Bringing vibrational imaging to chemical biology with molecular probes

Bringing vibrational imaging to chemical biology with molecular

probes

Jiajun Du

Haomin Wang

Lu Wei

1,*

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

California 91125, USA

Abstract

As an emerging optical imaging modality, stimulated Raman scattering (SRS) microscopy

provides invaluable opportunities for chemical biology studies using its rich chemical information.

Through rapid progress over the last decade, the development of Raman probes harnessing the

chemical biology toolbox has proven to play a key role in advancing SRS microscopy and

expanding biological SRS applications. In this perspective, we first discuss the development of

biorthogonal SRS imaging using small tagging of triple bonds or isotopes and highlight their

unique advantages for metabolic pathway analysis and microbiology investigations. The potential

opportunities for chemical biology studies by integrating small tagging with SRS imaging are

also proposed. We next summarize the current designs of highly sensitive and super-multiplexed

SRS probes, as well as provide future directions and considerations for next-generation functional

probe design. These rationally designed SRS probes are envisioned to bridge the gap between

SRS microscopy and chemical biology research and should benefit their mutual development and

applications.

Graphical Abstract

Corresponding : lwei@caltech.edu.

HHS Public Access

Author manuscript

ACS Chem Biol

. Author manuscript; available in PMC 2023 November 26.

Published in final edited form as:

ACS Chem Biol

. 2022 July 15; 17(7): 1621–1637. doi:10.1021/acschembio.2c00200.

Author Manuscript

Introduction

Vibrational spectroscopy, including infrared (IR) absorption and Raman scattering (Figure

1a), reveals molecular information through probing the inherent vibrations of chemical

bonds. It has been extensively used for characterizing molecules

, deciphering reaction

mechanisms

, and interpreting molecule-environment interactions

. Raman spectroscopy

was first discovered by the physicist Sir C.V. Raman in 1928, where the inelastic Raman

scattering shifts a very small portion of photons to lower frequency upon interacting with

molecules

. The changes of the photon energy reflect the vibrational energy levels of the

chemical bonds within the molecules and thus carry rich chemical information. Although

both IR and Raman spectroscopy contain vibrational information of the molecules, they have

different selection rules. For example, water has very high IR absorption but weak Raman

scattering. In addition, Raman typically utilizes UV to near-infrared light (200 – 1100

nm) while IR relies on mid-infrared light (2500 – 50000 nm), thus the spatial resolution

(scaling inversely with the light wavelength) is much higher for Raman (Figure 1a). Both the

subcellular spatial-resolution and the minimal water background make Raman scattering as a

better-suited technique for the biological Raman applications.

However, the utility of spontaneous Raman is largely limited by its feeble signals (~

smaller than fluorescence), which requires a long acquisition time and is easily

overwhelmed by the auto-fluorescence background from the biological samples. To address

both issues, the coherent anti-Stokes Raman Scattering (CARS) microscopy (Figure 1a)

Du et al.

Page 2

ACS Chem Biol

. Author manuscript; available in PMC 2023 November 26.

Author Manuscript

was invented. CARS microscopy significantly boosted the imaging sensitivity and speed

and pushed Raman spectro-microscopy to be more compatible with biological imaging

However, CARS signals suffer from severe nonresonant background and have non-linear

concentration dependence, which sacrifices imaging quality and adds complexity for

imaging annotation

. Around 2008, stimulated Raman scattering (SRS) microscopy was

introduced, providing even better sensitivity than CARS and tackling the above issues

present with CARS

–

. SRS utilizes two spatially and temporally overlapped laser pulse

trains (pump and Stokes, Figure 1b), which enhances the otherwise weak Raman transitions

by up to 10

-fold through stimulated emission quantum amplification. This two-photon

excitation feature also yields SRS intrinsic 3D optical sectioning capability for deep

tissue imaging. In addition, the technical implementation of the high-frequency modulation

transfer scheme also pushes the detection sensitivity of SRS close to the theoretical limit

and removes the potential fluorescence and other interfering background. The readers are

encouraged to check more technical details of SRS in other reviews

It is now widely recognized that SRS is the most suitable far-field Raman imaging

modality for live biological studies with provided image quality comparable to fluorescence

microscopy. Prominently, SRS also achieved a high imaging speed up to video-rate (110

frames/s)

–

and a spatial resolution within 100 nm with recent instrumentation and

sample-expansion strategies

–

. It would be informative to compare the key technical and

application features of SRS microscopy with the popular one- and two-photon fluorescence

microscopy. Below, we provide the energy diagram and a table summarizing key imaging

parameters for the three techniques (Figure 1b). Compared to fluorescence microscopy,

the absence of electronic excitation provides SRS with general imaging capability of all

types of molecules with minimum photobleaching or environmental quenching. In addition,

SRS typically uses near-infrared excitation lasers (800–1100 nm) and thus offers less

phototoxicity compared to one-photon fluorescence with visible laser excitation. This near-

infrared excitation together with the intrinsic 3D sectioning makes the penetration depth

of SRS into thick tissues similar to that obtained by two-photon fluorescence. Moreover,

the common utilization of pico-second lasers in SRS renders a much smaller laser peak

power compared to that from femto-second lasers for two-photon fluorescence, resulting in

less nonlinear photodamage. However, the μM-mM detection sensitivity is still the major

challenge in SRS that limits its application to detect molecules with low abundance in

live biological samples. Recent developments in probe engineering such as the creation of

highly sensitive MARS and polyynes dyes has largely improved the sensitivity down to nM

and broken the traditional color barrier in fluorescence microscopy with super-multiplexed

imaging.

