Photoinduced electron transfer (PET)
• An excited electron is transferred from the
excited state of a donor molecule to acceptor
• Leads to a reduction in
the excited state
lifetime of D* and
therefore emission
intensity
“Photomolecular Science”, ©Trevor Smith, 2021
229
PET
• Describe where electrons reside as electron
bands in bulk materials and electron orbitals
in molecules.
• Can occur intra- or inter-molecularly
• Can be between chromophore and solvent
• Can sometimes be described as a charge
separated electron-hole pair
“Photomolecular Science”, ©Trevor Smith, 2021
230
PET
What is different with photoinduced ET ?
• Photoinduced charge separation (CS):Photoinduced (CS):
• Photoexcite donor (or acceptor)
D + A* → D.+ + A.−
E
LUMO
D + A* → D.+ + A.−
HOMO
•
D
D.+
A*
A.-
DA*
E
D+ADA
Vauthey
“Photomolecular Science”, ©Trevor Smith, 2021
231
PET
• Electronic excitation leaves a vacancy in the ground state orbital that can
be filled by an electron donor (exciton).
• This produces an electron in a high energy orbital which can be donated to an
electron acceptor.
• A photoexcited molecule can act as a good oxidising agent or a good reducing
agent.
• Photoinduced Oxidation
• [MLn]2+ + h → [MLn]2+*
• [MLn]2+* + donor → [MLn]+ + donor+
• Photoinduced Reduction
• [MLn]2+ + h → [MLn]2+*
• [MLn]2+* + acceptor → [MLn]3+ + acceptor−
“Photomolecular Science”, ©Trevor Smith, 2021
232
Weller* equation
• An estimate of the “driving force” for photoinduced charge separation (-ΔGCS = -ΔETG0) in a solvent with
relative permittivity εs (dielectric constant) can be made using the equation for the “Gibbs energy of
photoinduced electron transfer” (ΔETG0 written here for neutral starting species)
• The Gibbs free energy change for electron transfer can be divided into two terms.
0
0
ΔETG = (e[Eox
(D +. /D) − Ered
(A/A −.)] − E00) + X
• 1st term is the limiting value of ΔGET at infinite separation and is the polar driving force
• X is a modifying term accounting for Coulombic effects (accounts for stabilisation of the ionic product) includes the finite
donor-acceptor separation (Rc), ionic radii (r+, r-) and solvent dielectric constant (εs)):
X=−
e2
e2
1
1
1
1
+
+
−
4πϵ0ϵsrAD 8πϵ0 ( rD rA ) ( ϵs ϵref )
• Calculation of ΔGET requires the oxidation potential of the donor and reduction potential of the acceptor
which can be determined from electrochemical measurements. E00, the lowest excited singlet state energy
can be obtained from the first absorption band energy
“Photomolecular Science”, ©Trevor Smith, 2021
233
• This requires the donor (D) and acceptor (A) standard electrode
potentials (E0(D+./D) and E0(A/A-.) [in e.g. V vs SCE in a solvent with
dielectric constant εEC]
• e denotes the elementary charge) and the singlet or triplet state energy [in eV]
(1ΔE0,0 and 3ΔE0,0)
• using the 3ΔE0,0 can give estimates for the energetics of the electron transfer process starting from
the triplet state, knowledge about the centre to centre distance (Rc [in Å]) and of the effective ionicradii of the donor and acceptor radical cation and anion (r+ respectively r- [in Å])).
“Photomolecular Science”, ©Trevor Smith, 2021
𝜈
𝜈
234
Effect of solvent polarity
⎞
2 ⎛
Esolv = − e ⎜⎜1− ε1 ⎟⎟
8πε0r ⎝
s⎠
The ionic product is stabilised by solvation energy:
Weller equation for any solvent:
ΔGET = e ⎡⎣ Eox ( D ) − Ered ( A ) ⎤⎦ − E * +C + S
e2 ⎛ 1 1 ⎞ ⎛ 1
1 ⎞
+ ⎟⎜ −
⎟
⎜
8
πε
r
r
ε
ε
⎝
⎠
• ET reactions are favoured in0 polar
⎝ s
D
Asolvents
s, pol ⎠
S=
exciplex formation
in apolar solvents.
When this work was initiated, the following questions seemed
of particular significance:
(a) Does the above model account quantitatively for the
growth and decay of AQ* and the decay of A* subsequent to
pulsed excitation of A ?
(b) What is the enthalpy of the formation reaction of AQ*
from A* and Q and how does the value obtained from d In
(k3/k4)/dT-' compare with that calculated from steady state
data in the conventional manner with a so-called Stevens-Ban
plOt43I' [In (@E/@.M[Q]) VS. 1/T]?
(c) Critically related to (b) is the question, does k5 vary with
temperature?
(d) Do the steady state and transient measurements give
a self-consistent quantitative account of the photokinetic behavior of the system?
(e) If we write k4 = A4 exp(-(AEd*/RT)), how do the
parameters A4 and AE4* compare with the other two systems
reported in the literature7,*and is the correlation suggested by
Figure 6, ref 12, borne out by more data?
(f) Is the enthalpy of the formation reaction of AQ* from
A* and Q consistent with known oxidation and reduction potentials and optical transition e n e r g i e ~ ? ~ , ~ . ~ . ~ ~
“Photomolecular
Science”,
Smith,
This
system is©Trevor
also of interest
in 2021
view of the recent work of
Chuang and EisenthalI2 using picosecond laser techniques to
follow the growth of the anthracene-DMA exciplex. In order
to explain their data they found it necessary to explore corrections for the effects of diffusion and in fact obtained good
agreement with the standard continuum model. Thus in
seeking answers to the questions posed above the possibility
of diffusion effects entering into the formulation was regarded
as a distinct possibility. Chuang and Eisenthal also obtained
the interaction distance for the forward quenching step which
is an important parameter for corrections to the kinetic
equations needed to describe the exciplex and monomer time
evolution as well as the steady state quenching.
It is not uncommon for the rates of various processes in an
exciplex system to be such that one cannot answer all of the
above questions; the reason being an inability to obtain individual rate constant^.'^ A critical test of the kinetic model is
frequently impossible. However, the aromatic hydrocarbonamine exciplexes are more amenable to comprehensive kinetic
studies because of the presence, generally, of both monomer
and exciplex emission sufficiently separated in average energy
to permit the independent study of the behavior of both species,
and because the monomer and exciplex lifetimes are generally
quite different. A typical set of spectra is shown in Figure 1,
The anthracene-DMA system is one such combination of a
donor and acceptor.
ET reactions are favoured in polar solvents.
11. Experimental Section
a. Transient Measurement. Fluorescence decay measurements were
made on a time-correlated single photon counting instrument, details
of which have been described e l ~ e w h e r e . ' ~The
- ' ~ excitation source
was a Photochemical Research Associate's Nanosecond Flashlamp
System. An Oriel (3-772-3900 sharp cut-off filter was used in the
measurement of the unquenched anthracene decay and also in the
cases where it was not necessary to separate the monomer and exciplex
fluorescence. A Balzers K - 1 broadband interference filter was used
to measure monomer decay in the presence of quencher. Although this
filter transmits a small amount of exciplex emission, the characteristics
of the photomultiplier photocathode (bialkali type DU) reduce detection of exciplex emission even further. Finally, the exciplex emission
was isolated by using an Oriel G-772-3900 filter followed by a Kodak
Wratten No. 12 filter. The function of the first filter is to cut down
short-wavelength radiation which might induce unwanted fluorescence
in the Kodak-Wratten filter. All samples were excited at 340 nm.
Temperature was regulated to f O . l 'C using a thermostated cell
holder.
As mentioned earlier, the exciplex scheme predicts that the decay
law for the monomer
fluorescence intensity should be of the form
2
8aa
4030
5030
4719
i
Figure 1. Corrected fluorescence spectra of anthracene + N,N-dimethylaniline in cyclohexane at 25 'C.
Ale-f/Tl + A2e-f/rZ
and
that foret
theal.
exciplex
should 98
be
Ware
JACS
(1976) 4718
A 3 ( e - f / " 235
- e-r/T2)
when k3 is time independent. The constants Ai are related to the Ci
through the appropriate radiative rate constants.
Therefore, even if the filtering system transmits some exciplex
emission together with the monomer emission, the values obtained
for 71 and 72 should not be affected; only the pre-exponential factor
would change. However, in later sections it will be shown that k ) is
in fact time
As long as the initial portion of the decay
curve was avoided in the analysis, the T I value obtained (short-lived
component) was independent of whether the Oriel (3-772-3900 filter
(monomer some exciplex) or the Balzers K - 1 filter (mostly monomer) was used. Since the Oriel filter transmits much more light than
the Balzers filter, it was used in many of the experiments.
Two time scales on the Time to Amplitude Converter (Ortec 437)
were used. A short time scale was used when 71 was to be accurately
measured. In this case, 72 could also be obtained, but only with a very
large standard deviation. Therefore, a long time scale was used when
7 2 was to be accurately measured. Here, the decay law was assumed
to be single exponential and only the later portion of the decay curve
to which 71 does not contribute was analyzed. 72 was independent of
which of the three filtering systems was used.
