Toward photoswitchable electronic pre-resonance stimulated Raman probes

J. Chem. Phys. 

154

, 135102 (2021); 

https://doi.org/10.1063/5.0043791

154

, 135102

© 2021 Author(s).

Toward photoswitchable electronic pre-

resonance stimulated Raman probes

Cite as: J. Chem. Phys. 

154

, 135102 (2021); 

https://doi.org/10.1063/5.0043791

Submitted: 11 January 2021 . Accepted: 15 March 2021 . Published Online: 02 April 2021

 Dongkwan Lee

, Chenxi Qian

,

 Haomin Wang

, Lei Li

, Kun Miao

, Jiajun Du

, Daria M. Shcherbakova

,

Vladislav V. Verkhusha

,

 Lihong V. Wang

, and 

 Lu Wei

COLLECTIONS

Paper published as part of the special topic on 

2021 JCP Emerging Investigators Special Collection

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Toward photoswitchable electronic

pre-resonance stimulated Raman probes

Cite as: J. Chem. Phys.

154

, 135102 (2021); doi: 10.1063/5.0043791

Submitted: 11 January 2021

•

Accepted: 15 March 2021

•

Published Online: 2 April 2021

Dongkwan Lee,

Chenxi Qian,

Haomin Wang,

Lei Li,

Kun Miao,

Jiajun Du,

Daria M. Shcherbakova,

Vladislav V. Verkhusha,

3,4

Lihong V. Wang,

and Lu Wei

1,a)

AFFILIATIONS

Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA

Division of Engineering and Applied Science, California Institute of Technology, Pasadena, California 91125, USA

Department of Anatomy and Structural Biology, and Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine,

Bronx, New York 10461, USA

Medicum, Faculty of Medicine, University of Helsinki, Helsinki, Finland

Note:

This paper is part of the 2021 JCP Emerging Investigators Special Collection.

Author to whom correspondence should be addressed:

lwei@caltech.edu

ABSTRACT

Reversibly photoswitchable probes allow for a wide variety of optical imaging applications. In particular, photoswitchable fluorescent probes

have significantly facilitated the development of super-resolution microscopy. Recently, stimulated Raman scattering (SRS) imaging, a sen-

sitive and chemical-specific optical microscopy, has proven to be a powerful live-cell imaging strategy. Driven by the advances of newly

developed Raman probes, in particular the pre-resonance enhanced narrow-band vibrational probes, electronic pre-resonance SRS (epr-SRS)

has achieved super-multiplex imaging with sensitivity down to 250 nM and multiplexity up to 24 colors. However, despite the high demand,

photoswitchable Raman probes have yet to be developed. Here, we propose a general strategy for devising photoswitchable epr-SRS probes.

Toward this goal, we exploit the molecular electronic and vibrational coupling, in which we switch the electronic states of the molecules to

four different states to turn their ground-state epr-SRS signals on and off. First, we showed that inducing transitions to both the electronic

excited state and triplet state can effectively diminish the SRS peaks. Second, we revealed that the epr-SRS signals can be effectively switched

off in red-absorbing organic molecules through light-facilitated transitions to a reduced state. Third, we identified that photoswitchable pro-

teins with near-infrared photoswitchable absorbance, whose states are modulable with their electronic resonances detunable toward and away

from the pump photon energy, can function as the photoswitchable epr-SRS probes with desirable sensitivity (

M) and low photofa-

tigue (

40 cycles). These photophysical characterizations and proof-of-concept demonstrations should advance the development of novel

photoswitchable Raman probes and open up the unexplored Raman imaging capabilities.

Published under license by AIP Publishing.

https://doi.org/10.1063/5.0043791

I. INTRODUCTION

Photoswitchable probes are molecules whose signals can be

turned on and off reversibly upon irradiation of light. The devel-

opment of such optical-highlighter probes could greatly expand the

range of questions that can be investigated, particularly in biology.

For example, the emergence of photoswitchable fluorophores has

allowed unique imaging of protein dynamics in cells, sensing of

subcellular environment, and optical data writing and storage.

1–5

By precisely activating and deactivating fluorescence in space and

time, these probes have also largely facilitated the development of

the ground-breaking super-resolution microscopy, pushing the spa-

tial resolution of optical imaging to tens of nanometers.

6–8

Promi-

nently, utilizing the non-fluorescent states (i.e., the OFF state) of

photoswitchable fluorescent proteins, which have a much longer

lifetime (

ms) compared to the fluorescence lifetime (

∼

ns) of

excited states, RESOLFT (reversible saturable optical fluorescence

transitions) has addressed the high-power photo-damage issues in

J. Chem. Phys.

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, 135102 (2021); doi: 10.1063/5.0043791

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STED (stimulated emission depletion) microscopy and allows an

eight-order-of-magnitude smaller illumination intensity than STED

for super-resolution live-cell imaging.

9–13

In addition to fluorescence microscopy, Raman imaging, which

targets the vibrational transitions of chemical bonds, has shown its

promises to be a powerful biomedical imaging modality that offers

complementary information when interrogating biological systems.

In particular, stimulated Raman scattering (SRS) microscopy, which

harnesses the stimulated emission amplification principle, could

accelerate the vibrational transitions by 10

times compared to spon-

taneous Raman [Fig. 1(a)]. Overcoming the low sensitivity issue in

conventional spontaneous Raman imaging, SRS has achieved sub-

cellular imaging with speed up to the video rate.

14,15

It allows detec-

tion of endogenous biomolecules in a label-free fashion and also

offers bioorthogonal chemical imaging of small metabolites and

drugs in live cells, tissues, and animals with tiny Raman tags.

16–18

However, no photoswitchable Raman probes have been reported

so far.

Recently, by bringing the pump photon energy close to, but still

slightly detuned away from, the electronic absorption maximum of

the red-absorbing dyes, electronic pre-resonance SRS (epr-SRS) has

been invented, enhancing the sensitivity of Raman imaging down to

250 nM. Such a sensitivity level is close to that offered by typical

confocal fluorescence microscopy.