Label-free vibrational imaging

Since Raman signals originate from chemical bonds instead of conjugated fluorophores,

SRS offers general imaging applicability for versatile molecules. Since its inception, SRS

microscopy has established itself as a powerful label-free bioimaging modality. Targeting

the fingerprint region (500 – 1700 cm

−1

) or high wavenumber carbon-hydrogen (C-H)

stretching region (2800 – 3100 cm

−1

), nucleic acids, proteins, lipids, carbohydrates, neuron-

transmitters and other endogenous biomolecules carrying O-P-O, C=O, C=C, C-H

, C-H

Du et al.

Page 3

ACS Chem Biol

. Author manuscript; available in PMC 2023 November 26.

Author Manuscript

bonds are readily imaged with subcellular resolution (Figure 2a). Additionally, hyperspectral

SRS

adds another layer of information for subcellular spectral analysis. With these

capabilities, label-free SRS paves the way for various applications, including sensing

environmental cues

, studying lipid metabolism and identifying druggable targets

–

tracking drug delivery and distribution

–

, multicolor cell sorting

, fast diagnosis

of tumors

, and investigations of amyloid plaques in neurodegenerative diseases

The current imaging speed, throughput and detection sensitivity are still being continuously

improved with rapid instrumental innovations

–

. In parallel, emerging data processing

approaches, particularly the machine learning algorithms, further upgrade image quality and

enable data mining with rich chemical information

–

Despite the success of label-free SRS imaging, there are several fundamental limitations.

First, the specificity of targets is often compromised since endogenous biomolecules tend

to share multiple chemical bonds and therefore overlapping spectra. Second, the detection

limit of SRS is still relatively low compared with fluorescence - at the scale of millimolar

for most biomolecules. Thus, label-free SRS is more suited for investigating relatively

abundant molecules including proteins, lipids, nucleic acids and carbohydrates. Third, label-

free imaging is not capable of tracking many dynamic processes such as uptake, synthesis,

catabolism, and intracellular-to-extracellular interactions. These limitations largely restrict

the applications of SRS imaging but can be greatly diminished through the implementation

of Raman probes.

Labeling with bioorthogonal probes

Driven by the need of higher specificity, sensitivity, and functionality, which are

fundamentally limited in the label-free approaches, Raman labels have been introduced

to shift SRS imaging from the label-free to the labeling paradigm

. Fortunately, the cell-

silent spectral region (1800–2800 cm

−1

), where there are no endogenous Raman signals

from cells, leaves spacious spectral room for background-free Raman labeling and imaging

(Figure 2a). Bioorthogonal chemical bonds, including alkynes (C

≡

C), nitriles (C

≡

N) and

carbon-deuterium bonds (C-D) are small, nontoxic and Raman active in this clean region,

making them well suitable for tagging with low perturbation to the biological systems

As such, biorthogonal SRS is especially beneficial for live-cell interrogations of small

molecules including metabolites and drugs whose label-free vibrational signatures are

overwhelmed by cellular background and whose physiological functions are perturbed by

conventional fluorophore labeling.

Furthermore, by harnessing the narrow linewidth of Raman peaks (50–100 times narrower

than fluorescence peaks), the development of highly sensitive Raman probe palettes

followed. The matching dye palettes enable super-multiplexed (more than 20 channels)

optical imaging for organelles or protein profiling with sensitivity down to 250 nM, bridging

the optical imaging’s subcellular spatial resolution with system biology’s high information

throughput

–

. Moreover, chemically activatable and photochromic SRS probes have also

been developed lately for intracellular sensing and multiplexed tracking, empowering the

functional SRS imaging

–

Du et al.

Page 4

ACS Chem Biol

. Author manuscript; available in PMC 2023 November 26.

Author Manuscript

All these recently established Raman probes have greatly expanded the application boundary

of vibrational imaging. Efforts in the past decade have proven that probe development plays

a central role in driving the next frontiers of SRS microscopy. The growing chemical biology

toolbox inspires the development of new SRS imaging functionalities. In turn, the SRS

platform also finds a unique niche for chemical biology studies. Exploring new opportunities

for merging SRS imaging with chemical biology is worth brainstorming. In this perspective,

we first review recent notable advances in the development of Raman probes and their

biological applications. On this basis, we further provide an outlook to further expand

multiplexing, enhance Raman signals and utilize SRS imaging to decipher new biology.