The decay curves were analyzed with the iterative c o n v o l ~ t i o n ~ ~ ~ ~ ~
method using the Marquardt algorithm22for least-squares estimation
of nonlinear parameters consistent with the minimum on the x 2 hypersurface. We believe that this is the best method presently available
to analyze decay data from single photon counting instruments. (This
will be discussed in detail in a later paper.22)To recover the shape of
the response of the system to &pulse excitation from the observed
fluorescence decay data and the observed lamp profile, the Exponential Series Method of Ware, Doemeny, and Nemzek was used.I6
The only modification was that the Marquardt method, rather than
the usual analytical method, was used to search the parametric hypersurface. This appears to yield an improvement over the older
methodI6 as far as accuracy is concerned. To obtain actual rate parameters, a decay law of the form A l e - f / T l A2e-'Ir2 was assumed.
The Marquardt algorithm was used again to find the best fitting values
for A ] , T I ,A2, and 7 2 , by iterative convolution with the lamp profile.
b. Steady State Measurement. Fluorescence spectra were measured
on a conventional 90', two-monochromator spectrofluorimeter
equipped with a thermostated cell holder. @ ~ o / @ and
w @E/@M values
were obtained with the same instrument. The intensities at 378 and
500 nm were taken as a measure of monomer and exciplex emission
respectively to avoid interference from spectral overlap. Initially, the
intensities were obtained from the spectra. Subsequently, the output
from the micromicroammeter was fed directly to a digital voltmeter.
With this modification, the measurements could be made within a
relatively short time interval, thus avoiding the problem of lamp instability.
c. Chemicals. Zone-refined anthracene was used in all experiments.
Additional purification by column chromatography produced no
+
+
Barrier to electron transfer - Marcus theory
• Energy represented in only two
dimensions:
G=
1
1
1
1
+
−
⋅
⋅ (Δe)
( 2r1 2r2 R ) ( ϵopt − ϵs )
Hui, Ware
/ Fluorescence Quenching of Anthracene
The ordinate is the Gibbs free
energy. ΔG(0)‡ = λo/4 i.e. the
reorganisation energy at Δe = 0.5,
it corresponds to the activation
energy of the self-exchange
reaction.
• where r1 and r2 are the radii of the spheres
and R is their separation, εs and εopt are
the static and high frequency (optical)
dielectric constants of the solvent, Δe the
amount of charge transferred.
• The graph of G vs. Δe is a parabola
transferred amount of charge
“Photomolecular Science”, ©Trevor Smith, 2021
R. M. Williams
236
Introduction to Electron Transfer
Furthermore values for the barrier to charge separation (ΔG#) can be estimated
via the classical Marcus equation.
ΔG# = (ΔG + λ)2/ 4λ
with λ = λi +λs
For the estimation of the solvent reorganization term (λs) the Born-Hush
approach can be used:
λs = e2/4πεo (1/r - 1/Rc)(1/n2 - 1/εs)
and the internal reorganization energy (λi) can be estimated using the charge
transfer absorption maximum and the charge transfer emission maximum in a
non-polar solvent (where λs ≈ 0) of the electron donor-acceptor system studied
(or one that shows great resemblance to the system). The energy difference between these two maxima equals 2λi. Values range from 0.2 to 0.7 eV. An
estimate of the Gibbs free energy change for charge recombination (ΔGcr =The
- Marcus Predictions
ΔGcs - 1ΔE0,0) can also be given. By using the harmonic approximation together
energy
(λ) is the
energywas
needed
• Reorganisation
with the quantities
described above
the Marcus
equation
derived. (it took 25
distort [Closs
the product
state 1988]
and its surroundings
years to prove ato theory
& Miller,
that was derived with high
to reach
theisequilibrium
the
school mathematics!
There
still hope configuration
for you!). Theofrelation
between these
reactant state (i.e. while staying in the potential
quantities and the
parabola
given
below. state)
energy
wellisof
the product
Marcus/Hush theory of e transfer
Inverted Region
(D-A)*
D+-A-
∆G° is zero and
∆G≠ equals λ/4
!G#
"
∆G° < zero and
∆G≠ decreases
(normal intuition)
∆G° is quite negative
and ∆G≠ becomes zero
!G
The barrier to charge separation (ΔG#), the
overall Gibbs free energy change (ΔG) and the
The dot traces the energy of the transition
State as ∆G° becomes more negative
Fig. 5.
Representation
of the potential energy curves used in electron
total reorganisation energy (λ)
“Photomolecular Science”, ©Trevor Smith, 2021
transfer theory. The barrier to charge separation (ΔG#), the overall Gibbs free
energy change (ΔG) and the total reorganization energy (λ) are indicated (from
Kaletas, B. K. Thesis 2004).
8
∆G° is even more negative
and ∆G≠ becomes positive
again (!)
237
• Calculations of the points of
intersection of the parabolas leads
to expression for Gibbs free energy
of activation (barrier to CS):
ΔG ‡ =
(λ + ΔG )
4λ
o 2
λ = λinternal + λsolvent
‡
• Assuming Arrhenius behaviour
• Leads to:
ket =
2π
| HRP |2
ℏ
1
4πλkBT
exp −
(λ + ΔG )
4λkBT
0 2
ket α exp −
(λ + ΔG )
4λkBT
0 2
“Photomolecular Science”, ©Trevor Smith, 2021
238
Classical Marcus theory
Solvent dependence of ET
A-D+
AD
barrierless region
• Vary the solvent and
monitor changes in ket
λ
• Different solvent
reorganisation energies
ΔGET
1.0
(c)
0.9
kET /kmax
0.8
−ΔGET < λ
0.7
0.6
(d)
0.5
(b)
0.4
-0.5
λ
0.0
0.5
1.0
ΔG /λ
1.5
2.0
2.5
ET
λ
inverted region
normal region
ΔGET
ΔGET
“Photomolecular Science”, ©Trevor Smith, 2021
−ΔGET = λ
kET
239
⎡ ( ΔGET + λ )2 ⎤
∝ exp ⎢ −
⎥
4 λ kBT ⎥⎦
⎢⎣
−ΔGET !"##$%&!'(&"%)
>λ
Ar
Ar
N
3
N
a
Ar
Ar
Ar
Ar
O
N
N Zn N
N
O
N
N
O
Ar
Ar
4
O
N
N Zn N
N
7
b,c
N
OMe
H
H
OMe
H
H
O
N
Ar
Ar
N
O
d,e
5
6
N
OMe
N
N
N Zn N
N
N
OMe
Ar
Ar
syn-8 + anti-8
f
syn-8
g
Ar
Ar
Porphyrin-MV2+
N
ET
O
OMe
O
N
N
Ar
N
H
N
N M N
N
Ar
CH3
OMe
H
H
N
N
2PF6
M = Zn: syn,syn-9 + syn,anti-9
h
i
f
• Long-lived CS due to structural changes following
ET (ratio of the rates of charge separation to
charge recombination is greater than 1400)
M = 2H: syn,syn-10 + syn,anti-10
syn,syn-10
+
H
j, k
syn,syn-9
+
CH3
syn,syn-2·2PF6
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“Photomolecular Science”,
©Trevor
2021
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but contributes significantly to the dyad spectrum at
shorter wavelengths. There is no spectral evidence
for any interchromophore electronic interactions in
the dyad studied in this work in contrast to observations on a much shorter bridged porphyrin-fullerene
reported previously [11].
Quenching of porphyrin fluorescence in the dyad
compared to the Pz, model chromophore is observed
in both non-polar (toluene, e~ = 2.38) and polar
(benzonitrile, e~ = 25.2) solvents. In toluene the
quenching of porphyrin fluorescence is accompanied
by an enhancement in C60 emission with maxima at
720 nm and 800 nm [11] (cf. Fig. 2). In this solvent
the fluorescence excitation spectrum recorded when
Energy & electron transfer
224
T.D,M. Bell et aL / Chemical Physics Letters 268 (1997) 223-228
30
:~
%~
×
Pzn model -'.,['----
1. . . . . . . . . . . . . . . .
2s~
C6o model
i
r ~--~-r-...
!
"'k
20 :i
ts F
o ~
, ~ ]
",
x':"
600
650
700
750
gO0
Wavelength (nm)
Ar = 3,5-di-tert-butylphenyl
Fig. 2. Fluorescence emission spectra of the Pz~-9cr-C6o dyad
(
) and model Pzn ( - - - ) and C6o ( - - - - - ) compounds
in toluene with excitation at 550 nm. The quenched dyad emission
intensity has been normalized to the porphyrin model fluorescence
maximum at 618 nre to erephasise sensitized C6o emission above
650 nm. Coo model emission intensity enhanced for comparison.
monitoring in the region of C60 emission shows a
contribution due to porphyrin absorption confirming
that singlet-singlet excitation energy transfer is occurring from PZn to C60. The rate constant for the
additional non-radiative process in the dyad, k,r, can
be obtained using
knr = 1 / / T d y a d - l / T m o d e 1.