19,20

Compared to conventional

non-resonance SRS [Fig. 1(a)], epr-SRS obtains an up to 10

-fold

signal boost while keeping the electronic-resonance-related back-

ground to a minimum level [Fig. 1(b)]. With a newly developed

and synthesized probe palette, epr-SRS has enabled optical super-

multiplex imaging for up to simultaneous 24-color visualization of

biological targets.

These probes, which incorporate narrow-band

and isotope edited nitrile (

∼

11 cm

−

) or alkyne (

∼

14 cm

−

) moi-

eties to their conjugation systems, share similar electronic absorp-

tion peaks, but show distinctly separated Raman bands in the desired

cell-silent Raman spectral region (1700–2700 cm

−

19–21

Herein, we explore and develop the photoswitchable epr-SRS

probes. Since epr-SRS probes manifest strong coupling between elec-

tronic and vibrational transitions, we use light-induced transitions

from one electronic state to another as a general strategy to switch

epr-SRS signals on and off. Specifically, we adopt an additional exci-

tation beam to induce electronic state transitions of the molecules

[Fig. 1(c), green arrow]. As the excitation beam depletes the ON

state population to the OFF state, the SRS laser pair [Fig. 1(d),

the Stokes beam fixed at 1031.2 nm and the pump beam tunable

around 830–880 nm for epr-SRS imaging] is utilized to probe the

depleted ON state epr-SRS vibrational modes. In principle, the epr-

SRS signals could possibly be switched off and on with and with-

out the excitation from the additional laser, respectively [Figs. 1(c)

and 1(d)]. One envisioned application with such photoswitchable

molecules is super-multiplex (i.e.,

10 plex) super-resolution imag-

ing, which has remained as a highly challenging but long sought-

after goal.

Since epr-SRS probes all share similar absorption peaks,

only a single doughnut depletion laser would be required to switch

off the periphery signals and leave the spectrally-separated epr-

SRS signals in the center (Fig. S1). As a comparison, if STED

or RESOLFT were to achieve this goal of super-multiplex super-

resolution, an additional pair of excitation and depletion beams is

required for each extra color.

9–13

This is highly challenging due to

two main reasons. First, the added laser lines and optics would

largely increase the complexity for precise instrumentation align-

ment. Second, the existing color-barrier in fluorescence (i.e., the

spectral overlap) would typically limit the number of possible colors

to 3–5.

23–25

Guided by the rationales above, we study the photophysics

of different molecular electronic states to evaluate whether they

can serve as an OFF state for the ground-state epr-SRS excita-

tions. We first investigated the excited state and the triplet state

to implement the ground-state depletion photoswitching strategy.

We found that transitions to the first excited state and triplet state

result in vanishing epr-SRS peaks but also induce large electronic

background. Guided by the Albrecht A-term pre-resonance approx-

imation equation (see the supplementary material, Scheme 1), we

then revealed that a thiol-promoted long-lived dark state of organic

dyes and an absorption-detuned transition state of near-infrared

(NIR) proteins could effectively eliminate the epr-SRS signals and

serve as the OFF states for cyclic photoswitching. We envision this

work to motivate further research in developing and optimizing the

FIG. 1

. Principle and design of photoswitchable electronic pre-resonance SRS (epr-SRS) via electronic state transition. [(a)–(c)] Energy diagrams of SRS (a), epr-SRS (b),

and the proposed electronic-state modulated epr-SRS with an additional electronic excitation laser (green) (c). (d) Experimental scheme of the electronic-state modulated

epr-SRS by introducing a third excitation beam (green) to a conventional SRS microscope. EOM, electro-optic modulator; REF, reference; and X, in-phase X-output of the

lock-in amplifier.

J. Chem. Phys.

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photoswitchable epr-SRS probes and to expand the capabilities of

optical imaging.

II. RESULTS

A. Photoswitching by ground-state-depletion

epr-SRS

First, we explored the possibility of harnessing electronic

excited states as the OFF state for epr-SRS signals [Fig. 2(a)]. It

is known that bond properties (e.g., force constants, bond orders)

change between the ground and excited states.

Recent studies

have also shown that Raman spectra of molecules in the excited

state exhibit shifted peaks compared to those of the ground state

for certain vibrational modes in the molecules.

27,28

We hence rea-

soned that shifting the population to the excited state could likely

deplete the ground-state Raman signals. To test this for epr-SRS,

we adopted Rhodamine 800 (Rh800), a near-infrared-absorbing dye

peaked around 680 nm with high and well-characterized epr-SRS

signals [the structure shown in Figs. 2(b) and 2(c)].

19–21

For elec-

tronic excitation, we integrated and aligned a 660 nm continuous

wave (CW) laser into the SRS system for steady state excitation

[Fig. 1(d)]. We planned to excite Rh800 by the 660 nm excitation

beam and simultaneously probe the ground-state epr-SRS signals by

the SRS beams [Fig. 2(a), OFF state, dashed laser lines for pump and

Stokes]. We would then compare the resulting epr-SRS spectra with

and without 660 nm excitation for signal suppression analysis. To

ensure over 80% excitation of the population from the ground state

to the excited state, we applied up to 34 mW of the 660 nm excitation

beam (Table S1).

Since excited-state Raman peaks are possibly shifted from those

of the ground state, we expected to observe a decrease in epr-SRS

signals for electronic pre-resonance enhanced peaks from Rh800

upon 660 nm excitation. We, indeed, observed a gradual decrease

in the epr-SRS peaks for both the double bond [Fig. 2(b) and Fig.

S2(a)] and triple bond [Fig. 2(c) and Fig. S2(b)] of Rh800 with

increasing excitation beam powers. However, we simultaneously

detected a large increase in the broad background [Figs. 2(b) and

2(c)]. The increase in background shows strong resemblance to the

SRS spectra in the rigorous resonance regime.