Small-molecule Raman tagging

Triple bonds (e.g. C

≡

C, C

≡

N) and stable-isotope-substituted chemical bonds (e.g. C-D,

N-D, O-D) vibrate in the cell-silent region (Figure 2a). Among these chemical bonds, C

≡

has the highest Raman signals (Figure 2b). One representative molecule tagged by C

≡

is 5-ethynyl-2

′

-deoxyuridine (EdU), the well-adopted thymidine analogue with an SRS

detection limit of 200 μM

, and is now frequently used as a benchmark for Raman intensity

quantification. Pioneered by the click chemistry field, chemists have developed a suite

of alkyne-tagged molecular handles for bio-labeling, many of which can now be directly

detected by Raman without the subsequent click reactions. With these available and newly

developed Raman-tailored alkyne probes (Figure 2c), the dynamic metabolic processes

including DNA synthesis, choline and glucose uptake can be readily visualized in live cells

and tissues (Figure 2d with the corresponding analog structures shown in Figure 2c)

–

Additionally, drugs bearing native alkynes (e.g. ponatinib) or nitriles (e.g. paxlovid) or upon

proper alkyne derivatization such as diyne-ferrostatin (Figure 2c&d) can be quantitatively

imaged for intracellular distribution with well-maintained pharmacokinetics

. Inspired

by the colorful fluorescent protein palette, alkyne “vibrational colors” are also tunable

through an isotope-editing strategy based on the dependence of Raman frequency on the

bond mass. A set of

Cedited probes were developed that enabled multicolor SRS imaging

of DNA, RNA and lipids in the same set of live cells

(Figure 2e).

Compared to C

≡

C, C

≡

N has lower (about 40%) Raman cross-sections (Figure 2b).

However, as the peak frequencies of C

≡

N are sensitive to the physical environment

(particularly the electrostatic interactions

), they provide additional functions as

vibrational sensors. Additionally, the C

≡

N vibration occupies the higher frequency region

(2200 – 2300 cm

−1

), which separates well from that of C

≡

C (2100 – 2200 cm

−1

). Therefore,

nitriles with similar isotope-editing for frequency shifting could be combined with alkynes

for expanded imaging multiplexing.

As the stable isotope of hydrogen, deuterium-labeled chemical bonds (exemplified by the

C-D) have unmatched advantages. Most importantly, since stable isotopes have almost

the same physicochemical properties as their counterparts, the labeled molecules could be

processed by cells’ natural machineries with minimal perturbation to the native biological

functions. In addition, compared to the label-free imaging of C-H, C-D yields an improved

SRS detection limit due to the absence of interfering cellular background. Although the

Raman cross section of C-D is smaller than that of the triple bonds (Figure 2b), what is

Du et al.

Page 5

ACS Chem Biol

. Author manuscript; available in PMC 2023 November 26.

Author Manuscript

lacking in cross section could be compensated by the large labeling number. For example,

palmitic acid, the most common long-chain saturated fatty acid in mammalian cells, can

have up to 31 deuteriums per molecule as d

palmitic acid. Owing to these features,

deuterium plays a significant role in Raman labeling and has been applied to interrogating

a wide range of uptake and metabolic dynamics for targets including amino acids

glucose

, fatty acids

, choline

, cholesterol

, water

, solvents

and drugs

(Figure

2f). For instance, the employment of deuterated amino acids allows imaging of complex

protein metabolism, including synthesis, degradation, and analysis of temporally defined

populations

(Figure 2g). Similarly, d

palmitic acid enabled quantitative SRS visualization

of fatty acid uptake and their metabolic incorporation

; and the deuteration of propylene

glycol (PG) allowed the capture of real-time 3D penetration for this common pharmaceutical

cosolvent/excipient across the mice stratum corneum

(Figure 2g).

Deuterium labeling can also obtain protein-specific imaging in live cells for certain

targets. For example, the aggregation-prone mutant Huntington (mHtt) protein harboring

polyglutamine (polyQ) expansions has been shown to be specifically labeled by deuterated

glutamine for their enrichment in the polyglutamine expansions

(Figure 2h). This

approach enabled the first quantitative analysis of mHtt and non-mHtt proteins inside the

same protein aggregates in live cells without the need of any fluorescent labels. To expand

the library of labeled targets apart from proteins, similar selective labeling strategies may be

developed, such as the selective labeling of glycogen by deuterated glucose in live cancer

cells

. The concept of harnessing such repeating units toward higher sensitivity is also

adopted for C-D and triple-bond containing polymers, which could amplify the Raman

signals by up to 10

fold

–

(Figure 2i). The versatile applications of Raman-tagged

cellular imaging are still expanding, and would benefit from easy synthetic accessibility to

new vibrationally-tagged molecules. Toward this front, we envision that the recent synthetic

advances in late-stage functionalization

–

could provide convenient synthetic routes to

deuterium or triple-bond tagged molecular targets beyond currently available pools.

