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2 Spectroscopy 32(4) April 2017 w w w. s p e c t r o s c o p y o n l i n e .

c o m

Pump–Probe Microscopy: Theory,


Instrumentation, and Applications
Excited-state dynamics provides an intrinsic molecular contrast of samples examined. These dynam-
ics can be monitored by pump–probe spectroscopy, which measures the change in transmission of
a probe beam induced by a pump beam. With superior detection sensitivity, chemical specificity,
and spatial–temporal resolution, pump–probe microscopy is an emerging tool for functional imag-
ing of nonfluorescent chromophores and nanomaterials. This article reviews the basic principle,
instrumentation strategy, data analysis methods, and applications of pump–probe microscopy. A
brief outlook is provided.

Pu-Ting Dong and Ji-Xin Cheng

A s a pioneer of femtochemistry, Nobel laureate Ahmed


Hassan Zewail (1–3)recorded the snapshots of chemi-
cal reactions with sub-angstrom resolution through
an ultrafast femtosecond transient absorption (TA) technique.
In a transient absorption experiment, a laser pulse pumps a
copy to measure fluorescence lifetime (13). In 2007, the Warren
group reported pump–probe imaging with a high-frequency
modulation scheme (14). Their work demonstrated the fea-
sibility of imaging melanin by using two-color two-photon
absorption (TPA) or excited state absorption (ESA) processes.
molecule into an excited state. The excited state itself exhibits Since then, extensive research has been conducted by har-
relaxation dynamics on the femtosecond or picosecond tim- nessing the merits of pump–probe microscopy. A majority of
escale. A second laser pulse then probes the population in the the research focused on nonfluorescent chromophores such
excited state at different temporal delays with respect to the as hemoglobin and cytochromes, which absorb light but do
excitation. This analysis method reveals the dynamics of the not emit fluorescence efficiently (15). Fu and colleagues used
excited state and is termed as pump–probe spectroscopy. two-color absorption to measure the degree of oxygenation
Pump–probe microscopy, also known as transient absorp- based on the different decay constants of deoxyhemoglobin
tion microscopy, is an emerging nonlinear optical imaging and oxyhemoglobin (16). Pump–probe microscopy can effi-
technique that probes the excited state dynamics, which is re- ciently discern hemoglobin and melanin, the two major ab-
lated to the third-order nonlinearity (3,4). Pump–probe mi- sorbers in a biological tissue. Based on their signatures from
croscopy is an attractive spectroscopic imaging technique with the time-resolved curves, hemoglobin shows a purely positive
the following advantages: First, it is nondestructive to cells and response because of excited state absorption, whereas melanin
tissues and can be performed without tissue removal (5). Thus, (eumelanin and pheomelanin) demonstrate a negative (ground
it can be used as a repeatable diagnostic tool. Second, it is a state bleaching) signal when the pump beam and probe beam
label-free technique and doesn’t need an exogenous target (4). spatially and temporally overlap (5). In addition, pump–probe
Third, as a nonlinear optical technique, pump–probe micros- microscopy enables the discrimination of melanomas by de-
copy can image endogenous pigments with three dimensional termining the ratio between eumelanin and pheomelanin.
(3-D) spatial resolution (6). Fourth, unlike linear absorption, Melanin play an important role in skin and hair pigmentation
which suffers from scattering in a tissue sample, the pump– and melanomas (17). Without external staining, pump–probe
probe technique only measures absorption at the focal plane, imaging yielded novel insight into the differentiation of eu-
which offers optical sectioning capability (6). Fifth, compared melanin and pheomelanin among thin biopsy slices and has
to scattering measurements, this absorption-based method been used to probe the metastatic potential of melanocytic
has a weaker dependence on the particle and thus is highly cutaneous melanomas (16). Besides applications to pigments
sensitive to nanoscale subjects (8–11). Sixth, pump–probe in biological tissue, pump–probe microscopy has also been
microscopy with near-infrared laser pulses permits biological applied to distinguish various kinds of pigments in arts based
applications with an enhanced penetration depth and a lower on their decay differences (18–21).
level of tissue damage (12). Another significant application of pump–probe microscopy
In 1990s, Dong and coworkers used pump–probe micros- is for characterization of single nanostructures including gold
w w w. s p e c t r o s c o p y o n l i n e . c o m April 2017 Spectroscopy 32(4) 3

nanorods (22)and single-wall nanotubes


(SWNTs) (23–26). Specifically, Jung and
coworkers for the first time deployed the
phase of the pump–probe signal as a
contrast to distinguish semiconducting Second excited state
carbon nanotubes from metallic ones
(25). Tong and colleagues further used
this contrast for imaging semiconduct-
ing and metallic nanotubes in living Pump
cells (26). By tuning the excitation wave- First excited state
Probe

length, which is resonant with the lowest Vibrational state

electronic transition in SWNTs, Huang


and colleagues exploited the band-edge
relaxation dynamics in isolated and
bundled SWNTs (23). Through as- Excited state absorption Stimulated emssion Ground state depletion

sembling SWNTs with CdS, Robel and


Ground state
colleagues demonstrated the charge-
transfer interaction between photoex- Figure 1: Three major processes in a pump–probe experiment: (a) Excited state absorption, (b)
cited CdS nanoparticles and SWNTs by stimulated emission, and (c) ground-state depletion. For ground-state depletion, the number of
transient absorption (24). the molecules in the ground state is decreased upon photoexcitation, consequently increasing
In this review, we summarize the con- the transmission of the probe pulse. For stimulated emission, photons in its excited state can
trast mechanisms and instrumentation be stimulated down to the ground state by an incident light field, thus leading to an increase of
strategies of pump–probe microscopy transmitted light intensity on the detector. In the case of excited-state absorption, the probe photons
and highlight some of these significant are absorbed by the excited molecules, promoting them to the higher energy levels.
applications. Because of space limita-
tions, we could not cover the entire lit-
erature and would recommend to the
readers other excellent articles in this Pump pulse train Motorized delay line