19,20,29

We, hence, rea-

soned that the observed background increase should originate from

the reduced energy gap between the first electronic excited state

) and the second electronic excited state (S

) compared to that

between the ground state (S

) and the first electronic excited state

) [Fig. 2(a), OFF state]. epr-SRS excitations for the excited-state

Rh800 would, therefore, invoke high rigorous-electronic-resonance-

involved background [Fig. 2(a), OFF state, solid laser lines for

pump and Stokes]. In addition to the peak shift that can induce

peak decrease at the original epr-SRS frequency channel as we

initially hypothesized, there are a few additional possible factors

that may likely underlie the decrease in the epr-SRS signals upon

660 nm excitation even without a peak shift. One possibility is

the population competition between the epr-SRS excitation and

the invoked rigorous-electronic-resonance-involved four-wave mix-

ing processes.

Second, since the frequency-independent K term

in the Albrecht A equation includes a quadratic dependence on

the oscillation strength of the molecular absorption (i.e.,

abs

), a

smaller

abs

for the S

–S

transition compared to that of the S

–S

transition may also lead to a much-lowered excited-state epr-SRS

peaks.

The invoked high electronic background from the excited state

would complicate the analysis for imaging applications. In princi-

ple, utilizing a frequency-modulation SRS scheme, which subtracts

SRS signals between on-resonance and off-resonance frequencies in

real-time

30,31

instead of the intensity-modulation SRS scheme in our

setup, should resolve this issue. Nonetheless, it is still desirable to

have a high signal-to-background ratio for straightforward imag-

ing interpretations. Since the background is potentially induced by

the S

–S

transition, we then aimed at investigating whether the

triplet state (T

) could serve as an OFF state for epr-SRS signals

with a decreased background [Fig. 3(a)]. To increase the T

popu-

lation, we added potassium iodide (KI) to the dye solution, which is

known to accelerate intersystem crossing by the heavy atom induced

FIG. 2

. Photoswitching of epr-SRS signals via transition to the electronic excited state. (a) Energy diagrams of the proposed ON (left, the green shade indicates the ground

state as the ON state) and OFF (right, the gray shade indicates the excited state as the OFF state) states for epr-SRS signals. (b) epr-SRS spectra of the conjugated double

bond mode (i.e., highlighted red in the molecular structure) of Rh800 at 0, 2, 8, 20, and 34 mW of 660 nm excitation beam irradiation. (c) epr-SRS spectra of the triple bond

mode (red colored in the molecular structure) of Rh800 at 0, 2, 8, 20, and 34 mW of 660 nm excitation beam irradiation.

J. Chem. Phys.

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FIG. 3

. Photoswitching of epr-SRS signals via transition to the triplet state. (a) Energy diagrams of the proposed ON (left, the green shade indicates the ground state as the

ON state) and OFF (right, the gray shade indicates the triplet state as the OFF state) states for epr-SRS signals. (b) epr-SRS spectra of the double-bond mode (red-colored in

the molecular structure) of Rh800 at 0, 0.4, 2, 8, and 20 mW of 660 nm excitation beam power in the presence of potassium iodide (KI). (c) epr-SRS spectra of the triple-bond

mode (red colored in the molecular structure) of Rh800 at 0, 0.4, 4, 12, and 34 mW of 660 nm excitation beam power in the presence of (KI).

spin–orbit coupling.

32–34

The increase in triplet state population was

confirmed by fluorescence intensity measurements (Fig. S3). Simi-

lar to the excited state SRS spectra, the double bond [Fig. 3(b) and

Fig. S4(a)] and triple bond [Fig. 3(c) and Fig. S4(b)] peaks disap-

peared when the Rh800 molecules were further shifted to the triplet

state in the presence of KI. Surprisingly, while we still detected a

large background increase for the triple-bond peaks, we observed a

large negative background signal across the double-bond frequency

range.

These negative signals indicate an increase in pump photons,

since we detected the stimulated Raman loss (i.e., the pump pho-

ton loss) as SRS signals (see the Methods section). Although a

complete understanding of the molecular pathways would require

further studies, a plausible reason for such an increase in pump

photons is the population depletion in the presence of Stokes pho-

tons due to the excitation competition for the T

–T

transitions.

Since the pump beam wavelength for the triple-bond excitation (i.e.,

838 nm) is further blue shifted from that for the double bond (i.e.,

880 nm) and from the Stokes beam (i.e., 1031.2 nm), it is possible

that 838 nm light falls out of the T

–T

transition range, and hence

do not induce a significant negative background. Here, we success-

fully demonstrated that both the excited state and the triplet state

could effectively deprive the epr-SRS signals for both the double

and the triple bond of the Rh800 molecules. However, both induced

negative and positive electronic backgrounds, which could intro-

duce artifacts in analyzing the epr-SRS images. In addition, inducing

transitions to the excited and triplet state would lead to increased

photobleaching. We hence continued to explore two other molecular

states as the effective OFF state.

B. Photoswitching by modulating the epr-SRS

enhancement: Organic dyes

The third electronic state we aimed at exploiting was the long-

lived reversible dark state (

∼

100 ms to s in an oxygenated environ-

ment). This photo-reduced state in the presence of electron donors

for organic fluorophores has been heavily explored in STORM and

d-STORM super-resolution fluorescence microscopy. For example,

oxazine and rhodamine dyes are known to form semi-reduced rad-

icals (F

⋅

) or leuco (FH) structures in buffers containing primary

thiols (RSH) upon irradiations to the triplet state (

F) [Fig. 4(a),

gray box, RS-indicates the thiolate anions].

Similarly, cyanine dyes

have also been shown to form a cyanine-thiol adduct under similar

excitation and buffer conditions.

A photophysical change associ-

ated with this photochemical reduction is the diminishment of the

electronic absorption peaks. Since epr-SRS signals strongly depend

on the oscillation strength of the molecular absorption (see the

supplementary material, Scheme 1, parameter K),

20,37

the disappear-

ance of absorption peaks in these photo-reduced states indicates that

they would be ideal candidates to serve as the OFF state for epr-SRS

excitations [Fig. 4(a) OFF state, gray box].

We tested this hypothesis with ATTO680, a red-absorbing

oxazine dye that falls into the desired electronic pre-resonance exci-

tation region under our SRS laser excitation and was reported to

undergo light-induced transitions to the above-mentioned long-

lived dark state [Figs. 4(a) and 4(b)].