Metabolic pathway analysis with deuterium labeling: from cell metabolism to microbiology

Various small Raman labels offer a wide range of applicability especially for investigating

different aspects of cellular metabolism. To assay the uptake of metabolites (e.g. glucose),

triple bonds are usually the top choice for their higher detectability

. However, even

triple-bond tagged metabolic analogs usually stop at the early catabolic steps in the

metabolic pathway. In this case, stable isotope tagged metabolites are superior for tracing

transformations into the downstream metabolic products from live cells to organisms with

minimum toxicity

. For example, with deuterated fatty acid labeling, lipid synthesis and

mobilization could be non-invasively probed in live

Caenorhabditis elegans

(

C. elegans

) by

SRS with high throughput

(Figure 3a).

C. elegans

with daf-11 mutants were discovered

to have no changes in the rate of lipid synthesis, but have a significant reduction in the rate

of lipid catabolism

Comprehensive cellular metabolism beyond metabolite uptake or distribution can be

probed with suitable deuterium labeled probes. Glucose is the primary energy source

for mammalian cells as well as an important precursor of downstream metabolites

Du et al.

Page 6

ACS Chem Biol

. Author manuscript; available in PMC 2023 November 26.

Author Manuscript

including amino acids, lipids, nucleic acids, glycogen and adenine dinucleotide phosphate

(NADPH)

. While 3-OPG (i.e. alkyne-tagged glucose, Fig. 2d) is able to capture the

glucose uptake in live cells and tissues, it stops at the phosphorylation step when going

into the glucose metabolism pathway

. Recently, with deuterated glucose (i.e. d

-glucose)

labeling, diverse downstream products, such as DNA/RNA, proteins, lipids and glycogen,

have been shown to be sparsely labeled with deuterium through each corresponding

metabolic pathway

(Figure 3b). these sparsely labeled downstream products are spectrally

separatable with varied features due to the different chemical environments surrounding

the deuterium (Figure 3b&c)

. A linear combination algorithm can then be utilized

to quantitatively retrieve the relative C-D enrichment maps in each identified species

(Figure 3d). Alternatively, site-specific deuterated glucose instead of d

-glucose allows for

tracing specific metabolic pathways. For example, 3-D-glucose ([3-D]Glc) was shown to

monitor NADPH-mediated lipid synthesis through oxidative pentose phosphate pathway

(oxPPP) by targeting lipid droplets

Metabolic reprogramming serves as a unique hallmark for cancer and neurodegenerative

diseases. This Raman-based imaging platform for complex glucose metabolism hence forms

a live-cell spatially resolved assay with subcellular resolution. Indeed, cancers cells have

been shown to exhibit different levels of glucose uptake rate versus metabolism rate

Subcellular glycogen accumulation through d

-glucose labeling was also discovered in

cancer cells as a potential indicator for their resistance to glucose deficiency

. A similar

spectral tracing strategy can be applied to cost-effective heavy water labeling (DO-SRS)

Since water is the most abundant molecule in biological systems, the incorporation of

deuterium from D

O to C-D in macromolecules is highly efficient even at low and

biologically-safe D

O concentrations (e.g. 20% D

O). The resulting distinct spectral

signatures of C-D enable visualizing both lipid and protein metabolism in animals with

long-term incubation (26 days)

Assaying microbial metabolism is another application field that can be empowered by

the metabolic Raman platform. The role of microbiota is increasingly recognized in

human health. One of the most important problems associated with microbes is antibiotic

resistance. Current standard antimicrobial susceptibility testing (AST) requires 16 – 24 h

for multiple cell cycle growth. By culturing the microbes in 70% D

O medium and tracing

the metabolic incorporation of deuterium into the biomass, varied metabolic responses to

antibiotics can be probed in as short as 10 min, the fastest method to date

. The C-D

signals indicated that

Pseudomonas aeruginosa

(

P. aeruginosa

), a common cause of hospital-

acquired infection, exhibits distinct metabolic rate under different common antibiotics

(gentamicin and cefotaxime) treatment (Figure 4a&b). As such, SRS imaging provides

a rapid and cost-effective AST assay. In a more clinical-relevant

P. aeruginosa

biofilm

system, 3D metabolic activity deep inside the film was visualized by SRS with 50% D

medium labeling

. SRS’s inherent optical sectioning capability provides a convenient way

to visualize 3D metabolic activity without the need of traditional paraffin embedding and

sectioning (Figure 4c). The active metabolism in the hypoxic deep region was revealed

to be supported by the small redox metabolite phenazine (phz) in the optical sections

(Figure 4d top, WT vs Δphz, peak 2). This finding was validated by paraffin sections

((Figure 4d bottom, WT vs Δphz, peak 2). In different

Staphylococcus aureus

(

S. aureus

)

Du et al.