T.D.M. Bell et al Chemical Physics Letters 268 (1997) 223-228
extended conformation
folded
conformation
“Photomolecular Science”, ©Trevor
Smith,
2021
241
Fig. 1. Structure of the zinc tetraarylporphyrin-C6o dyad (Pzn-9O'-C60) illustrating the extended and folded conformations.
solvents electron transfer occurs with high efficiency
in this system resulting in a charge separated state
with a remarkably long lifetime of 420 ns.
2. Experimental
The zinc tetraarylporphyrin-{9 sigma bond
bridge}-C60 (Pz,-9o'-C6o) (Fig. 1) and unlinked model
Pz, and C60 chromophores were synthesized as described elsewhere [12]. The norbornylogous bridged
dyad system is not completely rigid and flexing of
the cyclohexene ring gives rise to two conformations, extended and folded, the latter predicted to be
only 0.2 kcal mol -I less stable than the former
(using the AM1 theoretical model [13]) in the case of
the non-arylated analogue of the dyad. The calculated centre-to-centre interchromophore separations
of the extended and folded conformations of the
non-arylated systems are 20.4 ,~ and 15.3 ,~, respectively.
Solvents were distilled before use and solutions of
the dyad (absorbance < 0.2) were degassed by
freeze-pump-thaw cycles. Measurements were carried out at 298 K. Fluorescence spectra were recorded
Optical microscopy
on a Hitachi 4010 or Spex Fluorolog 2 spectrofluorimeter. Fluorescence decay times were obtained by
the time correlated single photon counting technique
as outlined previously [14] using either the frequency
doubled output of a pulse-picked femtosecond Mira
900F (Coherent) Titanium:sapphire laser (400 nm) or
a synchronously mode-locked and cavity-dumped dye
laser (Spectra Physics Model 3500, 590 nm) as
excitation sources. Transient absorption studies were
performed using a flash photolysis apparatus that
incorporated a Nd:Yag laser (Continuum NY-61,532
nm, 7 ns pulse width) for excitation. Transient species
generated by the absorption of 5 millijoule laser
pulses in 1 cm cuvettes were monitored at right
angles to the excitation using a pulsed 150 watt high
pressure xenon lamp, monochromator (Jobin-Yvon
H-10) and photomultiplier tube (Hamamatsu R928).
Decays recorded on a digital oscilloscope (Tektronix
TDS-520) were transferred to a personal computer
for analysis.
Ab initio calculations were carried out on model
C60 and zinc-porphyrin chromophore systems using
the GAUSSIAN 94 [15] program. Optimized geometries of the neutral C60 and zinc-porphyrin species,
both constrained to adopt C s symmetry, were ob-
• Need probe processes on small spatial domains
• Biology
• Chemistry
• Physics
• Time-resolved fluorescence microscopy
• “Super-resolution” optical techniques
•
•
•
•
•
Total Internal Reflection microscopy
Single molecule spectroscopy
Localisation microscopy (PALM/STORM)
Stimulated emission microscopy
Multi-photon imaging
“Photomolecular Science”, ©Trevor Smith, 2021
242
Wide field vs scanned microscopy
• Widefield
• rapid, direct image acquisition
• resolution limited by pixel density
72% QE, 100 fps, 0.9 e- read noise,
0.14 e-/pix/sec darkcurrent,6.5 µm
pixels, 2560 x 2160 pixels
• Scanned
• stepper stage
• galvo scanner
• resonance scanners
• piezoelectric scanners
• acousto-optic deflectors
• spinning disk
“Photomolecular Science”, ©Trevor Smith, 2021
(1)
From the fluorescence lifetime of the PZ, model
0"model= 1.24 ns) and the dyad chromophore 0"dyaa=
190 ps) in toluene, the rate constant for the energy
transfer process is estimated to be 4.4 x 10 9 s e c - l .
With benzonitrile as the solvent the porphi'yin fluorescence lifetime is reduced from 1.16 ns in the
model compound to 90 ps in the dyad, corresponding
to a quenching rate constant of 1 >< 10 ~° see -I, and
there is no evidence of any C6o emission.
Laser flash photolysis studies of solutions of the
model PZn and C60 compounds indicatedl the presence of long lived ( > 10 microseconds)transients
which were quenched by oxygen and assigned to
triplet states. In toluene the major transient species
observed upon excitation of Pzn in the PZn-9tT-C60
dyad was the C60 triplet (absorption maximum at
710 nm [6,11], triplet state lifetime 15 microseconds).
This is consistent with the fluorescence observations
reported above of singlet-singlet energy transfer
from Pzn to C60 followed by intersystem ¢rossing to
the fullerene triplet. However in benzonittile excitation of the dyad leads to new transient species and
kinetic behaviour. The transient spectrum recorded at
10 nm intervals in benzonitrile following ]aser excitation at 532 nm is shown in Fig. 3 and is idominated
243
microscopy, and the growing number of applications in
cell biology that rely on imaging both fixed and living
cells and tissues. In fact, confocal technology is proving
Principles of Confocal Microscopy
to be one of the most important advances ever achieved
in optical microscopy.
The confocal principle in epi-fluorescence laser
In a conventional widefield optical epi-fluorescence scanning microscope is diagrammatically presented in
microscope, secondary fluorescence emitted by the Figure 2. Coherent light emitted by the laser system
specimen often occurs through the excited volume and (excitation source) passes through a pinhole aperture that
obscures resolution of features that lie in the objective is situated in a conjugate plane (confocal) with a scanning
focal plane (25). The problem is compounded by thicker point on the specimen and a second pinhole aperture
specimens (greater than 2 micrometers), which usually positioned in front of the detector (a photomultiplier
exhibit such a high degree of fluorescence emission that tube). As the laser is reflected by a dichromatic mirror
most of the fine detail is lost. Confocal microscopy
provides only a marginal improvement in both axial (z;
parallel to the microscope optical axis) and lateral (x and
y; dimensions in the specimen plane) optical resolution,
but is able to exclude secondary fluorescence in areas
removed from the focal plane from resulting images
(26-28). Even though resolution is somewhat enhanced
with confocal microscopy over conventional widefield
techniques (1), it is still considerably less than that of
the photons
transmission electron microscope. In this regard,
of
confocal microscopy can be considered a bridge between
these two classical methodologies.
Illustrated in Figure 1 are a series of images that
compare selected viewfields in traditional widefield and
laser scanning confocal fluorescence microscopy. A
thick (16-micrometer) section of fluorescently stained
mouse hippocampus in widefield fluorescence exhibits
a large amount of glare from fluorescent structures
located above and below the focal plane (Figure
1(a)). When imaged with a laser scanning confocal
microscope (Figure 1(b)), the brain thick section reveals
a significant degree of
structural detail.
Likewise,
244
“Photomolecular
Science”,
©Trevor Smith, 2021
widefield fluorescence imaging of rat smooth muscle
fibers stained with a combination of Alexa Fluor dyes Figure 2. Schematic diagram of the optical pathway and
principal
components
in
a
laser
scanning
confocal
microproduce blurred images (Figure 1(c)) lacking in detail,
while the same specimen field (Figure 1(d)) reveals a scope.
highly striated topography when viewed as an optical
section with confocal microscopy. Autofluorescence and scanned across the specimen in a defined focal
in a sunflower (Helianthus annuus) pollen grain tetrad plane, secondary fluorescence emitted from points on the
produces a similar indistinct outline of the basic external specimen (in the same focal plane) pass back through the
morphology (Figure 1(e)), but yields no indication of dichromatic mirror and are focused as a confocal point at
the internal structure in widefield mode. In contrast, the detector pinhole aperture.
a thin optical section of the same grain (Figure 1(f))
The significant amount of fluorescence emission
acquired with confocal techniques displays a dramatic that occurs at points above and below the objective focal
difference between the particle core and the surrounding plane is not confocal with the pinhole (termed Outenvelope. Collectively, the image comparisons in Figure of-Focus Light Rays in Figure 2) and forms extended
1 dramatically depict the advantages of achieving very Airy disks in the aperture plane (29). Because only a
Confocal Laser Scanning Microscopy
• Out of plane scattered
light or emission is
rejected by use of
pinholes
• wasteful
• Compare with wide-field
3
Widefield vs Confocal images
http://www.olympus uoview.com/theory/confocalintro.html
“Photomolecular Science”, ©Trevor Smith, 2021
245
Time-resolved fluorescence
• Through fluorescence methods we have the
ability to temporally resolve the signal
• The time scale of fluorescence (ps-ns) is ideally
suited to report on a range of processes:
Energy transfer (FRET)
Electron/proton transfer reactions
Aggregation
Chromophore, segmental rotation - depolarisation
Isomerisations (photochromic reactions)
Solvent relaxation
“Photomolecular Science”, ©Trevor Smith, 2021
fl
•
•
•
•
•
•
246
Time-Resolved Microscopy
• Enhance signal over undesired fluorescence or
scatter (Raman, Rayleigh, SHG)
• Sensitivity of time-resolved signal to environment
• More information is available than just intensity
• (e.g. speciation,
f
variations etc.)