Exciting ATTO680 solu-

tions containing primary thiol

-mercaptoethylamine (MEA) with a

660 nm excitation beam could indeed transform the color of the

solutions into transparent [Fig. 4(c) vs Fig. 4(d), cuvette in the

inset, before and after 660 nm illumination]. Subsequent absorption

measurement confirmed the disappearance of the corresponding

absorption peak for ATTO680 [Fig. 4(c) vs Fig. 4(d)]. The rem-

nant absorption after irradiation [Fig. 4(d)] was due to a layer of

unconverted molecules at the interface between the solution and

the airspace of the cuvette. After gentle shaking of the cuvette to

facilitate dissolution of oxygen in the headspace, both the color

and the absorbance peaks of the same solution were fully recovered

[Fig. 4(e)], which indicates that the molecules are relaxed back to the

ground state (

). These results confirm that the absorption peaks

of ATTO680 can be switched on and off, as reported.

We next examined whether the same excitation and oxida-

tion steps can switch the epr-SRS signals on and off. We followed

the same excitation procedure but contained the sample solution

in a glass chamber typically used for SRS measurement [Fig. 5(a)].

Indeed, we successfully observed reversibly switchable epr-SRS sig-

nals targeting the double bond peak of ATTO680 at 1661 cm

−

[Fig. 5(b), solid line, red]. The concurrent switching of the fluores-

cence over multiple cycles was also observed [Fig. 5(b), solid line,

blue]. The reduced epr-SRS signals could reach as low as to 5% of

the original epr-SRS intensity. Control experiments in the absence

J. Chem. Phys.

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FIG. 4

. Proposed strategy to photo-

switch epr-SRS signals via transition to

the absorption diminished long-lived dark

state. (a) Energy diagrams of the pro-

posed ON (left, the green shade indi-

cates the ground state as the ON state)

and OFF (right, the gray shade indi-

cates the long-lived dark state as the

OFF state) states.

, singlet ground

state;

, singlet excited state;

F, triplet

state; F

⋅

, semireduced radical state; FH,

fully reduced leuco state; ISC, intersys-

tem crossing; red, reduction; and RS

−

thiolate anions. (b) Molecular structure

of ATTO680. [(c)–(e)] Absorption spectra

of ATTO680 before (c) and after (d) irra-

diating with a 660 nm excitation beam,

and after agitating the cuvette to facilitate

oxidation (e). Insets show the images

of solution color change in the same

cuvette.

of MEA showed no effect of such photoswitching [Fig. 5(b), dotted

line, red for epr-SRS and blue for fluorescence signals]. Together,

these data confirm that shifting the molecules between the ground

state and the photo-reduced dark state could serve as an effective

strategy to photoswitch epr-SRS signals.

Although we have demonstrated the recovery of epr-SRS sig-

nals by mechanically accelerating the oxidation through shaking or

pipetting the solutions, it is more appealing to utilize light to recover

SRS signals for the precise control of the activation kinetics. As the

oxidation of semi-reduced radicals is known to be accelerated by

irradiation of UV light,

we asked whether we could turn the epr-

SRS signals on from the dark state by illumination with a 405 nm

laser instead. We observed that the 405 nm laser irradiation could

increase the epr-SRS signals by 1.7 times compared to that from the

OFF state [Fig. 5(c), green vs pink]. Fluorescence signals also showed

a similar level of recovery (Fig. S5). We note that such a recovery

was not observed in the absence of the 405 nm activation [Fig. 5(d)],

implying that the recovery was not caused by other processes such

as diffusion.

Going beyond the solution characterization, we further con-

firmed this photoswitching effect in epr-SRS imaging. 5-ethynyl-2

′

deoxyuridine (EdU) was incorporated into newly synthesized DNA

of dividing HeLa cells and was then click-labeled by ATTO680

azide. The labeled cells immersed in a MEA-containing buffer were

subsequently imaged by epr-SRS, targeting the 1661 cm

−

peak of

ATTO680 [Fig. 5(e)]. After sequential irradiation of the excitation

beam and the 405 nm beam, the epr-SRS signals from ATTO680

(1661 cm

−

) were first decreased to 50% and then recovered back

to 70% of the original signals [Figs. 5(e)–5(h)]. The limited deple-

tion of the SRS signals in cell samples compared to solutions is likely

due to the restricted transport of the thiolate anions in cells. We note

that the limited recovery of the SRS signals after 405 nm irradiation

[Figs. 5(c) and 5(h), green] should be due to the fact that ATTO680

has a high electron affinity and accepts another electron to form a

leuco dye (FH) [Fig. 4(a)].

As this leuco dye is more stable than

the semi-reduced radical (F

⋅

), the oxidation is not easily facilitated

by the 405 nm laser. Screening other rhodamine and oxazine dyes

with a stable dark-state in the semi-reduced radical form should

further increase the activation efficiency. However, such currently

known structures (e.g., ATTO532) mostly fall outside the desired

epr-SRS excitation regime (640–790 nm). Shifting the wavelength

of SRS lasers to the bluer region, as recently reported, should

facilitate screening of better performing photoswitchable epr-SRS

probes.

39,40

C. Photoswitching by modulating the epr-SRS

enhancement: Photoswitchable proteins

Based on the pre-resonance Raman approximation,

19,20,37

epr-SRS signals are nonlinearly dependent on another photo-

physical parameter, the detuning between the pump photon

energy and the molecular electronic resonance [see supplementary

material, Scheme 1, parameter

(

−

pump

)

]. The epr-SRS signals

would decrease over 10

-fold when the pump laser energy (

pump

)

is detuned away from the molecular electronic transition energy

(

Therefore, molecules with switchable electronic resonances

closer to and further away from the pump laser energy could also

serve as ON and OFF epr-SRS states, respectively, with a decent

ON-to-OFF ratio [Fig. 6(a)].

We tested a recently engineered

truncated version of a reversibly switchable far-red absorb-

ing soluble bacterial phytochrome photoreceptor (BphP) from

Deinococcus radiodurans

, DrBphP-PCM [Fig. 6(b)].