field (27–32).
tau

Pump–Probe Theory
In a typical pump–probe measurement, Probe Pump
the pump-induced intensity change
of the probe is measured by a lock-in Scanning mirrors

amplifier referenced to the modulated


AOM
Modulation frequency

pump pulse. Then this change is nor-


Transferred modulation

tau
malized by the probe beam intensity
Loss
Pump pulse train Objective
Delay stack
to generate ΔIpr/Ipr (33). To express this Lock-in amplifier
Gain
Sample
Condenser

process at molecular level, we define the Ref Out In


Photodiode

absorption coefficient for an electronic Filter

transition between level “i” and level “j”


as

αij(ω) = σij(ω) (Ni – Nj) [1] Figure 2: Schematic illustration of pump probe microscopy.

where σij(ω) is the cross section from


electronic state i to j, and Ni and Nj are beam: observed: When the probe pulse is res-
the populations of the initial and final ΔIpr onant with i→j transitions (i ≠ 0), then
states, respectively. Conventionally, α is = −∑ αij(ω)ΔNj d [2] the probe pulse is absorbed by the mol-
Ipr i,j
positive for absorption and negative for ecule, reducing the transmission of the
gain (33). where d is the sample thickness. The probe pulse. This negative ΔIpr/Ipr signal
The pump pulse acts on the sample expression is derived from the Lambert- change is therefore called excited state
by changing the energy level popula- Beer relation within the small signal ap- absorption (ESA). When the probe pulse
tion, N→N + ΔN. As a consequence, the proximation. The “j” term describes all is resonant with 0→j transmission, the
population of excited states will increase possible excited states (33). probe transmission is enhanced upon
at the expense of that of the ground state. Depending on the probe energy, three pump excitation. This positive ΔIpr/Ipr
Such change is measured by the probe effects on the transmitted pulse can be phenomenon is called ground-state de-
4 Spectroscopy 32(4) April 2017 w w w. s p e c t r o s c o p y o n l i n e . c o m

pletion (GSD). When the lowest excited


state is dipole-coupled to the ground
(a) Pump-probe AFM image (b) 1.6 1.6
state and the probe pulse is resonant
Pump

Fluorescence (a.u.)
with the transition, stimulated emission

(105 M-1 cm-1)


1.2 1.2
Probe

0.8 0.8
(SE) occurs. An increased transmission
is observed in a SE process.
0.4 0.4
These three major processes are illus-

3
0.0 0.0 trated in Figure 1. A detailed description
500 600 700 800
Wavelength (nm) is provided in the following sections.
STAM 100 30
Pump-probe
Signal intensity (a.u.)

STAM (x2.5)
Excited-State Absorption

∂∂P/P (10-6)
20
50
0
Mean molecule
No. in focus
1 2 3 4
Excited-state absorption (ESA) is a
10 0.4 process where the probe photons are
0 280 nm
285 nm
0.2
attenuated by excited states as shown
0
in Figure 1. Since the 1970s, picosec-
0.0

0.0 0.5 1.0 1.5 0.0 0.1 0.2

0 2 4 6 8 10
Position (μm)
Concentration (μM) ond laser–based ESA measurements
have been extensively used to measure
Figure 3: Pump probe microscopy with subdiffraction spatial resolution and single-molecule detection ground and excited-state dynamics
sensitivity. (a) Subdiffraction-limited imaging of graphite nanoplatelets. Image from conventional (34,35). Compared to two-photon ab-
transient absorption microscopy (top left) and AFM image of graphite nanoplatelets (top right). Image sorption, which goes through a virtual
from saturation transient absorption microscopy (bottom left) and intensity profiles along the lines intermediate state, excited-state absorp-
indicated by the dashed lines in pump–probe image and STAM image (bottom right). Adapted with tion significantly enhances the detection
permission from reference 47. (b) Ground-state depletion microscopy with detection sensitivity of sensitivity by bringing a resonance with
single-molecule at room temperature. Ensemble absorption and emission spectra of Atto647N in pH a real intermediate electronic state. The
= 7 aqueous solution (top).The wavelengths of pump and probe beams are indicated. Ground-state mechanism for this process (36) can be
depletion signal as a function of concentration of aqueous Atto647N solution (bottom). The power is described using the following equation:
350 µW for each beam. The blue frame shows the points at lowest concentrations, indicating single-
∫N σ [σ′ −σ ℏν]I I exp (− τ ( dz [3]
Δt
molecule sensitivity is reachable. Figures adapted with permission from reference 40. ΔIpr =−
0 pu pr pr pu pr

pv

where N0 is the molecular concentra-


tion at ground state; σpr and σ′pr are
0.04 1

Hb 0.8
the linear absorption cross sections of
0.02 Eumel 0.6 75
Eumel
the ground state and excited states for
the probe beam, respectively; νpu repre-
Intensity (a.u)