Fluorescence Intensity (counts)
• e.g. Fluorescence “lifetime”, f, depends on polarity, pH,
[fluorophore], [ion], quenching processes, aggregation
etc.
30000
22500
15000
7500
• Many phenomena can influence the fluorescence
intensity
• Correlate time-scales with physical processes
0
0
5
10
15
20
Time (ns)
• e.g. molecular motion/rotation, energy transfer
• Discriminate between species with similar
spectral but different temporal ( ’s) properties:
“Photomolecular Science”, ©Trevor Smith, 2021
247
Applications of FLIM
• Has been used for a range of
samples:
FIG. 7. FT-IR spectrum of the brownish deposits; absorption peaks of
gypsum (3543, 3407, 1683, 1621, 1128, and 672 cm!1) are prevalent;
small absorptions of calcium carbonate (1442 cm!1), weddellite (calcium oxalate dehydrate, 1324 cm!1), and quartz (1001, 798, and 780
cm!1) are also visible.
•
•
•
•
Biological samples
Statue of David
Gun shot residue
Polymers
This surface alteration should be ascribed to the outdoor exposition of David sculpture until 1873, when the
statue was placed inside the museum ‘‘Galleria dell’ Accademia’’. Deposits or surface patinas of this type (a
compact mixture of different minerals often containing
small amounts of organic compounds) have been formed
over the centuries. 23,24 A complete characterization of the
patina could not be achieved since the amount of material
FIG. 7. FT-IR spectrum of the brownish deposits; absorption peaks of
we were allowed to take from the statue was just enough
gypsum (3543, 3407, 1683, 1621, 1128, and 672 cm!1) are prevalent;
to perform vibrational spectroscopy, while gas chromasmall absorptions of calcium carbonate (1442 cm!1), weddellite (calcitography and mass spectrometry, which are more
umsuited
oxalate dehydrate, 1324 cm!1), and quartz (1001, 798, and 780
1
to the study of the organic fraction of the patina,
cm!were
) are also visible.
precluded. Nevertheless, some insight can be gained from
fluorescence images. In fact, in correspondence with the
patina, the fluorescence of the marble, mostly due to
the surface alteration should be ascribed to the outThis
underlying wax residues, is decreased to shorter lifetime
door exposition of David sculpture until 1873, when the
and lower amplitude. While the decrease
in amplitude can
D. COMELLI, G. VALENTINI,
R. CUBEDDU,
L.inside
TONIOLO,
Applied
Spectroscopy
59(9):1174-81
(2005)
Fplaced
IG. 8.andFluorescence
analysis
performed
on the lower
partAcof David’s
statue
the
museum
‘‘Galleria
dell’
be easily explained by the shielding effect of the
patinawas face:
(a) color picture of the area; (b) HSV map; and (c) fluorescence
cademia’’.
Deposits
patinas
of
this
type
(a under
248
“Photomolecular
Science”,
©Trevor
Smith,
2021
in the outermost surface, the change in lifetime can
lead
spectra
showingor
thesurface
different nature
of the spots
over the
lips and
the nostrils.
compact mixture
of different minerals often containing
to different interpretations. It can possibly be ascribed
either to the presence in the patina of a low amount
smallofamounts of organic compounds) have been formed
fluorescent compounds with a lifetime shorter than
overthat
the centuries. 23,24 A complete characterization of the
of the beeswax or to some kind of interaction between
aging:
(1)achieved
beeswax residues
small drops
patina could
not be
since theconcentrated
amount of in
material
the beeswax and the inorganic salts of the patina,we
leading
or permeated
the the
marble
surface;
(2) enough
salt deposits,
were allowed
to takeinto
from
statue
was just
to an increase in relaxation pathways of the excited states.
mainly composed of gypsum, calcium oxalates, and parto performticulate
vibrational
spectroscopy, while gas chromaFinally, Fig. 8 shows the HSV map of the lower part
matter; and (3) some organic contaminants, not
tography
and
mass
spectrometry,
which
are
more
of David’s face. A small red spot under the right nostril
precisely identified, located in small areas. suited
to the
of the
organic
fraction
the other
patina,
were was
presents the typical lifetime of wax residues ("6
ns),study The
FLIM
apparatus,
alongofwith
devices,
Nevertheless,
some insight
be gained
from
while the larger yellow spot above the lips has aprecluded.
slightly
also
tested to compare
differentcan
cleaning
methods
applied
shorter lifetime (5.8 ns). The two contaminants
also
to small
test In
areas
on in
thecorrespondence
statue. As an example,
fluorescence
images.
fact,
with cleaning
the
greatly differ in spectral features (Fig. 8c). In fact,
the
tests
were
performed
in
a
region
located
on
the
patina, the fluorescence of the marble, mostly due to left
the shin,
spectrum of the spot under the nostril correspondsunderlying
to that
characterized
by
the
presence
of
inorganic
deposits
wax residues, is decreased to shorter lifetime
of beeswax, as expected from its lifetime, while the
(mainly composed
of decrease
gypsum). in
Figure
9a shows
andspeclower amplitude.
While the
amplitude
can two
trum of the spot over the lip peaks at a definitely longer
patches that were treated with different cleaning proce-FIG. 8. Fluorescence analysis performed on the lower part of David’s
be easily explained
by the shielding effect of the patina
wavelength. The appearance of this spot led us to suppose
dures: the upper patch (G1) was cleaned with a deionizedface: (a) color picture of the area; (b) HSV map; and (c) fluorescence
surface,while
the change
in patch
lifetime
lead
that it should be made of organic material, but it in
wasthe
notoutermost
water poultice,
the lower
(G2)can
was
cleanedspectra showing the different nature of the spots over the lips and under
to differentwith
interpretations.
It can
possibly
ascribed life-the nostrils.
possible to assess its chemical structure, since microsamion exchange resin
(DES90).
Thebe
fluorescence
presence
a low
amount
of and
pling from the David’s face was not permitted. either to the
time
maps of in
thethe
twopatina
areas of
taken
before
(Fig. 9b)
The analysis of many other details of the David’s
surafter
(Fig. 9c) the
cleaning
are also
shown.
fluorescent
compounds
with
a lifetime
shorter
than that
face allow us to conclude that three main types of overThe or
increase
in the
lifetime
that takesaging: (1) beeswax residues concentrated in small drops
the beeswax
to some
kindfluorescence
of interaction
between
laid materials were largely mapped by fluorescence
implace
cleaningsalts
(red of
shift
the false
color map)or permeated into the marble surface; (2) salt deposits,
the beeswax
andafter
the the
inorganic
theofpatina,
leading
to an increase in relaxation pathways of the excited states.
mainly composed of gypsum, calcium oxalates, and parSPECTROSCOPY
Finally, Fig. 8 shows theAPPLIED
HSV map
of the lower part1179ticulate matter; and (3) some organic contaminants, not
of David’s face. A small red spot under the right nostril
precisely identified, located in small areas.
presents the typical lifetime of wax residues ("6 ns),
The FLIM apparatus, along with other devices, was
while the larger yellow spot above the lips has a slightly
also tested to compare different cleaning methods applied
shorter lifetime (5.8 ns). The two contaminants also
to small test areas on the statue. As an example, cleaning
greatly differ in spectral features (Fig. 8c). In fact, the
tests were performed in a region located on the left shin,
spectrum of the spot under the nostril corresponds to that
characterized by the presence of inorganic deposits
of beeswax, as expected from its lifetime, while the spec(mainly composed of gypsum). Figure 9a shows two
trum of the spot over the lip peaks at a definitely longer
patches that were treated with different cleaning procewavelength. The appearance of this spot led us to suppose
dures: the upper patch (G1) was cleaned with a deionized
that it should be made of organic material, but it was not
water poultice, while the lower patch (G2) was cleaned
possible to assess its chemical structure, since microsamwith ion exchange resin (DES90). The fluorescence lifepling from the David’s face was not permitted.
time maps of the two areas taken before (Fig. 9b) and
The analysis of many other details of the David’s surafter (Fig. 9c) the cleaning are also shown.
(a)
face allow
us to conclude that three(b)main types of overThe increase in the fluorescence lifetime that takes
HpIX
laid materials were largely mapped by fluorescence implace after
the cleaning (red shift of the false color map)
Residuals
Autocorr.
Confocal FLIM of various porphyrins
0.16
0.08
-0.08
-0.16
2.2
1.1
-1.1
-2.2
HpD
APPLIED SPECTROSCOPY
38.0K
90% free-base monomer (τf=14.8
ns) 8% free base dimer (τf=2.9
ns) (2% 0.2 ns)
30.4K
Counts
81% free-base monomer
(τf=5.8 ns),22.8K
(15% τf=1.0 ns,
4%15.2K
0.2 ns)
1179
7.6K
0.0
1.6
3.3
4.9
6.5
Time (nSec)
HpD - diffuse cytoplasmic staining throughout
cell with much from perinuclear region
(c)
HpIX - diffuse cytoplasmic localisation with
marked emission from plasma membrane
BOPP
(d)
58% free-base monomer
(τf=6.3 ns) (27% 1.8 and
15% 0.1 ns)
40% free-base monomer (τf=14.4 ns), 30%
porphyrin dication (τf=2.4 ns)
16% porphyrin monocation (τf=0.7 ns) (8%
0.2 ns)
BOPP - highly localized emission
from mitochondria
P.G. Spizzirri et al
C6 cerebral glioma cells
𝜏
249
𝜏
.