Page 7

ACS Chem Biol

. Author manuscript; available in PMC 2023 November 26.

Author Manuscript

biofilms, tagging antibiotic vancomycin with alkynes uncovered a non-uniform and limited

penetration of antibiotic into biofilm with preferential affinity of the antibiotic to the cells

instead of the extracellular polymeric matrix (EPM)

Proposition of SRS imaging with small tagging

The coupling of SRS with small vibrational tags has established itself as a powerful live-

cell imaging platform. Going beyond what has been achieved, below we discuss some

of our thoughts for further improvements of molecular probes and potential opportunities

of the platform in chemical biology. First, the wide cell-silent region remains spacious

with room for spectral multiplexing (Figure 2a). Other triple bonds (Figure 5a), including

metal-carbonyl (M-C

≡

, isothiocyanate (-N=C=S), diazo (-N

≡

N), isonitrile (-N

≡

C) and

thiocyanate (-S-C

≡

N), with strong vibrations in this region are worth in-depth investigations

and engineering for their biological utilities. If these additional tags are available,

the multiplexing of Raman-based profiling strategies could be largely expanded. New

applications, such as imaging-based glycans profiling of cells, could then be envisioned.

Glycans are oligosaccharides attached to biomacromolecules including proteins. They are

regarded as post-translational modifications for modulating cell functions, but are still

less understood due to the lack of studying methods

. A typical way to image glycan

directly from cells is through metabolic incorporation of unnatural monosaccharides with

small chemical reporters (e.g. azides or alkynes), which undergo sequential labeling

via biorthogonal chemistry

. However, vastly different kinetics, selectivity and the

scarce availability of the biorthogonal reactions limit the applicability of multiplexed

monosaccharide labeling. With SRS imaging, we envision that spatial glycan profiling of

monosaccharides covering N-acetylglucosamine (GlcNAc), mannose (Man), galactose (Gal),

sialic acid (Neu5Ac), fucose in live systems will be possible with proper isotope-edited

triple-bond tagging (Figure 5b) together with the expandable tag repertoire (Figure 5a).

In addition to metabolic labeling, site-specific labeling through genetic encoding, such as

utilizing unnatural amino acids (UAA) with genetic code expansion (Figure 5c), is another

direction that is worth exploring for protein-selective Raman imaging. Although numerous

UAAs carrying alkynes or nitriles have been developed (Figure 5c)

, SRS signals from

these single-UAA labeled proteins are not sufficient

. While technical innovations are in

urgent needs of sensitivity improvement

, new chemical or biological labeling strategies

to incorporate an increased number (> 10) of triple bonds in one protein would also be a

breakthrough for obtaining satisfying SRS signals for general protein imaging applications.

Towards higher sensitivity, aqueous Glaser-Hay bioconjugation

, which may extend the

terminal alkynes from UAAs into polyynes

in situ

, could be an alternative option to

reduce the required labeling number of UAA by serving as a sequential signal-amplification

method.

Compared to fluorophores, the above-shown small and multiplexed triple-bond Raman

tags allow wider accessibility due to much smaller tag sizes and require no sequential

labeling (e.g. click reaction) for highly-multiplexed applications. We hence expect that SRS

imaging will contribute to solving certain important chemical biology questions, such as

spatially resolving the proteome reactivity in live cells, in which the signal level is not

Du et al.

Page 8

ACS Chem Biol

. Author manuscript; available in PMC 2023 November 26.

Author Manuscript

a problem. The side chain reactivity of canonical amino acids together with the local

protein microenvironment builds up “hotspots” in the proteome

. The electron-rich side

chains of various amino acids including cysteine, lysine, aspartate, glutamate, tyrosine, and

methionine are naturally the targets for electrophiles (Figure 5d). These functional amino

acids are always catalytic residues or sites of post-translational modifications, and thus they

are the keys in modulating cellular functions. The side-chain reactivity of the amino acids

can be quantitatively analyzed by isotopic tandem orthogonal proteolysis activity based

protein profiling (isoTOP-ABPP) platform

. This mass spectrometry method has high

throughput and unmatched protein resolvability, but it lacks spatial information and cannot

track dynamic processes with intra- and intercellular interaction in complex biological

systems.

Recently, researchers comprehensively profiled the proteome-wide reactivity for a library

of 54 alkyne-bearing electrophiles

100

. They identified highly selective probes specific to a

total of 9 amino acids plus the N-terminus with different reactivities (several representative

probes are listed in Figure 5e). These alkyne-tagged electrophiles provide an effective site-

selective labeling tool for proteins or peptides (Figure 5f). The alkyne handles across these

electrophile probes (Figure 5e) could be substituted with color-resolvable isotope-labeled

triple bonds from the Raman probe repertoire (Figure 5a&5b). With this design, it is possible

to generate maps for amino acid reactivities in a complex biological system such as cancer

immunology and microbe-host environment. By supplying the probes into the systems, the

subsequent multiplexed SRS imaging would allow single-cell profiling

en masse

(Figure

5g). Enough signals should be expected with this strategy since similar proteome-wide new

protein synthesis using alkyne-tagged methionine (HPG, Figure 2c) labeling was already

demonstrated by SRS imaging in live cells

. This application would be highly challenging

for direct fluorescent labeling as the active amino acid residues are likely less accessible

to bulkier fluorophores without the sequential labeling step. Together with isoTOP-ABPP

and protein targeting labeling, proteome-wide SRS screening may contribute to annotating

specific proteins and elucidating their spatially-resolved dynamic activity changes.