The absorb-

ing core of DrBphP-PCM is composed of a photosensory core mod-

ule (PCM), which is shared by all BphPs, and a covalently attached

biliverdin IXa chromophore, which is the enzymatic product of

heme catabolism and present in all mammalian cells. Biliverdin

undergoes reversible

cis

–

trans

isomerization when irradiated with

different wavelengths of light, causing BphP transitions between two

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FIG. 5

. Photoswitching of epr-SRS signals via transition to the long-lived dark state. (a) A photo of ATTO680 solution containing 0.5M MEA at pH 9.5 in an SRS imaging

chamber before (left) and after (middle) excitation beam irradiation, and after oxidation (right). (b) Reversible switching of epr-SRS (red) and fluorescence (blue) signals

for multiple cycles of irradiation and oxidation (solid line). No switching was observed in the absence of MEA for both epr-SRS (red) and fluorescence (blue) (dashed line).

(c) epr-SRS signals before (black) and after (magenta) excitation beam, and after 405 nm activation (green) irradiation. (d) epr-SRS signals of ATTO680 solutions without

405 nm activation. [(e)–(g)] epr-SRS images of ATTO680-click-labeled DNA in HeLa cells before irradiation (e), after excitation beam irradiation (f), and after 405 nm laser

irradiation (g). (h) Quantification of epr-SRS signals from the arrowed cell in (e)–(g). Scale bars, 10

M. In (c) and (d), statistical significance was determined by the unpaired

two tailed

test. ns, not significant (p

>

0.05),

∗

<

0.05. Data are shown as mean

±

standard deviation (n = 3 replicates for each group).

absorbing states, Pr and Pfr [Fig. 6(b),

cis

–

trans

isomerization high-

lighted in the blue circle].

With purified DrBphP-PCM solutions,

we first confirmed their reversibly switchable absorptions, peaked at

750 nm [Fig. 6(c), magenta, the Pfr state in Fig. 6(b)] and 700 nm

[Fig. 6(c), black, the Pr state in Fig. 6(b)], upon illumination with

690 and 780 nm, respectively.

We next quantified its epr-SRS signal magnitude and reversibil-

ity. As the absorption peaks of DrBphP-PCM fall within the desired

epr-SRS excitation regime,

19,20

DrBphP-PCM in its epr-SRS ON

state (i.e., the Pfr state) shows an around 300 times signal magni-

tude to that of EdU, adopting the recent RIE (the relative intensity

to EdU) quantification metrics [Fig. 6(d)].

Such a signal size is

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FIG. 6

. Photoswitching of the purified near-infrared absorbing DrBphP-PCM protein. (a) Energy diagrams of the proposed ON (left, green shaded) and OFF (right, gray

shaded) states. (b)

Cis

–

trans

configuration change of the biliverdin chromophore in DrBphP-PCM upon irradiation. (c) Absorption spectra of DrBphP-PCM in the Pr (black)

and Pfr (magenta) conformation states. (d) Relative epr-SRS signals of DrBphP-PCM in the Pfr (1615 cm

−

, magenta) and Pr (both 1628 cm

−

, black; and 1615 cm

−

brown) states compared to the standard SRS signal of EdU (2124 cm

−

, green). (e) epr-SRS spectra of DrBphP-PCM in the Pr (black) and Pfr (magenta) states. (f) Cycles

of reversible photoswitching of DrBphP-PCM fluorescence, observed in the Pr state. (g) Cycles of reversible photoswitching of DrBphP-PCM epr-SRS signal at 1615 cm

−

the ON state (Pfr state) on-resonance channel. (h) Photoswitching of DrBphP-PCM at 1615 cm

−

for over 40 cycles demonstrates no detectable photofatigue.

equivalent to a detection sensitivity below 1

M for this probe.

When switched to the epr-SRS OFF state (i.e., the Pr state) by 780 nm

laser, DrBphP-PCM indeed presents a lowered epr-SRS signal

for the double bond mode with a Raman peak shifted from

1615 to 1628 cm

−

[Fig. 6(e), magenta and black, respectively]. This

peak shift is resulted from the change in

cis

–

trans

conformation of

the double bond between ring C and ring D of the biliverdin chro-

mophore [Fig. 6(b)].

Detecting the absolute intensity changes at

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the 1615 cm

−

channel yielded an about three-fold signal decrease

between the epr-SRS ON and OFF states [Figs. 6(d) and 6(e)]. The

residual 33% of epr-SRS signals in the OFF state [Figs. 6(d) and 6(e)]

originates from a combination of the remaining epr-SRS enhance-

ment from the OFF-state Raman peak and a slight electronic back-

ground by the pump laser from the relatively broad absorption

bands of DrBphP-PCM [Fig. 6(c)].

After the initial characterization of DrBphP-PCM, we tested

the robustness of the photo-switching for epr-SRS signals as the

resistance to switching fatigue is an important photophysical param-

eter in reversibly switchable probes. We first monitored the cycles

of reversibility for the DrBphP-PCM fluorescence signals using

the sequence of alternating 60 and 30 s illuminations by the

780 nm [Fig. 6(f), yellow] and 640 nm [Fig. 6(f), green] lasers.

The 780 nm laser switches the protein to the Pr state exhibiting

a weak fluorescence peak at 720 nm,

whereas the 640 nm laser

serves as both the readout laser and the deactivation laser that

shifts the protein back to the Pfr non-fluorescent state. We note

that the ON and OFF states for fluorescence signals are reversed

from those of epr-SRS signals as quantum yields of the Pr and Pfr

states are 2.9% and 0%, respectively.

Our observed fluorescence

depletion and recovery are similar to what were reported previously

[Fig. 6(f)].

42,46

We then probed the reversibility of the epr-SRS signals. Here,

a 780 nm laser was adopted as the deactivation laser, a 690 nm

laser was used as the activation laser, and the SRS beams were used

as the signal readout laser. Figure 6(g) shows the laser sequence

and the corresponding epr-SRS ON and OFF intensity. Similar to

that for fluorescence, clear photo-switching of epr-SRS signals was

observed over multiple cycles [Fig. 6(g)], whereas the epr-SRS sig-

nal levels remained unchanged in the absence of the 690 nm and

780 nm lasers [Fig. S6(a)]. In a separate control experiment, we

irradiated the protein solution with Stokes and pump beams for

30 s and allowed the molecules to diffuse and replenish for 60 s

[Fig S6(b)]. However, no SRS recovery was observed [Fig. S6(b),

dotted line]. In contrast, when the protein sample was irradiated

by a 690 nm activation laser, the SRS signal immediately increased

back to the original intensity [Fig. S6(b), green arrow]. This result

indicates that the role of diffusion is minimal and shows that

the recovery of SRS signals is not caused by diffusion of the ON

molecules into the focal volume, but from activation via the 690 nm

light.