“Photomolecular Science”, ©Trevor Smith, 2021
𝜃
Pc - diffuse emission associated with plasma
membrane
,
Pc
analyse individual particles at high spatial resolution provides
far more information than using methods of bulk analysis (see
Section 2.1) since primer and bullet related residues often have
characteristic morphologies that reflect the mode of formation
and their compositions vary considerably (Romolo and Margot,
2001). Furthermore, by observing the emission properties in
the time domain, simple differentiation of an excited state
fluorescence lifetime from instantaneous scattered light (or
other luminescent processes) from a sample can be achieved
by examining photon detection times relative to an input laser
pulse.
The results are given in Figs 4(a) and 4(b) (best viewed in
colour), which show two representative confocal time-resolved
images obtained from a single GSR particle. The resulting
images are reconstructed by summing the individual photon
counts in each time channel at each pixel to obtain intensity
and the process of false colour mapping is described in Section
3. The total acquisition time per image set (which comprises
256 × 256 pixels) was 90 s at average photon count rate of
approximately 0.4 MHz. In each case, attenuation of the optical
signal was required in order to reduce the photon count rate at
the detector to within a suitable range for the SPC830 TCSPC
electronics (approximately 0.5–1.0 MHz). In this range, longer
acquisition times are required in order to obtain a sufficient
number of photons for each of the histogram distributions
Time-resolved fluorescence imaging - Gunshot residue
Fig. 4(b). Representative confocal time-resolved image of a single GSR
particle recorded 5 µm into the sample relative to Fig. 4(a). The subdominant (a 2 ∼ 26%) fluorescence component is mapped between 0.9 ns
and 2.2 ns red-to-blue, which clearly highlights the spatial variation of the
excited state lifetime of the emitting species throughout the particle.
20
D. K . B I R D E T A L .
at each pixel to ensure accurate two-exponential curve
fitting.
A comparison of the images reveals distinct differences in
Single
GSR particle close to the surface of the
the temporal and spatial distribution of the emitting species,
which is likely
due toimage
a numberis
of factors
including
differences
sample.
The
almost
entirely
a result of
in the local microenvironment (both chemical and physical).
the
dominantimage
(a1given
∼ 96%)
short
timefrom
component,
The time-resolved
in Fig. 4(a)
was acquired
plane close the surface of the particle and is the result of
τaa focal
mapped
between 100 ps and 250 ps red-to1,
two-exponential
model fit. Analysis reveals that the image is
96%) by
an
extremely short
dominated
almost entirely
(a 1 ∼ due
blue,
which
is
likely
to
scattered
excitation
time component τ 1 , which has been colour mapped over the
range a
of 100
ps to 250
(red-to-blue). Given
that these time
and
variety
ofpsshort-lived
emitting
species.
22
D. K . B I R D E T A L .
analyse individual particles at high spatial resolution provides
far more information than using methods of bulk analysis (see
Section 2.1) since primer and bullet related residues often have
characteristic morphologies that reflect the mode of formation
and their compositions vary considerably (Romolo and Margot,
2001). Furthermore, by observing the emission properties in
the time domain, simple differentiation of an excited state
fluorescence lifetime from instantaneous scattered light (or
other luminescent processes) from a sample can be achieved
by examining photon detection times relative to an input laser
pulse.
The results are given in Figs 4(a) and 4(b) (best viewed in
colour), which show two representative confocal time-resolved
images obtained from a single GSR particle. The resulting
images are reconstructed by summing the individual photon
counts in each time channel at each pixel to obtain intensity
and the process of false colour mapping is described in Section
3. The total acquisition time per image set (which comprises
256 × 256 pixels) was 90 s at average photon count rate of
approximately 0.4 MHz. In each case, attenuation of the optical
signal was required in order to reduce the photon count rate at
the detector to within a suitable range for the SPC830 TCSPC
electronics (approximately 0.5–1.0 MHz). In this range, longer
acquisition times are required in order to obtain a sufficient
number of photons for each of the histogram distributions
Fig. 4(a). Representative confocal time-resolved image of a single GSR
particle close to the surface of the sample. The image is almost entirely
a result of the dominant (a 1 ∼ 96%) short time component, τ 1 , mapped
between 100 ps and 250 ps red-to-blue, which is likely due to scattered
excitation and a variety of short-lived emitting species.
scales are of the order of the measured instrument temporal
response of the optical system (approximately 230 ps), we
attribute the origin of this signal in part to scattering of
the incident illumination (i.e. the lower end of the measured
range). However, considering that convolution has been used
in the fitting procedure (allowing fast processes of the order
of approximately 50 ps to be resolved) the measured lifetime
values at the upper end of this range are clearly too large to
be attributed to scattered light alone, and are more suggestive
of the presence of a variety of short-lived fluorescent species
throughout the sample. This is further exemplified if one
considers the spatial variation of the lifetime of this short decay
across the entire image, which would not be expected if the
measured signal was entirely due to scattered excitation.
Fig. 1. Distribution of discharged gunshot residues on a target from a firing distance of 60 cm as observed under (a) ambient light and (b) a forensic light
T R F M O F G S R : A P P L I C AT I O N T O F O R E N S I C S C I E N C E 2 3
source.
Distributionofdischargedgunshotresiduesonatargetfroma ringdistanceof60cmasobser
vedunder(a)ambientlightand(b)aforensiclight source.
in an inverted microscope (IX-71, Olympus, Center Valley, PA,
USA) through a scanning unit (Olympus FV-300) containing
x and y galvanometer scan mirrors and via a transfer lens, L
producing a focused scanning spot moving across the nominal
back aperture of an imaging objective, O (Nikon, 60× 1.4 NA,
Melville, NY, USA). The de-scanned signal from the sample
Recorded 5 μm into the sample. The sub- A schematic diagram of the instrument used in our imaging oil,
was filtered, D 1 (450-DCLP, Omega Optical, Inc., Brattleboro,
VT, USA) and reflected by a mirror, M 1 through a confocal
experiments is given in Fig. 2. Individual GSR particles were
dominant (a2 ∼ 26%) uorescence
#
C 2007 The Authors
pinhole, P (200 µm) before impinging on an enhanced silver
excited with a 400 nm ultrashort-pulsed laser beam (τ p ∼
C 2007 The Royal Microscopical Society, Journal of Microscopy, 226, 18–25
Journal compilation #
component is mapped between 0.9 ns and
200 fs) produced from the frequency doubled output of a
mirror, M 2 mounted at 45◦ housed in a custom made turret
designed to slide into the standard filter block mount of the FVTi : Sapphire laser (Coherent, Mira, Santa Clara, CA, USA)
2.2 ns red-to-blue, which clearly highlights tuned
Fig. 5. Recorded photon-count histogram at pixel (67 155) in the time-resolved image of Fig. 4(a). The two-exponential decay model fit reveals a dominant
300 scan unit. This mirror reflects the collected photons onto
to 800 nm and operating at a repetition rate of 76
short component (τ 1 ∼ 0.1
ns), and a sub-dominant
(a 2 ∼ 4%) long component
(τ 2 ∼ 1.0 ns).
Recorded
photon-count
histogram
at fast
pixel(67155)
in
a peltier-cooled
photomultiplier tube (PMC-100-1, Becker
MHz. The illumination
was delivered to the sample
mounted
the spatial variation of the excited state
Hickl, Berlin, The
Germany),
which is masked with a blocking
the time-resolved image of &Fig.4(a).
twoFigure 5 shows the measured decay histogram from a
It should
pointed on
outan
that
there
is still likely
a significant
lifetime of the emitting species throughout
filter,
BF andbe
mounted
x-y
translation
stage
that aids
representative pixel (67,155)
of the time-resolved
image
shown tindegree
of scatter
and the
possibility
of shortexponential
decay
model
reveals
athedominant
shortof a number
optimizing
signal
level.
Alternatively,
the fluorescence
the particle.
in Fig. 4(a). Observation of Fig. 5 clearly reveals a dominant
lived fluorescence
processes
contributing
to the recorded
decay
signal
be directed
to an additional
on the IX-71
component
(τ1
∼ is0.1
a may
sub-dominant
(a2
∼andsidethatDport
short component in the
decay, however,
there
also ns),
clear and
function
atby
each
pixel ina Fig.
4(b),mirror,
the fluorescence
microscope
inserting
dichroic
2 into the beam
evidence of a (or possibly a distribution of) longer-lived emitting
emission
(in all three dimensions).