75 0.4 50
50 Hb
0
sents the pump frequency and τ is the
25 0.2
25
Pheomel 0
s

–0.02
Ink
–0.2 Pheomel lifetime of the excited state (assume this
–0.4 is a single-exponential decay); and Δt is
the time delay between pump beam and
–0.04 –0.6 Ink
–0.8
–0.06
0 0.5 1 1.5 2 2.5 3 3.5 4
–1
–1 –0.5 0
ω = π/2 THz
0.5 1
probe beam. Ipu and Ipr denote the in-
t (ps) g tensity of pump beam and probe beam,
1
(a) (b)
1
respectively. In the presence of a pump
0.8 Eumel pulse, excited-state population would
Normalized counts per pixel bin

0.6 Eumel

0.4
give birth to the transmission changes of
0.2 Hb
the probe. Equation 3 demonstrates that
Hb
0 Pheomel Pheomel
only at Δt = 0 when the pump beam and
s

–0.2 probe beam are spatially and temporally


–0.4 overlaid can ΔIpr have the biggest value.
–0.6
Ink
Ink
As Δt becomes longer, ΔIpr depicts as
–0.8
ω = π/2 THz ω = 1.4π THz
an exponential decay curve convoluted
–1
–1 –0.5 0
g
0.5 1 –1 –0.5 0
g
0.5 1
0
with an instrumental response function
that is a Gaussian function.
Figure 4: Phasor analysis to interrogate pump probe signal. Experimental transient absorption
spectra of hemoglobin (Hb), sepia eumelanin, syhthetic pheomelanin, and surgical ink (top left). Stimulated Emission
Phasor difference of mixtures of eumelanin and pheomelanin (eumelanin fraction of 75%, 50% When interrogating the short-lived
and 25%) along with their phasor locations on the s-g coordinate (top right). Cumulative histogram excited states in pump–probe experi-
phasor plot of 17 ocular melanoma samples at frequency /2 THz (bottom left) and 1.4 THz (bottom ments, the photons in the excited states
right). Adapted with permission from reference 54. are stimulated down to the ground
w w w. s p e c t r o s c o p y o n l i n e . c o m April 2017 Spectroscopy 32(4) 5

state by a time-delayed probe pulse as


shown in Figure 1. This process is called
stimulated emission (37). The absorption (a) (c) 2.0 Singlet
Triplet (e)

coefficient decreases with increasing 1.0

∆A (10-3)
excitation irradiance. The decrease in
Delay = 0 ps
0.0

absorption happens due to the annihi- –1.0


<100 ps
5 ns

lation of the number densities of both 550 600 650 700


Wavelength (nm)
750 850

the ground state and the state being ex- (b) (d) 1.0
2 μm
NW1
(f)

cited, this process can be portrayed as


NW2 1.0 10
2 μm
4 8

lS(√)l2

Count
Signal (norm.)
NW3 6

equation 4:
3 4

∆I/lx105
2 μm
0.5 2
0.5

∆I
0
NW1 (i-Si) 2 0 50 100 10 15 20 25
Frequency (GHz) Period (ps)
NW2 (i-Si)

( (
1

I exp − Δt
∫N σ σ I
0.0
0 pu pr pu pr τ dz [4] 0.0 0

ΔIpr =− ℏνpv
NW3 (n-Si)
0 50 100
Delay time (ps)
150 0 50 100
Delay time (ps)
150
0 200 400 600 800 1000 1200 1400
Pump-probe delay (ps)

From equation (4), we can tell at Δt =


0, strongest signal is achieved. As Δt be- Figure 5: Imaging nanomaterials by pump–probe microscopy. (a) TA imaging of graphene on glass
comes longer, the transmission change coverslip. 0 stands for defects, 1 is single layer graphene, 2 is double layer, 3 is triple layer, respectively.
of probe also demonstrates an exponen- Pump = 665 nm (1.10 mW) and probe = 820 nm (0.68 mW), respectively. Data adapted from reference
tial decay curve convoluted with Gauss- 70. (b) Transient absorption image of DNA-SWNTs internalized by CHO cells after 24 h incubation.
ian function. Based on the stimulated Gray, transmission of cells; green, S-SWNTs; red, M-SWNTs. Pump = 707 nm, probe = 885 nm. The
emission, Min and colleagues achieved laser power post-objective was 1 mW for the pump beam and 1.6 mW for the probe beam. Adapted
nanomolar detection sensitivity of non- with permission from reference 26. (c) Decay-associated spectra of the triplet (red) and singlet (black)
f luorescent chromophores (37). The excitons of tetracene obtained by global analysis of the ensemble transient absorption spectra with the
integrated intensity attenuation of the probe polarization to maximize triplet absorption. Data adapted from reference 75. (d) Pump–probe
excitation beam can also be expressed as microscopy decay kinetics following photoexcitation of a localized region in three different Si nanowires;
ΔIpu N0σ01 NW1 (red) and NW2 (green) are intrinsic, and NW3 (blue) is n-type. Curves are fit to a tri-exponential
Ipu
=− S ∼10–7 [5] decay. Inset shows the SEM image of three wires. Adapted with permission from reference 76. (e)
3D transient absorption microscopic images of gold nanodiamonds in living cells taken from eight
where S ~ 10 -9 cm 2 denotes the beam successive focal planes with 1-μm step. Scale bar: 20 µm. Data adapted from reference 77. (f) Transient
waist, and σ01 ~ 10-16 cm2 represents the absorption trace from a single Ag nanocube from a sample with an average edge length of 35.5 ±
absorption cross section from ground 3.4 nm (left). The inset shows the Fourier transform of the modulated portion of the data. Ensemble
state to the first electronic state. The transient absorption trace for the Ag nanocube sample (right). Inset gives a histogram of the measured
stimulation beam will experience a periods from the single-particle experiments, red line is the distribution calculated from the size
transmission gain after interaction with distribution of the sample. Adapted with permission from reference 78.
the molecules:
ΔIpr N0σ10
in 2007 (39). Similar to stimulated emis- time. The order of modulation depth of
Ipr
=− S ∼10–7 [6] sion, it presents as an out-phase signal the transmitted probe beam by a single
(Figure 1). The overall mechanism is molecule (Atto647N) is ~10 -7, which
consistent with other transient absorp- means we can still demodulate the sig-
N0IpuIprσ10σ1
ΔIpr∝ − S2 [7] tion mechanisms. If expressed in equa- nal from a lock-in amplifier. Based on
tion form, the GSD process has the the mechanism above, ground-state
From equation 7, we can conclude same expression as stimulated emission depletion microscopy could reach sin-
that the stimulated emission process in equation 4. The only difference lies gle-molecule detection (40). The ground
shows overall quadratic power depen- in the probe wavelength. For GSD, the state depletion method could also be ap-
dence, allowing three-dimensional op- probe is chosen close to the maximal ab- plied to localize fluorescence emission
tical sectioning. In addition, the linear sorption peak, whereas the probe beam from fluorophores bound to the surface
dependence upon the concentration of in the case of stimulated emission is se- of a nanowire, thus making it possible to
analyte allows for quantitative analysis. lected away from the absorption peak. map out the structure of a nanowire [41].
The detected sensitivity would be down Based on ground-state depletion, Zink and colleagues also showed how
to 10 -9 M if the incident irradiance of single-molecule detection at room tem- GSD microscopy can be applied to mea-
pump beam and probe beam are in the perature has been achieved (40). Under sure tubulin modifications in epithelial
range of megawatt cm-2 (37). the condition that both pump and probe cells (42). High sensitivity coupled with
beams (continuous-wave lasers) were optical sectioning capability makes
Ground-State Depletion chosen close to near saturation inten- ground-state depletion microscopy an
Ground-state depletion (GSD) micros- sity levels (350 μW at the focus for each important emerging technique.
copy is a form of super-resolution light beam), a shot noise limited sensitivity
microscopy suggested almost a decade is achieved. The detected sensitivity for Instrumentation
ago (38), and it was first demonstrated Atto647N is 15 nM with 1s integration A typical pump–probe imaging setup is
6 Spectroscopy 32(4) April 2017 w w w. s p e c t r o s c o p y o n l i n e . c o m