Interestingly, we observed a decrease in the epr-SRS signals

when the DrBphP-PCM solution was irradiated with SRS beams

[Fig. 6(g), black arrow]. We attribute this switching-off effect to

pump beam excitation (around 884 nm), which could excite at the

very tail of the absorption peak of the Pfr state [Fig. 6(c), magenta].

We note that this decrease is not due to photobleaching, as epr-SRS

signals always recovered back to 100% with 690 nm beam activa-

tion [Fig. 6(g)]. We further extended the epr-SRS switchable cycles

to more than 40 cycles with minimum photobleaching, demonstrat-

ing the robustness of the DrBphP-PCM protein as a photoswitchable

epr-SRS probe [Fig. 6(h)]. We reasoned that the minimal photo-

bleaching observed here should likely be due to two reasons. First,

the competing switching-off pathway from the SRS beams may have

likely helped in reducing the potential photobleaching kinetics from

either the one-photon or the two-photon excitation by the SRS

lasers. Second, the adopted picosecond SRS beams should contribute

much less to the higher-order multi-photon excitation induced

photobleaching, since their peak power is much smaller compared

to that of the femtosecond lasers.

III. DISCUSSION

This work presents a series of photophysical characterizations

and proof-of-principle observations toward a new type of opti-

cal imaging probes, i.e., the photoswitchable epr-Raman probes.

We explored the possibilities of four electronic states to serve

as the effective OFF state for the epr-SRS signals. In our first

two strategies, we induced transitions to the excited state and the

triplet states, which led to successful reduction of the epr-SRS

peaks. However, robust frequency-modulation SRS techniques are

required to remove the large electronic background (both the posi-

tive and negative ones) for further applications.

30,31

Guided by the

Albrecht A-term pre-resonance approximation equation (see the

supplementary material, Scheme 1), we subsequently explored

another two states, the long-lived dark state with a diminished

absorption peak from organic dyes and a tunable absorption tran-

sition state with modulable detuning to the pump photon energy

from photoswitchable proteins. We proved that all four states

together with the ground state could serve as the ideal candidates

for reversibly photo-switching the epr-SRS signals upon further

optimizations through additional engineering and designing of the

photoswitchable Raman probes.

As we indicated above, for red-absorbing organic molecules,

extensive screening of rhodamine and oxazine dyes with a stable

dark-state in the semi-reduced radical form should help improve the

activation efficiency with 405 nm laser. In addition, cyanine dyes

with a similar cyanine-thiol adduct may also offer new opportu-

nities. In parallel with the dye screening, doubling the frequencies

of the SRS lasers as recently demonstrated would offer the match-

ing excitation region for molecules across the entire visible absorp-

tion range, vastly expanding the pools for photo-switchable epr-SRS

probes.

39,40

We have also demonstrated that the DrBphP-PCM protein

shows high promise toward generating a new category of photo-

switchable Raman proteins. Further protein engineering efforts for

obtaining larger dynamic ranges of the ON-to-OFF signal ratios

are required. This would offer multiplexable epr-SRS peaks and

allow genetical encodability for future cell imaging applications.

First, slightly blue shifting the absorption peak (e.g., for 30–50 nm)

would minimize the electronic background. This would lead to a

clearer separation of Raman peaks between the epr-SRS ON and

OFF states for easier multiplexing. Second, larger shift in absorption

peaks between the epr-SRS ON and OFF states should also con-

tribute to enhancing the dynamic ranges of the ON-to-OFF ratios.

Third, mutagenesis of the amino acid residues around the biliverdin

binding pocket may shift its double bond vibrational frequency by

changing the interacting environment and, hence, creating more

colors.

Fourth, incorporation of a nitrile bond to the conjuga-

tion system of the biliverdin would significantly help expand the

epr-SRS color palette owing to the features of the narrow-band

and editable nitrile bonds that are ideal for multiplexing. Fifth, the

superior property of photoswitchable Raman protein-based probes

is their genetic encodability, which is critical for live cell imaging

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and is not offered by organic dyes. Moreover, it is worth noting

that whereas photoswitching of organic dyes frequently requires

UV light, which is phototoxic for cells, the Raman protein probes

derived from BphPs use non-cytotoxic photoswitching far-red and

near-infrared light, which penetrates much deeper in biological

tissues, thus enabling intravital imaging.

Further engineering

of distinct BphP-based Raman probes, along with their different

intracellular targeting, will allow super-multiplex epr-SRS imaging

in live cells.

Ultimately, with successful invention of a new category of

photoswitchable epr-SRS probes, super-multiplex super-resolution

optical imaging may be implemented, as we rationalized above.

Adopting a doughnut setup similar to RESOLFT but with only

one additional switching laser, super-multiplex imaging could be

brought into the super-resolution regime and offer a valuable new

addition to the toolbox of optical imaging in investigating biological

activities and functions.

SUPPLEMENTARY MATERIAL

See the supplementary material for experimental methods,

scheme, supporting figures, and tables.

ACKNOWLEDGMENTS

This work was supported by the grants from the National

Institutes of Health, Grant No. DP2 GM140919 (to L.W.) and Grant

No. R35 GM122567 (to V.V.), and by the start-up fund from the

California Institute of Technology (to L.W.).

DATA AVAILABILITY

The data that support the findings of this study are available

from the corresponding author upon reasonable request.

REFERENCES

R. Ando, H. Mizuno, and A. Miyawaki, “Regulated fast nucleocytoplasmic shut-

tling observed by reversible protein highlighting,” Science

306

(5700), 1370–1373

(2004).