Next-generation Raman probe palettes for highly sensitive and super-multiplexed imaging

Even with the signal amplification from SRS and the introduced Raman tags, there is still a

sensitivity gap between the SRS and the fluorescence microscopy. The SRS detection limit

of EdU is 200 μM in live cells while the most sensitive fluorophores offer single-molecule

sensitivity (< 10 nM). One central drive in SRS imaging is to narrow this sensitivity gap.

A promising solution that has been proven successful is the development of highly sensitive

Raman probes. As the Raman bands are inherently narrow (peak width about 10 cm

−1

~ 50–100 times narrower than that for fluorescent peaks), these Raman probes would

enable high-throughput super-multiplexed (> 20 channels) imaging once the requirement for

sensitivity is met. Therefore, Raman imaging holds the promise of breaking the color barrier

of fluorescence imaging and should greatly benefit systematic biology investigations.

Two sets of highly sensitive and multiplexed Raman palettes have been developed for

SRS imaging over the past five years. The first is xanthene-based electronic pre-resonance

enhanced Manhattan Raman scattering (MARS) dyes

(Figure 6a). By carefully tuning

Du et al.

Page 9

ACS Chem Biol

. Author manuscript; available in PMC 2023 November 26.

Author Manuscript

the absorption of the dyes (650 – 760 nm) to moderately close to the laser wavelength (~

900 nm), SRS intensities of the nitrile vibration from these dyes could be pre-resonantly

enhanced by up to 10

(detection limit up to 250 nM) with a well-maintained high signal-

to-background ratio (Figure 6b&c). Taking advantage of the much narrower (78 times, 13

vs 1020 cm

−1

of FWHM) SRS peak compared with the fluorescence absorption peak and

the multiplexing from isotope labeling as illustrated in the example of MARS2228 series

dyes (Figure 6c), MARS dyes have the inherent capability for super-multiplexed imaging.

With central atom (position 10) replacement, ring expansion and isotope editing, an SRS

dye palette with up to 24 plex was created for super-multiplexed imaging

(Figure 6d).

Through investigations in our lab, we later found that different scaffolds could present vastly

different pre-resonance SRS signals even with the same absorption wavelength, possibly

due to the complicated electronic–vibrational coupling strength. This indicates that finding

the right chemical scaffold is as important as physically modulating the absorption of the

electronic structures. To facilitate the development of new palettes, theoretical models and

computational tools are also urgently needed

101

. While the construction of near-infrared

chromophores is more challenging, the suitable electronic pre-resonance palettes could be

largely expanded with freely tunable laser sources into the visible range

102

103

The second established set of highly-sensitive Raman probes is the Carbow series, which is

a set of linear conjugated alkynes with aromatic capping

104

(Figure 6e). Different from the

electronic enhancement mechanism, the strong SRS signals of polyynes originate from the

amplified second-order hyperpolarizability (

) from conjugated alkynes. When the number

of conjugated alkynes increases from 2 to 6, the Raman intensity grows super-linearly with

an exponent of 2.77 (i.e.

2.77

, Figure 6f), offering a desirable detectability down to

630 nM for 4-yne. The increase of conjugation length is also accompanied by a desirable

Raman peak shift, offering more spectral resolvability for multiplexed applications. Further

combined with end-capping substitution and isotope editing, another 20-color CARBOW

palette was created (Figure 6g). Without the involvement of electronic excitation, polyynes

are free of photobleaching or environmental quenching. Although slightly smaller in Raman

cross section compared to the MARS palette, the Carbow palette has neutral scaffolds

and is more suitable for live-cell targeted imaging with lower non-specific background.

Theoretically, the longest conjugation length of polyynes could be over 6 with higher

sensitivity than MARS, but at the risk of decreased stability. Going beyond the polyyne

structures, other oligomers such as polytriacetylenes, oligo(1,4-phenyleneethynylene)s

105

polydiacetylenes

have also shown exponential power-law relationship between the second-

order hyperpolarizability (

and repeating units (

) with slightly increased sizes (Figure 7a)

106

, thus are promising candidates for next-generation strong Raman probes.