in the preceding sentence?] Therefore,


(a) 2.5
2.0 (c) this scheme is sensitive to artifacts. The
second type with high repetition rates
Oxyhemoglobin l
Oxyhemoglobin
2.0
Deoxyhemoglobin Deoxyhemoglobin
Signal intensity (μV)

1.5

allows for averaging more laser shots


1.5 stratum comeum

1.0
1.0

0.5 0.5
+1
per unit time. As a result, the detection
sensitivity of ~10-6 units of absorbance
+1
0.0 0.0
1
-2 0 2 4 6 -2 0 2 4 6
can be achieved (28). By employing high-
0 0
Delay (ps) Delay (ps)

(b) -1 2 -1 2
frequency (that is, megahertz) lock-in
720 nm pump
810 nm probe 0.8
Tissue principal
components
modulation, Hartland and coworkers

Signal (au)

Signal (au)
detected signals from isolated single-
0 720 nm pump
810 nm probe
0.4
Tissue region 1

walled carbon nanotubes with a sensi-


-1 Tissue region 2
Sepia eumelanin 0.0
Synthetic pheomelanin Component 1

50 μm
-2 -0.4

2
Component 2
Component 3 tivity of ΔI/I ~ 5 × 10-7 (44). Moreover,
in this scheme, the modulation provided
-1 0 2 4 6 8 10 -1 0 1 3 4 5
Interpulse delay (ps) Interpulse delay (ps)