G. U. Nienhaus, K. Nienhaus, A. Hölzle, S. Ivanchenko, F. Renzi, F. Oswald,

M. Wolff, F. Schmitt, C. Röcker, B. Vallone, W. Weidemann, R. Heilker, H. Nar,

and J. Wiedenmann, “Photoconvertible fluorescent protein EosFP: Biophysical

properties and cell biology applications,” Photochem. Photobiol.

82

(2), 351–358

(2006).

J. Lippincott-Schwartz and G. H. Patterson, “Photoactivatable fluorescent pro-

teins for diffraction-limited and super-resolution imaging,” Trends Cell Biol.

19

(11), 555–565 (2009).

Y.-T. Kao, X. Zhu, and W. Min, “Protein-flexibility mediated coupling between

photoswitching kinetics and surrounding viscosity of a photochromic fluorescent

protein,” Proc. Natl. Acad. Sci. U. S. A.

109

(9), 3220–3225 (2012).

X. X. Zhou and M. Z. Lin, “Photoswitchable fluorescent proteins: Ten years

of colorful chemistry and exciting applications,” Curr. Opin. Chem. Biol.

17

(4),

682–690 (2013).

T. A. Klar and S. W. Hell, “Subdiffraction resolution in far-field fluorescence

microscopy,” Opt. Lett.

24

(14), 954–956 (1999).

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S.

Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging

intracellular fluorescent proteins at nanometer resolution,” Science

313

(5793),

1642–1645 (2006).

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochas-

tic optical reconstruction microscopy (STORM),” Nat. Methods

3

(10), 793–796

(2006).

M. Hofmann, C. Eggeling, S. Jakobs, and S. W. Hell, “Breaking the diffraction

barrier in fluorescence microscopy at low light intensities by using reversibly

photoswitchable proteins,” Proc. Natl. Acad. Sci. U. S. A.

102

(49), 17565–17569

(2005).

M. Andresen, A. C. Stiel, J. Fölling, D. Wenzel, A. Schönle, A. Egner,

C. Eggeling, S. W. Hell, and S. Jakobs, “Photoswitchable fluorescent pro-

teins enable monochromatic multilabel imaging and dual color fluorescence

nanoscopy,” Nat. Biotechnol.

26

(9), 1035–1040 (2008).

S. J. Sahl, S. W. Hell, and S. Jakobs, “Fluorescence nanoscopy in cell biology,”

Nat. Rev. Mol. Cell Biol.

18

(11), 685–701 (2017).

F. Lavoie-Cardinal, N. A. Jensen, V. Westphal, A. C. Stiel, A. Chmyrov, J. Bier-

wagen, I. Testa, S. Jakobs, and S. W. Hell, “Two-color RESOLFT nanoscopy

with green and red fluorescent photochromic proteins,” ChemPhysChem

15

(4),

655–663 (2014).

I. Testa, E. D’Este, N. T. Urban, F. Balzarotti, and S. W. Hell, “Dual chan-

nel RESOLFT nanoscopy by using fluorescent state kinetics,” Nano Lett.

15

(1),

103–106 (2015).

C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. He, J. C. Tsai,

J. X. Kang, and X. S. Xie, “Label-free biomedical imaging with high sensitiv-

ity by stimulated Raman scattering microscopy,” Science

322

(5909), 1857–1861

(2008).

B. G. Saar, C. W. Freudiger, J. Reichman, C. M. Stanley, G. R. Holtom, and X. S.

Xie, “Video-rate molecular imaging in vivo with stimulated Raman scattering,”

Science

330

(6009), 1368–1370 (2010).

J.-X. Cheng and X. S. Xie, “Vibrational spectroscopic imaging of living systems:

An emerging platform for biology and medicine,” Science

350

(6264), aaa8870

(2015).

L. Wei, F. Hu, Z. Chen, Y. Shen, L. Zhang, and W. Min, “Live-cell bioorthog-

onal chemical imaging: Stimulated Raman scattering microscopy of vibrational

probes,” Acc. Chem. Res.

49

(8), 1494–1502 (2016).

F. Hu, L. Shi, and W. Min, “Biological imaging of chemical bonds by stimulated

Raman scattering microscopy,” Nat. Methods

16

(9), 830–842 (2019).

L. Wei, Z. Chen, L. Shi, R. Long, A. V. Anzalone, L. Zhang, F. Hu, R. Yuste, V. W.

Cornish, and W. Min, “Super-multiplex vibrational imaging,” Nature

544

(7651),

465–470 (2017).

L. Wei and W. Min, “Electronic preresonance stimulated Raman scattering

microscopy,” J. Phys. Chem. Lett.

9

(15), 4294–4301 (2018).

H. Xiong, L. Shi, L. Wei, Y. Shen, R. Long, Z. Zhao, and W. Min, “Stimulated

Raman excited fluorescence spectroscopy and imaging,” Nat. Photonics

13

(6),

412–417 (2019).

L. Möckl and W. E. Moerner, “Super-resolution microscopy with single

molecules in biology and beyond–essentials, current trends, and future chal-

lenges,” J. Am. Chem. Soc.

142

(42), 17828–17844 (2020).

L. Schermelleh, A. Ferrand, T. Huser, C. Eggeling, M. Sauer, O. Biehlmaier, and

G. P. C. Drummen, “Super-resolution microscopy demystified,” Nat. Cell Biol.

21

(1), 72–84 (2019).

M. Bates, G. T. Dempsey, K. H. Chen, and X. Zhuang, “Multicolor super-

resolution fluorescence imaging via multi-parameter fluorophore detection,”

ChemPhysChem

13

(1), 99–107 (2012).

J. Bückers, D. Wildanger, G. Vicidomini, L. Kastrup, and S. W. Hell, “Simulta-

neous multi-lifetime multi-color STED imaging for colocalization analyses,” Opt.

Express

19

(4), 3130–3143 (2011).

R. Wilbrandt, N. H. Jensen, P. Pagsberg, A. H. Sillesen, and K. B. Hansen,

“Triplet state resonance Raman spectroscopy,” Nature

276

(5684), 167–168

(1978).