(shown
greyed spatially
in Fig. 2) localized
where micro-spectroscopy
with a
4%) long component (τ2 ∼ path
1.0
ns).is highly
species (τ 2 ∼ 1.0 ns), albeit a small fractional component
Minimal adjustment
of the
microscope’s
translation
stage
high-resolution
(0.09 nm)
imaging
spectrograph
(Jobin Yvon,
(a 2 ∼ 4%). In order to investigate this feature further Fig. 4(b)
drastically
reducesNJ,
theUSA)
fluorescence
count rate to a
Triax
550, Edison,
equipped photon
with a liquid-nitrogen
was acquired from the same transverse coordinates on the
few tens
of (Jobin
kHz atYvon,
some spatial
locales. This
result
is somewhat
cooled
CCD
SpectrumOne)
can be
performed.
GSR particle but at an axial depth approximately 5 µm deeper
expected
and canimaging
be understood
if one considers
the complexity
Time-resolved
was carried
out by time-correlated
into the sample (relative to Fig. 4a). A two-exponential model
and inhomogeneous
formation
individual and
GSR Phillips,
particles,
single
photon counting
(TCSPC)of(O’Connor
D.K. Bird, K.M. Agg, N.W. Barnett and T.A. Smith, “Time-Resolved
Fluorescence Microscopy of Gunshot Residue:
decay function was again implemented that yielded accurate
which
is
addressed
in
the
discussion
below.
Fig. 4(b). Representative confocal time-resolved image of a single GSR
1984) using a complete electronic system for recording fast
particle recorded 5 µm into the sample relative to Fig. 4(a). The subAn Application to Forensic Science”, J. Microscopy,
fitting of226(Pt
the data 1),
that 18-25
forms the(2007)
basis of the time-resolved
light signals (SPC-830, Becker & Hickl). Two dimensional
dominant (a 2 ∼ 26%) fluorescence component is mapped between 0.9 ns
image. By contrast to Fig. 4(a), Fig. 4(b) exhibits a significant
fluorescence
images were collected by synchronizing photon
5. Discussion
and 2.2 ns red-to-blue, which clearly highlights the spatial variation of the
component in the exponential sum (a 2 ∼ 26%) of a longercounts (anode pulses from the photomultiplier tube) with the
excited state lifetime of the emitting species throughout the particle.
lived emitting species, τ 2 , which is mapped between 0.9 ns
in Sections
4.1
and 4.2 areLaser
particularly
xThe
and results
y laser presented
scanning signals
of the
microscope.
pulses
and 2.2 ns, red-to-blue. For comparison to Fig. 5, the measured
encouraging
the context
of forensic
are
detected byin
directing
a small
fractionapplications
of the outputsince
beamthey
of
at each pixel to ensure accurate two-exponential curve
decay histogram from the same representative pixel (67 155)
clearly
demonstrate
the feasibility
ofhigh-speed
a new non-destructive
the
Ti : Sapphire
laser onto
an external
photodiode
fitting.
has been taken from the time-resolved image of Fig. 4(b) and
method(PD-400,
of imaging
individual
GSRthese
particles,
further,
module
Becker
& Hickl) and
pulsesand
are used
to
A comparison of the images reveals distinct differences in
is given in Fig. 6 (i.e. the same transverse spatial location
offer a potential
method
unambiguously
the
determine
the detection
time oftoa photon
(an anodeidentify
pulse from
Fig. 2. Schematic diagram of the time-resolved scanning confocal
the temporal and spatial distribution of the emitting species,
but now microscope.
from the deeper
section).
the obvious
origin
of such particles
from
the
photomultiplier
tube) (i.e.
relative
toaa particular
laser pulse.firearm
Timing and/or
starts
fluorescence
M 1 , M 2 ,axial
M 3 : Mirrors,
D 1 , DGiven
2 : Dichroic Mirrors,
which is likely due to a number of factors including differences
profile
and the
associated
scale,
this
is clearly
ammunition
combination)
without
theafter
needthe
formeasured
extensive
on
receipt of a detected
photon
and stops
P:decay
Pinhole,
L: Transfer
Lens,
O: Imagingtime
Objective
and
BF:result
Blocking
Filter.
in the local microenvironment (both chemical and physical).
indicative of fluorescence and was confirmed by independently
sample preparation. However, determination of the exact
The time-resolved image given in Fig. 4(a) was acquired from
imaging all 8 samples of GSR particles formed from the same
source of the fluorescence emission is difficult
to allude to,
$
C 2007 The Authors
a focal plane close the surface of the particle and is the result of
ammunition/firearm type.
primarily
to the
molecular
complexity
of the226,
resultant
$
C 2007due
Journal compilation
The Royal
Microscopical
Society,
Journal of Microscopy,
18–25
a two-exponential model fit. Analysis reveals that the image is
dominated almost entirely (a 1 ∼ 96%) by an extremely short
time component τ 1 , which has been colour mapped over the
range of 100 ps to 250 ps (red-to-blue). Given that these time
scales are of the order of the measured instrument temporal
response of the optical system (approximately 230 ps), we
attribute the origin of this signal in part to scattering of
the incident illumination (i.e. the lower end of the measured
range). However, considering that convolution has been used
in the fitting procedure (allowing fast processes of the order
of approximately 50 ps to be resolved) the measured lifetime
values at the upper end of this range are clearly too large to
be attributed to scattered light alone, and are more suggestive
of the presence of a variety of short-lived fluorescent species
Fig. 6. Recorded photon-count histogram at pixel (67 155) in the time-resolved image of Fig. 4(b). By contrast to Fig. 5, the two-exponential decay model
throughout the sample. This is further exemplified if one
fit exhibits a significant contribution (a 2 ∼ 26%) from a longer-lived emitting species (average τ 2 ∼ 1.6 ns) in the exponential sum, which is clearly
indicative of fluorescence.
considers the spatial variation of the lifetime of this short decay
across the entire image, which would not be expected if the
measured signal was entirely due to scattered excitation.
"
C 2007 The Authors
Fig. 4(a). Representative confocal time-resolved image of a single GSR
particle close to the surface of the sample. The image is almost entirely
a result of the dominant (a 1 ∼ 96%) short time component, τ 1 , mapped
between 100 ps and 250 ps red-to-blue, which is likely due to scattered
excitation and a variety of short-lived emitting species.
were removed and individually mounted on glass microscope
slides, then sealed under a 0.17-mm cover slip suitable for
confocal microscopy.
3.2. Time-resolved confocal laser scanning microscopy
“Photomolecular Science”, ©Trevor Smith, 2021
250
C 2007 The Royal Microscopical Society, Journal of Microscopy, 226, 18–25
Journal compilation "
#
C 2007 The Authors
C 2007 The Royal Microscopical Society, Journal of Microscopy, 226, 18–25
Journal compilation #
The Journal of Physical Chemistry Letters
LETTER
em
Table 1. Peak Emission Wavelength (λmax
, see Figure 4), Fluorescence Decay Times, Relative Intensities (in Brackets) and
Amplitude Weighted Mean Lifetime Lifetimes, τm, for Three Representative Points in Each of the Three Solvent-Cast Films
fitted decay times (ps) and relative intensities
peak emission wavelength
chlorobenzene film
Conjugated polymer blends
toluene film
chloroform film
The Journal of Physical
Chemistry
Letters
X-T Hao
et al
Methods Appl. Fluoresc. 1 (2013) 015004
τ1 (nm)
τ2
τ3
τm
A
597
130 (90.5%)
301 (5.4%)
975 (4.1%)
174
B
582
178 (44%)
1062 (49%)
1443 (7%)
699
C
A
575 and 530
595
946 (56%)
122 (89%)
1208 (37%)
480 (7%)
1852 (7%)
1070 (4%)
1110
188
B
580
156 (50%)
911 (34%)
1378 (16%)
606
C
575 and 525
330 (22%)
1078 (68%)
1726 (10%)
974
A
596
125 (88%)
290 (7%)
131 (63%)
816 (29%)
1502 (8%)
423
240 (18%)
842 (65%)
1688 (17%)
879
LETTER
Distribution histogram deduced from uorescence lifetime
of the polymer. The current work identifies inhomogeneities in
the monomer/aggregate populations in the films, which will
affect the overall photophysical properties of the film.
The inherent aggregation characteristics of conjugated polymers due to the π!π electronic interaction among polymer
chains impose the existence of the spatial photophysical inhomogeneities in solid state films. The distribution histograms of
average fluorescence lifetime in the MEH-PPV films cast from
various solvents are depicted in Figure 3. The samples were
scanned over an area of 375 μm " 375 μm selected from the
representative regions of the films. The experiments were carried
out on three to five samples for polymers cast from each solvent.
Average fluorescence lifetimes from pixels shown in the lifetime
image recorded from the film cast from chlorobenzene solution
concentrate around 800 to 1000 ps with a peak at 905 ps, whereas
the average lifetime of the toluene film is rather more dispersed,
mainly in the range 450!950 ps with a peak at 715 ps and some
pixels in the 200 ps region. The longer average lifetimes, being
concentrated in a relatively small range of the chlorobenzene-cast
films, demonstrate the higher degree of uniformity of this film
compared with the broader lifetime distribution in toluene-cast
films. More aggregated clusters also can be seen in the inset
lifetime image of the film cast from toluene. The distribution
histogram of the chloroform-cast film is uniformly distributed in
a shorter lifetime range from 530 to 750 ps with a peak around
670 ps. The existence of a higher propensity for aggregate
formation is therefore inferred in the chloroform film.