by either an AOM or an electro-optic


Figure 6: Imaging microvascular and melanomas by pump–probe microscopy: (a) Pump–probe
modulator (EOM) operates at a high
microscopy is applied to differentiate oxyhemoglobin and deoxyhemoglobin. ESA signal from
frequency in the range of 100 kHz to 10
oxyhemoglobin and deoxyhemoglobin with pump = 810 nm (10 mW) and probe = 740 nm (6.4
MHz, where the noise approaches the
mW) (left). ESA signal from oxyhemoglobin and deoxyhemoglobin with pump = 740 nm (2.4 mW)
shot noise limit. One possible drawback
and probe = 810 nm (10 mW) (right). Adapted with permission from reference 36 (copyright 2008
of such setups is their high probability
Society of Photo-Optical Instruction Engineers). (b) Ex vivo imaging of microvasculature network
of detecting the accumulation of long-
of a mouse ear based on endogenous hemoglobin contrast. Red, blood vessel network; green,
lived species, such as triplet or charge-
surrounding sebaceous glands. Pump = 830 nm (~20 mW, two-photon excitation of Soret band),
separated states (27).
probe = 600 nm (~3 mW, one-photon stimulated emission of Q-band of hemoglobin). Adapted with
When it comes to the detection of
permission from reference 37. (c) Pump-probe image of a compound nevus at 0-fs (left) and 300-fs
pump–probe signal, a phase-sensitive
(right) interpulse delay (top). Regions containing eumelanin have positive signal (red/orange). Pump–
lock-in amplifier is usually indispens-
probe time delay traces comparing tissue regions of interest 1 and 2 (white boxes in top) with pure
ably used to demodulate the probe
solution melanins (bottom). The first three principal components found in tissue pump–probe signals
signal. Slipchenko and colleagues re-
(loadings plot, right). The first two components account for more than 98% of the variance. Pump =
ported a cost-effective tuned amplifier
720 nm, probe = 810 nm. Scale bar = 100 μm. Adapted with permission from reference 6.
for frequency-selective amplification
of the modulated signal. By choosing
shown in Figure 2. An optical paramet- intensity is detected by a photodiode. a pump beam of 830 nm and a probe
ric oscillator pumped by a high-intensity A phase-sensitive lock-in amplifier beam of 1050 nm, the tuned amplifier
mode-locked laser generates synchro- then demodulates the detected signal. can be used for pump–probe imaging of
nous pump and probe pulse trains. The Therefore, pump-induced transmission red blood cells. This lock-in free method
Ti:sapphire oscillator is split to separate changes of the sample versus time delay improved the single-to-noise ratio by
pump and probe pulse trains. Tempo- can be measured from the focus plane. one order of magnitude compared to
ral delay between the pump and probe This change over time delay shows dif- conventional detection based on a lock-
pulses is achieved by guiding the pump ferent decay signatures from different in amplifier (45).
beam through a computer-controlled chemicals, thus offering the origin of Spatial resolution is designated as
delay line. Pump beam intensity is mod- the chemical contrast. the distance between two points of the
ulated with an acousto-optic modulator Generally speaking, lasers applied sample that can be resolved individually
(AOM), and the intensity of both beams in pump–probe microscopy can be di- according to the Rayleigh criteria. The
is adjusted through the combination of vided into two types: systems working lateral (r0) and axial (z0) resolutions are
a half-wave plate and polarizer. Sub- with relatively high pulse energy (5–100 defined (46) as
sequently, pump and probe beams are nJ) and repetition rate of 1–5 kHz, and
collinearly guided into the microscope. systems using a low pulse energy (0.5– r0 = 0.61 λ and z0 = 2 n 2 λ [8]
• • •
NA (NA)
After the interaction between the pump 10 nJ) and >1 MHz repetition rate (27).
beam and the sample, the modula- With appropriate detection schemes where λ is the wavelength, n is the re-
tion is transferred to the unmodulated that involve multichannel detection on fractive index of the medium and NA is
probe beam. Computer-controlled scan- a shot-to-shot basis, the first type can the numerical aperture. By using spa-
ning galvo mirrors are used to scan the achieve the signal detection sensitiv- tially controlled saturation of electronic
combined lasers in a raster scanning ity of ~10 -5 units of absorbance over a absorption, diffraction limit in far-field
manner to create microscopic images. broad wavelength range (27). Neverthe- imaging of nonfluorescent species could
The transmitted light is collected by less, the presence of multiple excited be broken as shown in Figure 3a. Wang
the oil condenser. Subsequently, the states under high excitation density and colleagues designed a doughnut-
pump beam is spectrally filtered by an conditions leads to singlet-singlet an- shaped laser beam to saturate the elec-
optical filter, and the transmitted probe nihilation (43). [AUTHORS: Sense OK tronic transition in the periphery of the
w w w. s p e c t r o s c o p y o n l i n e . c o m April 2017 Spectroscopy 32(4) 7

focal volume, thus introducing modu-


lation only at the focal center. By ras-
(a) 0
(b)
ter scanning three collinearly aligned 0.1
-0.05
Synthetic
0 0.5
1

Probe transient absorption (arb. units)


0.05

beams, high-speed subdiffraction-lim-


−0.04
0
-0.1 ultramarine blue 0.2 0
in acrylic −0.08
ited imaging of graphite nano-platelets
-0.05 -0.15
−1
-0.1 -0.2 0 0.2
0 5 10 15 20 −0.12
was achieved (47). 0.4
0
−0.16 −0.2

Alternatively, Miyazak and colleagues 0.2 -0.5


Lapis lazuli 0 10 20 30
0
0 10 20 30
(48) demonstrated the use of annular in casein
0
-1
0.2 0.4
-0.2

beams in pump–probe microscopy to 0.2


-1.5
0 5 10 15 20 0.5
-0.4 Pump-probe delay (ps)

improve spatial resolution in the focal


0
0.1 0 0.2
−0.5
plane, since the point spread function −0.2

(PSF) in pump–probe microscopy is 0 0


0 10 20 30 0 10 20 30
23% (43%) smaller than the diffraction-
0.8 0.10
limited spot size of the pump (probe)
0.4
1
0.6
beam. The authors also used intensity 0.4
0 0.06
0

modulated continuous wave laser di- 0.2


−1
0.02
−0.4

odes in a balanced detection scheme to 0 0

achieve subdiffraction resolution with 0 10 20 30 0 10 20 30

shot-noise limited sensitivity (49,50).


Figure 7: Imaging artistic pigments by pump probe microscopy: (a) Transient absorption images of
Regarding the sensitivity, single-mol-
synthetic ultramarine in acrylic (golden artist colors GMSA 400, top) and lapis lazuli in casein (Kremer
ecule detection can be achieved through
pigments 10530, bottom) and the corresponding pump-probe delay traces in the indicated region of
pump–probe microscopy. Chong and
interest (white rectangle) where the line indicates double-exponential fits. Scalar bar = 100 µm. S/N
coworkers conducted ground-state
= 100. Adapted with permission from reference 19 (copyright 2012 Optical Society of America). (b)
depletion microscopy and achieved a
Graphs showing pump probe dynamics in test samples with the pigments lapis lazuli, vermilion, caput
detection limit of 15 nM with a 1-s inte-
mortuum, quinacridone, phthaloblue, and indigo. Adapted with permission from reference 21.
gration time, which corresponds to 0.3
molecules in the probe volume, indicat-
ing the detection of a single-molecule that is based on phase information and
( 2τσ − τt ( (1−erf ( σ 2−*σt **ττ (( [12]
2 2
absorption signal as shown in Figure 3b modulation frequency can be used as I(t) = exp 2