The ultimate goal of improving the signals of Raman probes is to achieve single-molecule

SRS imaging, meaning that even for the most sensitive MARS probes, there still needs a

sensitivity improvement of ~ 30–50 folds. Towards this goal, a possible design direction

for Raman probes is to combine MARS and Carbow scaffolds in the same molecules for

cooperative enhancement from electronic pre-resonance and amplified

(Figure 7b). That is

to replace the single alkyne in the current alkyne-containing MARS probes to be conjugated

polyynes (Figure 7b). In the case that the pre-resonance effect remains, the Raman signal is

Du et al.

Page 10

ACS Chem Biol

. Author manuscript; available in PMC 2023 November 26.

Author Manuscript

also expected to undergo exponential increase with the number of conjugated alkynes, thus

the resulting MA(RS)CAR(B)OW palette might have another dozens of times enhancement

compared with the MARS palette to meet the desired single-molecule detectability.

Functional Raman Imaging Probes

Probing biological systems through chemical probes is a central topic for chemical biology

study. Leveraging the live-cell compatibility, strict linear-concentration dependence and

non-quenching nature of Raman signals, SRS probes are preferred for quantitative analysis.

Although small vibrational probes have been extensively utilized for spectroscopic analysis

of the targeted cellular environment, such as electrostatic interactions, electrical currents,

and temperature

, the development of highly sensitive Raman imaging probes for

environmental sensing is still in its infancy. Benefiting from the unique super-multiplexed

Raman features, functional SRS imaging probes should open the door for comprehensive

investigations to elucidate the intricate intracellular and cell-to-cell interactions. Over the

past four years, we witnessed the rapid growth of the development of such highly sensitive

SRS sensors. Based on their Raman spectroscopic features, we categorized these Raman

sensors into four classes: sensing by peak shifts, peaks enhancement, peak generation,

and peak switching. Through the following discussions, we aim to summarize systematic

guidelines for designing new probes with tailored functions.

Most current Raman sensors are designed for sensing the targeted chemical environment

through Raman peak shifts (Figure 8a). The chemical reactions triggered by environmental

stimuli are designed to change the chemical structures adjacent to the Raman reporters

(e.g. alkynes or nitriles), therefore resulting in distinct Raman peak shifts. For example,

in a 2-yne scaffold, when the electron-withdrawing azide group on the end-phenyl cap is

reduced to the electron donating amine in the presence of the reductive hydrogen sulfide

species (such as H

S and NaSH), the alkyne Raman peak shows a 9 cm

−1

red shift

107

(Figure 8b), which is significant enough to be distinguished by SRS imaging (the typical

full-width-half-maximum (FWHM) for alkyne peak is 15 cm

−1

and the typical spectral

width of SRS lasers is ~ 12–15 cm

−1

). The demonstrated SRS ratiometric imaging indeed

showed a strong response to NaSH level changes in the mitochondria of live cells (Figure

8c). With similar targeted reactions, triple-bond Raman probes with the peak-shift principles

have also been rationally designed and developed for sensing pH

108

, fluoride

109

and metal

ions

110

. Recently, the isotope exchange reactions (especially the H/D exchange) have been

harnessed on terminal alkynes for both two-color imaging and cellular environmental

sensing applications

111

112

, taking advantage of the dramatic alkyne Raman peak shift (>

130 cm

−1

) due to both the large mass difference between D and H and the quantum

coupling between the alkyne and the adjacent C-D. In addition to chemical reactions, Raman

peak frequency can also be tuned by the surrounding physical environment. A well-known

example is that the hydrogen-bounding and electrostatics can shift the peak frequencies

of triple bonds especially nitriles, an effect known as vibrational solvatochromism. By

specifically mapping the peak frequency of nitrile-bearing MARS Raman dyes in live cells,

the bound-water percentage in cytoplasm was revealed to be around 60% while that in

nucleus was about 30%

113

114

. Recently, the SRS peak of voltage-sensitive rhodopsin has

also been shown to shift upon voltage changes

115

Du et al.

Page 11

ACS Chem Biol

. Author manuscript; available in PMC 2023 November 26.

Author Manuscript

Intensity enhancement represents another less explored category of design principle for

SRS sensors (Figure 8d). Recently, modulation of electronic pre-resonance SRS effect

was implemented for intracellular enzyme activity sensing. The nitrile xanthene scaffold,

once caged by amides structures (9CN-JCP probes), will absorb in the visible region (506

nm, electronic non-resonance) with almost invisible SRS signals. However, the enzymatic

conversion of amide structures to amine structures (9CN-JCPs) will dramatically boost

SRS signals by shifting the absorption to the near-infrared region (630 nm, electronic

pre-resonance) (Figure 8e). With isotope editing on the nitrile to generate multiple colors,

this elegant intensity-modulation principle was successfully exploited to create a 4-color

enzymatic sensing probe palette

(Figure 8e). In this case, simultaneously detecting the

activities of four distinct enzymes was demonstrated for effective profiling of different

cancer cell phenotypes (Figure 8f). With the super-multiplexed MARS dye palette, this

enzyme-activatable sensor design holds high promises for profiling more than ten enzymes

simultaneously.