S. Rieger, M. Fischedick, K.-J. Boller, and C. Fallnich, “Suppression of resonance

Raman scattering via ground state depletion towards sub-diffraction-limited label-

free microscopy,” Opt. Express

24

(18), 20745 (2016).

P. Kukura, D. W. McCamant, and R. A. Mathies, “Femtosecond stimulated

Raman spectroscopy,” Annu. Rev. Phys. Chem.

58

(1), 461–488 (2007).

J. Chem. Phys.

154

, 135102 (2021); doi: 10.1063/5.0043791

154

, 135102-9

Published under license by AIP Publishing

The Journal

of Chemical Physics

ARTICLE

scitation.org/journal/jcp

L. Shi, H. Xiong, Y. Shen, R. Long, L. Wei, and W. Min, “Electronic resonant

stimulated Raman scattering micro-spectroscopy,” J. Phys. Chem. B

122

(39),

9218–9224 (2018).

D. Zhang, M. N. Slipchenko, D. E. Leaird, A. M. Weiner, and J.-X. Cheng,

“Spectrally modulated stimulated Raman scattering imaging with an angle-to-

wavelength pulse shaper,” Opt. Express

21

(11), 13864–13874 (2013).

D. Fu, W. Yang, and X. S. Xie, “Label-free imaging of neurotransmitter acetyl-

choline at neuromuscular junctions with stimulated Raman scattering,” J. Am.

Chem. Soc.

139

(2), 583–586 (2017).

A. Chmyrov, T. Sandén, and J. Widengren, “Iodide as a fluorescence quencher

and promoter—Mechanisms and possible implications,” J. Phys. Chem. B

114

(34), 11282–11291 (2010).

E. Gatzogiannis, X. Zhu, Y.-T. Kao, and W. Min, “Observation of frequency-

domain fluorescence anomalous phase advance due to dark-state hysteresis,”

J. Phys. Chem. Lett.

2

(5), 461–466 (2011).

J. Widengren, U. Mets, and R. Rigler, “Fluorescence correlation spectroscopy

of triplet states in solution: A theoretical and experimental study,” J. Phys. Chem.

99

(36), 13368–13379 (1995).

S. van de Linde, A. Löschberger, T. Klein, M. Heidbreder, S. Wolter, M.

Heilemann, and M. Sauer, “Direct stochastic optical reconstruction microscopy

with standard fluorescent probes,” Nat. Protoc.

6

(7), 991–1009 (2011).

G. T. Dempsey, M. Bates, W. E. Kowtoniuk, D. R. Liu, R. Y. Tsien, and

X. Zhuang, “Photoswitching mechanism of cyanine dyes,” J. Am. Chem. Soc.

131

(51), 18192–18193 (2009).

A. C. Albrecht and M. C. Hutley, “On the dependence of vibrational Raman

intensity on the wavelength of incident light,” J. Chem. Phys.

55

(9), 4438–4443

(1971).

S. van de Linde, I. Krsti

c, T. Prisner, S. Doose, M. Heilemann, and M. Sauer,

“Photoinduced formation of reversible dye radicals and their impact on super-

resolution imaging,” Photochem. Photobiol. Sci.

10

(4), 499–506 (2011).

H. Xiong, N. Qian, Y. Miao, Z. Zhao, and W. Min, “Stimulated Raman excited

fluorescence spectroscopy of visible dyes,” J. Phys. Chem. Lett.

10

(13), 3563–3570

(2019).

Y. Bi, C. Yang, Y. Chen, S. Yan, G. Yang, Y. Wu, G. Zhang, and P. Wang,

“Near-resonance enhanced label-free stimulated Raman scattering microscopy

with spatial resolution near 130 nm,” Light: Sci. Appl.

7

, 81 (2018).

H. Fujioka, J. Shou, R. Kojima, Y. Urano, Y. Ozeki, and M. Kamiya, “Multicolor

activatable Raman probes for simultaneous detection of plural enzyme activities,”

J. Am. Chem. Soc.

142

, 20701 (2020).

L. Li, A. A. Shemetov, M. Baloban, P. Hu, L. Zhu, D. M. Shcherbakova, R. Zhang,

J. Shi, J. Yao, L. V. Wang, and V. V. Verkhusha, “Small near-infrared pho-

tochromic protein for photoacoustic multi-contrast imaging and detection of

protein interactions in vivo,” Nat. Commun.

9

(1), 2734 (2018).

H. Yamakoshi, K. Dodo, A. Palonpon, J. Ando, K. Fujita, S. Kawata, and

M. Sodeoka, “Alkyne-tag Raman imaging for visualization of mobile small

molecules in live cells,” J. Am. Chem. Soc.

134

(51), 20681–20689 (2012).

L. Wei, F. Hu, Y. Shen, Z. Chen, Y. Yu, C.-C. Lin, M. C. Wang, and W. Min,

“Live-cell imaging of alkyne-tagged small biomolecules by stimulated Raman

scattering,” Nat. Methods

11

(4), 410–412 (2014).

C. Kneip, P. Hildebrandt, W. Schlamann, S. E. Braslavsky, F. Mark, and

K. Schaffner, “Protonation state and structural changes of the tetrapyrrole chro-

mophore during the P

→

phototransformation of phytochrome: A resonance

Raman spectroscopic study,” Biochemistry

38

(46), 15185–15192 (1999).

V. V. Lychagov, A. A. Shemetov, R. Jimenez, and V. V. Verkhusha, “Microflu-

idic system for in-flow reversible photoswitching of near-infrared fluorescent

proteins,” Anal. Chem.

88

(23), 11821–11829 (2016).

S. D. Fried, S. Bagchi, and S. G. Boxer, “Extreme electric fields power catalysis in

the active site of ketosteroid isomerase,” Science

346

(6216), 1510–1514 (2014).

D. M. Shcherbakova, O. V. Stepanenko, K. K. Turoverov, and V. V. Verkhusha,

“Near-infrared fluorescent proteins: Multiplexing and optogenetics across scales,”

Trends Biotechnol.

36

(12), 1230–1243 (2018).

J. Chem. Phys.

154

, 135102 (2021); doi: 10.1063/5.0043791

154

, 135102-10

Published under license by AIP Publishing