filter holder.
Adjust the count rate within the pile-up limit by adjusting the flashlamp iris or inserting
neutral density filters into one of the filter holders. If repeat measurements are to be
performed then it may be desirable to prepare a scatter sample that gives a similar count
rate to the decay sample without the need to adjust the count rate.
22
Start the Prompt measurement. The icon associated with the Prompt item on the
Measurement Tree will animate and the prompt profile will accumulate on the
Measurement Chart. When the measurement is finished, press OK. The Decay and Prompt
data items can be saved as a single DAS file. A dialogue window will appear allowing you to
choose the location and name of the DAS file to be saved.
To collect more prompts or more decays simply right click on the white space around the
Measurement Tree and select the desired type of measurement item from the pop-up
menu. Try collecting another prompt and another decay, then select FileSave As from the
menu again. This time you will be asked to select which prompt and which decay is to be
included in the DAS file. Only one decay and one prompt can be included in the DAS file, but
DataStation gives you the flexibility to choose any measurement items in the tree. Figure
3.10 illustrates how to save the second prompt and decay that you have just measured. Comment [MR24]: Where is this? Will it be
included in this document or is just in the manual?
If you measure decay data into a prompt measurement item, or vice versa, it is necessary to
correct this before saving the DAS file. Right-click the item to be corrected and select the
Properties option on the pop-up menu and change the “Type” property to suit the data. It is
also a good idea to update the “Name” property at this time.
6. Practical Aspects: Advanced Techniques

6.1. Phosphorescence Measurements
Talk about the details of phosphorescence measurements.
6.2. Fluorescence Anisotropy Measurements

Talk about the details of anisotropy measurements.
6.3. Time Resolved Emission Spectroscopy (TRES)

Talk about the details of TRES measurements.
6.4. Advanced TCSPC Measurements: Sequential TCSPC

Talk about the details of sequential TCSPC measurements.
6.5. Multi-Channel Scaling (MCS) Lifetime and Fast MCS
Talk about the details of MCS mode measurements.

6.6. Miscellaneous Measurements: Steady State, Gated Steady State
Talk about brief details of other types of measurements available.

Comment [E25]: A section on calculations
would be nice (e.g. how to calculate quantum yield,
etc.)
7. References
23
1
Based on: Estrada, L. A. PhD Dissertation, Bowling Green State University, Bowling Green, OH, 2010.
2
Tolman, R. C. The Principles of Statistical Mechanics. Oxford University Press, London, UK, 1938.
3
Hollas, J. M. Modern Spectroscopy. 3rd Ed. John Wiley & Sons. West Sussex, England, 1996.
4
Born, M.; Oppenheimer, R. Ann. Phys. 1927, 84, 457.
5
(a) Franck, J. Trans. Farad. Soc. 1926, 21, 536. (b) Condon, E. Phys. Rev. 1928, 32, 858. (c) Coolidge, A. S.;
James, H. M.; Present, R. D. J. Chem. Phys. 1936, 4, 193.
6
Franck-Condon Principle at the Wikipedia Home Page. http://en.wikipedia.org/wiki/Franck%E2%80%93Condon_
principle (Accessed June 9, 2010).

7
Gilbert, A.; Baggott, J. Essentials of Molecular Photochemistry. Blackwell Scientific Publications, Oxford,
England, 1991.
8
Kasha, M. Disc. Faraday Soc. 1950, 9, 14.
9
Lackowicz, J. R. Principles of Fluorescence Spectroscopy. 3rd Edition, Springer, New York, 2006.
10
Turro, N. J. Modern Molecular Photochemistry. University Science Books, Sausalito, CA, 1991.
11
Medinger, T.; Wilkinson, F. Trans. Faraday Soc. 1965, 61, 520.
12
Lamola, A. A.; Hammond, G. S. J. Chem. Phys. 1965, 43, 2129.
13
This section is based from Chapter 4 of Reference Error! Bookmark not defined..
14
Kubista, M.; Sjöback, R.; Eriksson, S.; Albinsson, B. Analyst 1994, 119, 417.
15
Based on Horiba Scientific Fluorolog®-3 Spectrofluorometer. Operation Manual. J81014 rev. D.
16
Based on Horiba Scientific FluoroHub Single Photon Counting Controller. User Guide.
IBH.190.UG.2735 rev. C.
17
Based on Horiba Scientific DataStation v. 2.4 Software for Single Photon Counting Data Acquisition.
User Guide. IBH.370.UG.2717 rev. G.
24

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Fluorolog FL3C-21

User Guide and Tutorial for Using the Fluorolog FL3C-21

Georgia Institute of Technology

This guide is not supposed to act as a replacement of textbooks on Fluorescence

2. Theoretical Aspects of Radiative Decay1

ˆ fi   f μ i   f μ i   f i  S f Si (1) Comment [W1]: The  integral at the right

kobs  k0  k1 f e  f v  f S  (2)

Orbital Spin-Orbit FC factor

3. The Fluorimeter System

An illustration of the basic elements

4. Practical Aspects: Fundamentals

Additionally, Figure 7 summarizes common errors related to

increased 2-3 fold. Usually, samples

3.Turn on the SpectrAcq controller

a.Turn on the Hub Station

d.If needed, turn on the Hamamatsu InGaAs NIR controller (key).

a.Switch on the host computer

b.Choose excitation and click next.

structured signal from 400-500 nm. In these, the

maximum peak should be located at 4671 nm. 0.07

If this is the case, the excitation monochromator 0.06 Lamp Spectrum

is calibrated and no further measures need to be

charge of the instrument. A representative 0.02

excitation spectrum is presented in Figure 10. 0.01

e.A new system should display a water Raman 80 Water Raman

signal with a peak intensity of at least 450,000

counts/s. This peak should be centered at 3971 450W XE OFR

nm. If this is the case, the excitation 40

monochromator is calibrated and no further

Figure 11. Water Raman emission spectra. The

b.Make sure of the following:

ii.Shutter mode is set in auto.

5.2. Excitation Spectra15

To acquire an excitation spectrum perform the following steps:

2.In the monochromator menu ( ), Formatted: Bullets and Numbering

5.3. Time Resolved Fluorescence16,17

Figure 13. Signal connections on

In reverse mode the connections to the TAC Start and TAC

Figure 14. TCSPC Schematic

2.In the Hardware Tree:

a.Go to the System Optics menu and:

Close the iris on the flashlamp (if applicable).

Select a detection wavelength which is not at the peak of the emission.

Open the iris on the flashlamp (if applicable).

Increase the concentration of the sample.

6. Practical Aspects: Advanced Techniques

Talk about the details of phosphorescence measurements.

6.2. Fluorescence Anisotropy Measurements

6.3. Time Resolved Emission Spectroscopy (TRES)

6.4. Advanced TCSPC Measurements: Sequential TCSPC

6.5. Multi-Channel Scaling (MCS) Lifetime and Fast MCS

Talk about the details of MCS mode measurements.

Talk about brief details of other types of measurements available.

principle (Accessed June 9, 2010).

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