Because the fluorescence decay profiles at different points of
the films are distinct from each other, they may also be expected
to exhibit diverse fluorescence spectral behaviors. The confocal
microscope coupled to a high-resolution Ocean Optics spectrometer allows us to record the emission spectra of specific points
by holding the excitation laser beam stationary at those points.
The sampled volume is significantly smaller than the colored
areas in the emission images of Figure 2. Fluorescence emission
spectra at different points of the film cast from various solvents
are illustrated in Figure 4 ((a) chlorobenzene, (b) chloroform,
and (c) toluene). It is clear that these spectra are significantly
different from those recorded from bulk areas of the film in the
conventional fluorimeter. For point A, which is considered as a
highly aggregated region in the chlorobenzene-cast film, the peak
position of the emission spectrum is located at 597 nm, and the
shape closely resembles that of the corresponding (bulk film)
spectrum in Figure 1. The spectrum is blue-shifted with the band
imagesand
of the
MEH-PPV
cast from various
“monomer-like”
contain
weaklylms
interacting
chains solvents
and those
scanning over an area of 375 μm 375 μm. The lifetime images
that are “aggregate-like”
and contain strongly interacting
are shown in the insets.
chains”.18 Collison et al.26 also reported time-resolved emission
studies of MEH-PPV in solutions of different solvent quality
suggesting that isolated segments with shorter conjugation
lengths existed in good solvents. The long-lived fluorescence
decay was attributed to the singlet state following back-transfer
from nonemissive interchain excited states to intrachain excited
states. It is worth noting that the decay times reported here are
shorter than those that have been attributed elsewhere to excited
state complexes27,28 with “excimer-like” species having emission
lifetimes
significantly
longer and
thanT.A.
theSmith,
∼0.8 “Nanomorphology
to 1 ns radiative of
◦C
P3HT:
PCBM
lm
annealed
at
150
X. Hao,
L.M. Hirvonen
Figure 2. (a) Intensity-based and (b) time-resolved scanning confocal fluorescence image of the P3HT: PCBM film annealed at 150 C for
The diverse fluorescence
decay
lifetime
of the monomer.18 bulk-heterojunction
1 h; (c) fluorescence decays of selected regions of the time-resolved image.
polythiophene-fullerene
lms investigated
behaviors
in micrometer
regions optical
of filmsimaging
cast from
solutions
by structured
illumination
andall
time-resolved
X. Hao, L.J. McKimmie and T.A. Smith, “Spatial Fluorescence
show confocal
the inhomogeneous
microscopic
microscopy”, structures
Meth. Appl.existing
Fluor. 1,on
015004/1-8
(2013)
Inhomogeneities
Light Emitting
Polymer
Films”, J.
Phys.Figurelaser
scales
depending
on
chain
conformation
and
packing
configuraand large-scale nanowires have
also been in
observed
using Conjugated
Optical
microscopy,
including
scanning
confocal
2. Fluorescence lifetime images (left panel) for MEH-PPV thin films cast from various solvents (a) chlorobenzene, (b) chloroform, and
2, 1520–1525
(2011)microscopy (LSCM),
(c) toluene
the fluorescence
decay profiles (right panel) recovered from the lifetime images (d) chlorobenzene, (e) chloroform,tion
and of
(f) conjugated
toluene.
polymers.
fluorescence
can and
provide
additional
AFM measurements from P3HT/PCBM filmsChem.
[37].Lett.
These
251
“Photomolecular
This suggests that the “uniformly coated” and small regions
important for the identi- Science”, ©Trevor Smith, 2021
fibres are interpreted as regions of crystalline PCBM. The spectral and temporal information,
correspond to these differently sized aggregates (points A!C,
chlorobenzene, there is little evidence of the short-lived (∼few
(e.g., points B and C) correspond to areas in which any
AFM, electron
microscopy
surface of these fibres should therefore provide a large fication of species, to complement
hundred picosecond) component at all in the largely nonaggrerespectively).
The decays
are clearly nonexponential, and we
aggregates
formed
are
small,
whereas
the
large
regions
(e.g.,
microscopy
(STXM)
interfacial region with the surrounding polymer through and scanning transmission x-ray
gated region C. The shortest of the decay times we observed are
have analyzed
them in terms
of multiexponential decay functions,
region
A) correspond to larger aggregates with correspondingly
LSCMto of
in general agreement with those typically reported
for MEH-PPV
although we do not intend
attribute specific decay compowhich electron/hole exchange between the polymer and measurements. Conventional (intensity-based)
shorter
fluorescence decay times. In most work on bulk films
solutions and films recorded in large area illumination
(ensemble
nents to individual
species.
The decay times, relative intensities
the previous
observaPCBM could occur. Surrounding the PCBM crystals is a P3HT:PCBM films (figure 2(a)) confirm
21
(i.e.,with
the functional
form of conjugated polymer ), the measuredecay
averaged) measurements.19,23,24 The components
and amplitude weighted-average lifetimes for three representaregion, 10–20 nm thinner and of lower mass density than tion [10] that, in addition to the expected weak emission from
times in the nanosecond regime resemble those
reported
ments
reflectbyfilms that consist largely of highly aggregated forms
tive points
in each exists
of the three
solvent-cast films are tabulated in
emission
around
the bulk of the film, corresponding to a PCBM-depleted the bulk film, a region of enhanced
Rothberg et al.25 for PPV that were attributed to relaxed
Table 1. is also evidence of
domain resulting from diffusion of the PCBM towards the the rods themselves. We note thatInthere
intrachain excitons formed via interchain excitons, with the more
each case, the regions representing the larger aggregated
1523
some enhanced emission at the tips
some
of the
the shortest
rods. This
areasof(A)
show
emission decay compared with
usually observed picosecond range fluorescence decay resulting
generation of the crystalline needles. This region is almost
enhanced emission is somewhatregions
surprising
it regions
would C
beare generally dominated by a
B andsince
C, and
from the rapid diffusion by F€orster transfer from short to long
pure, and highly crystalline, P3HT [10]. Surrounding this
expected that the emission be most
highly quenched
when regime.
decay component
in theInorganic
nanosecond
The contribution of
conjugation segments. Sherwood et al.18 used time-resolved
Chemistry
Article
region is the remaining ‘bulk film’, which has been shown
the slower
decay contact
component
increases as the aggregate size
fluorescence measurements to study the aggregate size and
the polymer is closest to (i.e. when
in direct
with)
to be P3HT/PCBM with fine phase segregation on the the PCBM. In the LSCM images
decreases
for all films
cast[10]
from the
different solvents, consistent with
dynamics of MEH-PPV using model oligomers of various
of Swinnen
et al
Time-Resolved Fluorescence. Fluorescence lifetime
order of tens of nanometres [38]. STXM has indicated bright region surrounding the PCBM
the findings
of Sherwood
et al.18 The films cast from chloroform
lengths. They discuss several explanations for their observations
needles
was interpreted
imaging
can provide valuable information complementary to
and toluene show similar fluorescence decay behavior for the
but found the most consistent was the formation
of a “core-shell”
that in the case of MDMO-PPV:PCBM blended films also, as indicating that little or no fluorescence
quenching due to
that core
obtained
structure in which “there are areas of the aggregate
that are using conventional confocal fluorescence
respective regions in the emission image, whereas in the case of
the PCBM clusters are surrounded by polymer in which electron/hole separation is observed
in the PCBM-depleted
intensity-based imaging particularly relating to the molecular
‘The PCBM-rich domains are actually 100% pure PCBM, region, which extends several micrometres from the needles.
1522
dx.doi.org/10.1021/jz200540j |J. Phys. Chem. Lett. 2011, 2, 1520–1525
nature of the fluorescent species. The free ligand H2L1, CuIIL1,
while the composition of the surrounding polymer-rich phase Intensity-based emission imaging, of course, only reports
and ZnIIL1 emit at similar wavelengths (λem ≈ 510 nm) (Figure
contains roughly equal proportions by weight of polymer on the quantity of light detected, so it could be argued
5) despite their differences in quantum yield, so it is not
and methanofullerene’ [39]. The dynamics and mechanism of that the bright signal observed around the PCBM needles
related phase changes during annealing have been studied in might be attributable to other factors such as (i) a small
possible to distinguish between them inside cells by confocal
MDMO-PPV/PCBM blends by AFM measurements [6].
level of scattered light being detected, (ii) emission from the
microscopy. However, both the ZnII and CuII complexes have
4
Figure 8. (a) Live cell confocal image of primary cortical neurons,
isolated from E14 mice, treated with CuIIL1 (green, λex = 488 nm),
Lysotracker (red, λex = 568 nm), and neutral lipid stain (blue, λex = 640
nm). Scale bar = 10 μm. (b) Magnified section indicated by a white
box in part a showing CuIIL1. (c) CuIIL1 overlay with Lysotracker,
showing partial colocalization with CuIIL1 and lysosomes. (d) CuIIL1
overlay with neutral lipid stain, showing partial colocalization with
CuIIL1 and lipid droplets. (e) Overlay of CuIIL1 (green), Lysotracker
(red), and neutral lipid stain (blue). Scale bar = 3 μm.