(40). In their work, the sample was il- is discussed below. [AUTHORS: Sense
luminated by two tightly focused laser OK in the preceding sentence?] where erf (x) is the error function, a
beams where the pump beam and the standard function in most mathematical
probe beam have different wavelengths Multiexponential Fitting software packages. For single exponen-
but both are within the molecular ab- Multiexponential fitting, as the name tial decay, the mathematical equation for
sorption band of the analyzed sample. implies, fits the time-resolved curves the time-resolved decay curve is
In this case, the pump beam only excites with an exponential decay model. This
a molecule so that it only stays in its method is easy to conduct and under- ( ((
I(t) = I0+A * exp σ −2* t * τ * 1− erf σ − t * τ
( (( [13]
2 2

2 * τ2 2*σ* τ
ground state, and, hence, photons from stand. The time-resolved intensity is
the probe beam can’t be absorbed. Fast regarded as the conjugation between where τ is the decay constant and I0 is
on-off modulation of a strong, saturat- the instrumental response R(t) and the the signal from background. A similar
ing pump beam leads to the modulation response from sample S(t): equation can be used for double expo-
of transmitted probe beam at the same nential decay. After fitting with this
modulation frequency. ∫
I(t) = R(t – t′)S(t′)dt′ [9] model, we obtain the real decay constant
τ along with the laser pulse width σ.
Data Analysis Methods Suppose the time resolution of the de- Through the deconvolution approach,
Generally, two methods can be used tector is modeled by a Gaussian function we could resolve the time constant
to analyze a decay curve. The easier with a full width half maximum as σ: purely from decay of chemicals without
method is multiexponential fitting to the effect of laser response function.
get the decay constants. However, a R(t) = A1 exp − ( t2
2 * σ2 ( [10] However, the drawback of this method
drawback is that its accuracy is relatively is that it is sensitive to the initial input
low. The other method is called phasor In this case, pump–probe decay is mod- parameters, and therefore its accuracy
analysis, a method that needs neither eled by an exponential decay with decay is relatively low.
any assumptions regarding the physical constant τ:
model nor integration fitting to deter- Phasor Analysis
mine the lifetimes of multiexponential
signals (51–53). When dealing with a
( (
S(t) = A2 exp − t
τ
[11] Phasor analysis is a method that trans-
lates the time-resolved decay curve into
long lifetime (~1 ns), another method Then the convolution integral is a single point at a given frequency in the
8 Spectroscopy 32(4) April 2017 w w w. s p e c t r o s c o p y o n l i n e . c o m

Table I: Applications of pump–probe microscopy parts of the signal after Fourier trans-
form of the time-resolved curve:
Authors Topic Application References
Single metal g(ω) =
∫I(t) cos (ωt) dt [14]
Muskens et al. Nanomaterial 65
nanoparticle ∫∣I(t)∣dt
Davydova et al. Nanomaterial PtOEP crystal 66
Hot carrier dynamics s(ω) =
∫I(t) sin (ωt) dt [15]
Xia et al. Nanomaterial 67
in HfN and ZrN ∫∣I(t)∣dt
Cui et al. Nanomaterial WSe2 68
Any multicomponent signal can be
Graphene of different described as
Li et al. Nanomaterial 70
layers and defects
Hot photon dynamics
Gao et al. Nanomaterial
in graphene
61 Itot(t) = ∑f I (t)
i
i i
[16]
Lauret et al.
Gao et al. where fi is the fraction of each indepen-
73, 74, 60, dent species contributing to the total
Koyama et al. Nanomaterial SWNTs 62, 63
Ellingson et al. signal:
Kang et al.
Phase of semiconductor-
gtot=
∫∣I (t)∣dt
i
gi [17]
Jung et al.
Nanomaterial SWNTs and metallic- 25, 26 ∑f ∫∣I i

(t)∣dt

Tong et al. i
tot
SWNTs
Chirality grown
Gao et al. Nanomaterial 74 By plotting g(ω) against s(ω) at a given
of SWNTs
Singlet fission frequency, we can map the distribution
Wan et al. Nanomaterial 75
of tetracene of different chromophores with distinct
Carrier motion in lifetimes in the semicircle coordinate.
Gabriel et al. Nanomaterial 76
silicon nanowires Here ω is a free parameter depending
Chen et al. Nanomaterial
Nonfluorescent
77 on the separation efficiency. Robles and
nanodiamond colleagues demonstrated its capability to
Hartland et al. Nanomaterial Silver nanocube 78 discriminate eumelanin, pheomelanin,
Lo et al. Nanomaterial Single CdTe nanowire 79 and ink by phasor analysis as shown in
Mehl et al. Nanomaterial Single ZnO rods 80 Figure 4 (54).
Optoelectronic The phasor representation of lifetime
Cabanillas et al. Nanomaterial 33
semiconductor images has become popular because it
Wong et al. provides an intuitive graphical view of
Polli et al. 83, 81, 84, the fluorescence lifetime content with-
Polymer Polymer blends
Guo et al. 85
Yan et al.
out any prior knowledge. Meanwhile, it
Guo et al.
significantly improves the overall sig-
Semiconducting
Simpson et al. materials Perovskite film 82, 69 nal-to-noise ratio when used for global
Fu et al. Deep-tissue imaging analysis. Besides that, the region of in-
Hemoglobin 36, 37
Min et al. of blood vessels terest selected in the phasor plot can be
Differentiation mapped back to its corresponding image
Fu et al.
Piletic et al.
Melanin between eumelanin 5, 15, 87 to realize segmentation (56).
and pheomelanin
Samineni et al. Historical pigments Lapis lazuli 19 Frequency Domain Approach
Villafana et al. Historical pigments
Quinacridone red and
20 The frequency domain approach is more
ultramarine blue suitable for long-lived excited state. In
this method, the lifetime information
phasor space. One of the most advanta- the fluorescence spectrum at each pixel is extracted through a phase-sensitive
geous features of phasor analysis when (55,56). Fu and colleagues further ap- detection. A simple model tan ϕ = ω *
applied to fluorescent-lifetime imaging plied this analysis method to hyperspec- τ is applied to calculate the lifetime on
microscopy (FLIM) (52,53) is that it has tral stimulated Raman scattering data. the basis of phase change correspond-
the capability to quantitatively resolve It allows the fast and reliable cellular ing to different modulation frequency.
a mixture of fluorophores with differ- organelle segmentation of mammalian When a modulated pump beam I1(t) =
ent lifetimes. Phasors from those mix- cells, without any a priori knowledge of I1(1+cos ωt) is incident on the sample,
tures display linearly across the phasor their composition or basis spectra (57). the excited state population is given by
plot (54). For the first time, Fereidouni The basic mechanism for this method Miyazaki and colleagues (58) in the fol-
and colleagues proved spectral phasor is described through mapping the real lowing equation:
analysis was powerful for the analysis of parts of the signal against the imaginary
w w w. s p e c t r o s c o p y o n l i n e . c o m April 2017 Spectroscopy 32(4) 9

other techniques such as high-resolution exhibit transient absorption signals


σ1 I1
P(t) =
hν1s
{A(ω) cos [ωt+ϕ(ω)]+1} [18] transmission electron microscopy, scan- with opposite phases. Figure 5b shows
ning electron microscopy, and scanning the transient absorption image of DNA-
where tunneling microscopy proved to be cum- SWNTs internalized by CHO cells,
bersome in sample preparation (70). In where gray represents the transmission
1
A(ω) =
1+(ω * τ)2
[19] their work, they achieved high speed (2 image. The different colors in the image
μs/pixel) imaging of graphene on vari- result from different phases represent-
ous substrates under ambient condition ing two different kinds of SWNTs: green
ϕ(ω) = tan–1(ω * τ) [20] and even in living cells and animals. In- represents semiconducting SWNTs and
terestingly, the intensity of the transient red represents metallic SWNTs. Gao and
Here, σ1 is the absorption cross sec- absorption images is found to linearly colleagues reported transient absorption
tion, ν1is the frequency of the pump, h is increase with the number of layers of microscopy experiments on individual
the Planck constant, s is the beam waist graphene. In Figure 5a, in the TA image semiconducting SWNTs with known
area at the focal point, ϕ is the phase of graphene, we can clearly observe the chirality grown by chemical vapor de-
calculated from the x and y channel sig- location of graphene defects and that of position (CVD) with diffraction-limited
nals, and τ is the excited-state lifetime. different layers. It only takes a few sec- spatial resolution and subpicosecond
In the case of a long excited-state life- onds to acquire a TA image of graphene. temporal resolution (74)].
time, equation 20 suggests an efficient In addition, with polyethylene glycol Pump–probe microscopy has also been
method: tan ϕ = ω * τ. This equation used to functionalize graphene oxide, extensively applied to study nanopar-
demonstrates the linear relationship be- these well-dispersed particles have ticles and nanowires. Figure 5c presents
tween tan ϕ and modulation frequency shown the capability for in vitro and ex visualization of singlet fission by observ-
ω and the corresponding phase images. vivo imaging in Chinese hamster ovary ing the decay-associated spectra of the
The slope of this equation yields the (CHO) cells (70). triplet (red) and singlet (green) excitons
lifetime of the excited state. It is worth Pump–probe microscopy is also ex- of tetracene. The curves in Figure 5c
noting that because of the relatively ploited to study SWNTs. Carbon nano- were obtained by global analysis of the
larger shot noise at lower modulation tubes, especially single-wall carbon ensemble transient absorption spectra
frequency, the standard deviation is very nanotubes, have attracted much at- (75). As shown in Figure 5d, pump–
high (59). tention in the last two decades (71,72). probe microscopy has been used to
The excellent properties of SWNTs demonstrate the spatial kinetics of sili-
Applications of in thermal conductivity, electronics, con nanowires (76). In addition, nano-
Pump–Probe Microscopy optics, and mechanics make them ap- diamonds and nanocubes are of great
With its superior detection sensitivity, pealing. Pump–probe microscopy has interest to researchers. Figure 5e dem-
chemical specificity and spatial-tempo- proved to be a powerful tool to explore onstrates 3D transient absorption mi-
ral resolution, pump–probe microscopy the intrinsic photochemical properties croscopic images of gold nanodimonds
has been used to study pigmentation of single-wall carbon nanotubes. Ac- in living cells (77). Figure 5f shows a fast
(14), microvasculature (14), ultrafast curate detection of carrier dynamics decay of silver nanocubes resulting from
relaxation in SWNTs (23–26,60–63), in these nanostructures is essential for electron-phonon coupling and subse-
single semiconductor and metal nano- understanding and developing their op- quent modulations from the coherently
structures (64,65), and other nanomate- toelectronic properties. Lauret and col- excited breathing mode (78).
rials (66–69). Table I summarizes repre- leagues reported for the first time the Pump–probe microscopy examines
sentative applications in various areas. time-resolved study of carrier dynam- intrinsic excited state dynamics of
These applications are reviewed in more ics in single-wall carbon nanotubes by semiconductors. Lo and colleagues
detail in the following sections. means of two-color pump–probe ex- demonstrated transient absorption
periments under resonant excitation measurements on single CdTe nanow-
Semiconducting with a selective injection of energy in ires, and they showed for the first time
Nanomaterials and Graphene the semiconducting nanotubes (73). that acoustic phonon modes were fast
Pump–probe microscopy provides a Jung and colleagues for the first time because of the efficient charge carrier
vivid image of graphene with high sen- exploited the phase of the pump–probe trapping at a lower excitation intensity
sitivity. Muskens and colleagues have signal as a contrast to study SWNTs (25). (79). Mehl and coworkers (80) reported
demonstrated the study of a single Later Tong and coworkers showed that pump–probe microscopy of the indi-
metal nanoparticle by combining a transient absorption microscopy offers a vidual behaviors of single ZnO rods at
high-sensitivity femtosecond pump– label-free approach to image both semi- different spatial locations. Dramatically
probe setup with a spatial modulation conducting and metallic SWNTs in vitro different recombination dynamics were
microscope (65). Besides metal nanopar- and in vivo in real time with submi- observed in the narrow tips compared
ticles, Zhang and colleagues also imaged crometer resolution, by choosing appro- with dynamics in the interior. Cabanil-
graphene with single-layer sensitivity priate near-infrared wavelengths (26). las-Gonzalez and colleagues (33) high-
through transient absorption, whereas Semiconducting and metallic SWNTs lighted the contribution of pump–probe
10 Spectroscopy 32(4) April 2017 w w w. s p e c t r o s c o p y o n l i n e . c o m