dx.doi.org/10.1021/jz200540j |J. Phys. Chem. Lett. 2011, 2, 1520–1525
dramatically different fluorescence lifetimes when compared to
the free ligand. Time-resolved emission measurements were
carried out, using time-correlated single-photon counting, on
the ligand and the complexes in bulk solution in order to
characterize their fluorescence lifetimes (Figure S1, Supporting
Information). The ligand H2L1 displays a fluorescence decay
profile adequately fitted by a single-exponential decay function,
giving a fluorescence lifetime of 2.83 ns. In comparison, fitting
the fluorescence decay curves of the Zn and Cu complexes
requires double-exponential decay functions. CuIIL1 has a
signature dominant (76%) initial short decay component of τ1
≈ 250 ps along with a relatively minor contribution from a
longer lived decay component of τ2 ≈ 2.79 ns. Similarly, ZnIIL1
has a dominant (83%) short decay component of ∼170 ps and
a minor (17%) long decay component of τ2 ≈ 2.55 ns. These
findings are in general agreement with those of Dilworth et al.
on related but structurally different bis(thiosemicarbazone)
complexes conjugated to a BODPY fluorophore through a twocarbon aliphatic linker except that for the complexes presented
here, ZnIIL1 and CuIIL1, the longer lifetime component does
not exceed that of the free ligand.38 The dominant short decay
component of the fluorescence of both complexes is attributed
to the quenched emission of the ligand, as its magnitude
relative to the free ligand correlates well with the degree of
• Intracellular Distribution
of Fluorescent Copper
and Zinc
Bis(thiosemicarbazonato)
Complexes
Figure 9. Fluorescence lifetime images with color scales mapping the mean fluorescence lifetime for (a) H2L, (b) CuIIL1, and (c) ZnIIL1, respectively,
in M17 cells. (d, e, and f) Corresponding fluorescence lifetime distributions/histograms for the average fluorescence lifetime, τav, τ1, and τ2.
Inorg. Chem. 2015,9562
54, 9556−9567
DOI: 10.1021/acs.inorgchem.5b01599
Inorg. Chem. 2015, 54, 9556−9567
fi
fi
fl
252
fi
fl
fi
“Photomolecular Science”, ©Trevor Smith, 2021
fi
171
587
575 and 530
Figure 3. Distribution histogram deduced from fluorescence lifetime
images of the MEH-PPV films cast from various solvents scanning over an
area of 375 μm " 375 μm. The lifetime images are shown in the insets.
Cellular imaging
836 (5%)
B
C
Botanical samples
MP-FLIM OF EUCALYPTUS SECRETORY CAVITIES
39
MP-FLIM OF EUCALYPTUS SECRETORY CAVITIES
• TREM of
essential oils
39
Fig. 4. Transmitted light (a) and fluorescence lifetime images (b and c) of enzymatically isolated E. polybractea secretory cavities. Pseudocolour mapping
represents mean fluorescence lifetime (τ m ). Lumen (L); secretory cells (SC); cell walls (CW); NVC; essential oil (EO). Secretory cell damaged during sample
preparation indicated with an arrow.
Furthermore, our estimate of the mean (±1 SE) percentage
volume occupied by the fluorescent region in the lumen 60 ±
3% is in the same range as that estimated by subtracting
the volume of oil extracted from isolated secretory cavities
from their total lumen volume (Goodger et al., 2010). It is
also apparent from the lifetime images that there is a narrow
region exhibiting shorter fluorescence lifetimes of between
0.7 and 1.3 ns at the boundary between the secretory cells
and the NVC of the lumen which is particularly apparent
in the optical section taken towards the top of the secretory
Fig. 4.Transmitted
Transmittedlight
light (a)
(a) and fluorescence
images
(b and
of c)
enzymatically
isolatedisolated
E. polybractea
secretory cavities.
Pseudocolour
uorescencelifetime
lifetime
images
(b c)
and
of enzymatically
E.polybractea
secretory
cavities. mapping
).mean
Lumen
(L);
secretorylifetime
cells
(SC);
cell
(CW);
NVC;
essential
oil (EO).
Secretory
cell(CW);
damaged
during sample
represents
mean fluorescence
lifetime
(τ malong
Pseudocolour
mapping
uorescence
(τsecretory
Lumen
(L);
secretory
(SC);
cell walls
NVC;
m ).walls
Fig. represents
5. Optical section
the z-axis of
an enzymatically isolated
E. polybractea
cavity. Pseudocolour
mapping
representscells
mean fluorescence
lifetime (τ m ). Depth along the z-axis relative to the surface from left to right: 20 µm, 40 µm, 60 µm, 80 µm. Secretory cells (SC); NVC; essential oil (EO).
preparation indicatedessential
with an
arrow.
oil (EO). Secretory cell damaged during sample preparation indicated with an arrow.
Furthermore, our estimate of the mean (±1 SE) percentage
volume occupied by the fluorescent region in the lumen 60 ±
3% is in the same range as that estimated by subtracting
the volume of oil extracted from isolated secretory cavities
from their total lumen volume (Goodger et al., 2010). It is
also apparent from the lifetime images that there is a narrow
region exhibiting shorter fluorescence lifetimes of between
0.7 and 1.3 ns at the boundary between the secretory cells
and the NVC of the lumen which is particularly apparent
in the optical section taken towards the top of the secretory
Journal of Microscopy 2012, 247, 33–42
Fig. 6. Representative©Trevor
histogram of photon Smith,
counts recorded for
a region of secretory cell wall taken from Fig. 5 fitted with a triple-exponential decay
“Photomolecular Science”,
2021
model.
253
!
C 2012 The Authors
C 2012 Royal Microscopical Society, 247, 33–42
Journal of Microscopy !
Fig. 5. Optical section along the z-axis of an enzymatically isolated E. polybractea secretory cavity. Pseudocolour mapping represents mean fluorescence
lifetime (τ m ). Depth along the z-axis relative to the surface from left to right: 20 µm, 40 µm, 60 µm, 80 µm. Secretory cells (SC); NVC; essential oil (EO).
Fig. 6. Representative histogram of photon counts recorded for a region of secretory cell wall taken from Fig. 5 fitted with a triple-exponential decay
model.
Abbe’s law
!
C 2012 The Authors
C 2012 Royal Microscopical Society, 247, 33–42
Journal of Microscopy !
• Ernst Karl Abbe (Jan. 23, 1840–Jan. 14, 1905)
• German physicist, optometrist, entrepreneur, and social
reformer.
• Together with Otto Schott and Carl Zeiss, he laid the
foundation of modern optics.
• ''The microscope image is the interference effect of a
diffraction phenomenon” (Abbe).
• A convergent lens produces at its back focal plane
the Fourier transform of the field distribution at its
front focal plane.
• One way to describe an imaging system (e.g. a microscope)
is in terms of a system of two lenses that perform two
successive Fourier transforms.
“Photomolecular Science”, ©Trevor Smith, 2021
254
Rayleigh criterion - diffraction limit
• The maximum resolving power
of a far-field optical microscope
is given by:
dmin = 0.61
λ
n.sinθ
(NA of lens)
€
fl
fl
“Photomolecular Science”, ©Trevor Smith, 2021
S1
Detector
Localisation
STED
Coherent X-ray imaging
Localisation
Fluorescence
“super-resolution”
microscopy
Structured
illumination
microscopySTED
- SIM Phase
Summary
Structured illumination microscopy - SIM
plate
Stimulated
emission
Excitation
Spontaneous
emission
PSfr
Coherent X-ray imaging
Fluorescence “super-resolution” microscopy
Summary
Stimulated
depletion
- STED
Stimulated
Emission
Depletion
microscopy
(STED)
S
Stimulated
emission
depletion
- emission
STED
0
SSTED
0
Exc. beam
Stimulated
emission
Excitation
Confocal
Detector
S1
Stimulated
emission
Exc. beam
mercial system
tion: ∼ 90 nm
Wavelength
Collected
emission
Exc. beam
STED beam
ntensity & pulsed
normally needed
Stefan Hell: imaging 40 nm beads.
Liisa Hirvonen
S0
STED
laser
Collected
emission
esol. < 20 nm
STED
Exc.
laser
laser
Liisa Hirvonen
Liisa Hirvonen
Exc.
laser
Microscopy at nm scale resolution using X-rays and visible light
Microscopy at nm scale resolution using X-rays and visible light
“Photomolecular Science”, ©Trevor Smith, 2021
Microscopy at nm scale resolution using X-rays and visible light
Wavelength
Liisa Hirvonen
Exc.
laser
Phase
plate
Phase
plate
Wavelength
roposed by
Hell in 1994
STED
laser
Detector
STED beam
S1
STED beam
Excitation
Collected
emission
Localisation
STED
Structured illumination microscopy - SIM
Spontaneous
emission
Coherent X-ray imaging
Fluorescence “super-resolution” microscopy
Summary
d emission depletion - STED
Spontaneous
emission
PSfr
PSfr
Microscopy at nm scale resolution using X-rays and visible light
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