spectroscopy to the understanding of the 650 nm, and successfully harvested two- microscopy, since the variety of pigments
elementary processes taking place in or- color TPA images of microvasculature in the artist’s palate is enormous com-
ganic based optoelectronic devices. They at different depths with a penetration pared with the biological pigments pres-
further illustrated three fundamental depth of ~70 µm. In their following-up ent in skin (20). This is a great approach
processes (optical gain, charge photo- study, they chose a longer probe beam of to extract microscopic information for a
generation and charge transport). This 810 nm to differentiate oxyhemoglobin broad range of cultural heritage applica-
work opens new perspectives for assess- and deoxyhemoglobin as shown in Fig- tions. Samineni and colleagues (19) were
ing the role of short-lived excited states ure 6a. Beyond two-photon absorption, the first to conduct a pump–probe study
on organic device operation. Polli and other procedures can also be applied to of lapis lazuli, a semi-precious rock, by
coworkers developed a new instrument observe microvessels. Min and colleagues analyzing the multiexponential decay
approach by combining broadband fem- conducted stimulated emission imaging behavior as shown in Figures 7a and 7b.
tosecond pump–probe spectroscopy and of microvasculature network in a mouse The ratio of amplitude for short decay
confocal microscopy, enabling simulta- ear based on the endogenous hemoglobin constant to that of long decay constant for
neously high temporal and spatial reso- contrast by choosing the pump beam as the synthetic ultramarine pigment is 6.6
lution (81). Guo and colleagues (82) re- 830 nm (two-photon excitation of Soret ± 0.35, while that for natural lapis lazuli
ported spatially and temporally resolved band) and probe beam as 600 nm (one- is 2.5 ± 0.05. Thus, they readily could be
measurements of perovskite by ultrafast photon stimulated emission of Q-band) distinguished.
microscopy. This work underscores the (see Figure 6b) (37).
importance of the local morphology Pump–probe microscopy could also Outlook
and establishes an important first step be used to differentiate different mela- Looking into the future, we predict the
toward discerning the underlying trans- nins. Melanins generally come in two following advancements of this emerging
port properties of perovskite materials. polymeric forms: eumelanin (black) technology. First, compact and low-cost
Pump–probe microscopy also provides and pheomelanin (red/brown). Their pump–probe microscopy will be devel-
new insight into the properties of poly- biosynthetic pathways involve the oxi- oped and made commercially available
mer blends by directly accessing the dation of tyrosine leading to the forma- for broad use of this technique by non-
dynamics at the interfacing between dif- tion of indoles and benzothiazines (87). experts. Second, handheld pump–probe
ferent materials (83).Guo and colleagues Pheomelanin is reddish yellow, and it imaging system will be developed to as-
(84) elucidated the exciton structure, exhibits phototoxic and pro-oxidant be- sist precision surgery in the clinic. Third,
the dynamics, and the charge genera- havior (88). Eumelanin is a brown–black important applications of pump–probe
tion in the solution phase aggregate of pigment that is substantially increased in microscopy will be identified, in which
a low-bandgap donor-acceptor polymer melanoma. Therefore imaging the mi- the decay kinetics are used to study cel-
by transient absorption. The technique croscopic distribution of eumelanin and lular development and disease stage.
enables important applications in con- pheomelanin could be used to separate These advances will make pump–probe
trolling morphology. Using ultrafast mi- melanomas from benign nevi in a highly microscopy an important member of the
croscopy, Yan and colleagues proved that sensitive manner (16). The differences of nonlinear optical microscopy family with
adding an amorphous content to highly the signals of these two different mela- broad use in biology, medicine, and ma-
crystalline polymer nanowire solar cells nins are shown in Figure 6c. Eumelanin terials science.
could increase the performance (85). has an abrupt positive absorption cor-
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