Peak generation allows background-free imaging and is highly beneficial for sensitive and

multiplexed detection (Figure 8g). The Raman intensity activation does not solely come

from the cellular or the chemical environment, but it can also originate from the photons.

In a recent design, the first photoactivatable SRS imaging probes were developed based

on the photocaged alkynes, the cyclopropenones (Figure 8h)

. Such rationally designed

cyclopropenone structures were optimized with live-cell compatibility and have been proven

to be well-suitable for multiplexed live-cell imaging and tracking

(Figure 8i). With the

high precision spatial-temporal control offered by photoactivation, this series of isotope

editing probes was demonstrated for multiplexed tracking from the subcellular to the

single-cell level. Upon further improvement of multiplexing, these new Raman sensors may

illuminate complex cell-to-cell interactions and facilitate massive-parallel cell profiling. For

example, combining photoactivatable SRS probes and single-cell RNA sequencing (scRNA-

seq) could likely enable spatially-resolved transcriptomics profiling

116

Raman peaks could also be reversibly switchable with light manipulations (Figure 8j).

Such features are crucial to a variety of biological investigations including tracking protein

dynamics, subcellular environment sensing and super-resolution imaging. In 2021, three

groups independently reported photophysical or photochemical approaches to achieve

photoswitchable SRS imaging

–

. To name one example, the alkyne-tagged diarylethene

showed impressive photo-switchable property in the cell-silent spectral window

(Figure

8k). The UV induced photoisomerization converts diarylethene from the open-ring state to

the closed-ring state while visible light would induce the reverse conversion, accompanied

by the switching of SRS peak intensities. These alkynes-tagged diarylethenes were

demonstrated for photo-rewritable patterning and mitochondria tracking (Figure 8l). It is

noteworthy that the SRS readout lasers would induce the off-switch pathway, competing

with the UV induced photo-cyclization, underscoring the careful choice of molecular

absorption when designing the new probes.

Du et al.

Page 12

ACS Chem Biol

. Author manuscript; available in PMC 2023 November 26.

Author Manuscript

Conclusion

Through the past decade we have seen that the SRS microscopy has transited from the

initial technical demonstration into a powerful method with the potential to answer many

biological questions in a way no other methods can (Figure 9). Such transformation

is impossible without appropriate Raman probes. With small triple-bond or isotope

tagging, SRS microscopy enables the visualization and analysis of dynamic metabolism

pathways in live cells and animals. From chemistry innovations, the construction of

highly sensitive MARS and Carbow palettes realized the full potential of Raman for super-

multiplexed imaging. The recently developed functional Raman sensors further expanded

the applications for multiplexed cellular environment sensing and high precision spatial-

temporal tracking. We also envision the design with further improved multiplexing and

sensitivity of the Raman probes as a next step towards interrogating more complex biology.

As the development of Raman probes continue to be a central topic for the SRS community,

we also hope to invoke brainstorming to borrow the wisdom from the chemical biology field

and to promote vibrational imaging to solve biological questions. In retrospect, much of the

recent progress of SRS probes was inspired by other fields: deuterium and other isotope

labeling are prevalent in mass spectrometry; alkynes are the most important biorthogonal

chemical group handles; MARS dyes originate from the design of commercially available

fluorophores; diarylethene is a class of well-established photoswitchable chromophores, etc.

The knowledge across fields should accelerate the advances of SRS probes into higher

selectivity, sensitivity, photostability, biocompatibility, and multiplexing capability. Together

with the developments of instrumentation, biorthogonal chemistry, molecular delivery and

labeling methods, data analysis and more, SRS microscopy will further develop into an

indispensable tool for chemical biology studies.

Acknowledgement:

L. Wei acknowledges the startup funds from California Institute of Technology and Grant No. DP2 GM140919

from National Institute of Health. We thank A.Colazo for helpful discussion.

Reference:

(1). Kneipp K; Kneipp H; Itzkan I; Dasari RR; Feld MS Ultrasensitive Chemical Analysis by Raman

Spectroscopy. Chem. Rev. 1999, 99 (10), 2957–2975. 10.1021/cr980133r. [PubMed: 11749507]

(2). Fang C; Frontiera RR; Tran R; Mathies RA Mapping GFP Structure Evolution during Proton

Transfer with Femtosecond Raman Spectroscopy. Nature 2009, 462 (7270), 200–204. 10.1038/

nature08527. [PubMed: 19907490]

(3). Kukura P; McCamant DW; Mathies RA Femtosecond Stimulated Raman Spectroscopy. Annu.

Rev. Phys. Chem. 2007, 58, 461–488. 10.1146/annurev.physchem.58.032806.104456. [PubMed:

17105414]

(4). Chattopadhyay A; Boxer SG Vibrational Stark Effect Spectroscopy. J. Am. Chem. Soc. 1995, 117

(4), 1449–1450. 10.1021/ja00109a038.

(5). Thomas GJ Raman Spectroscopy of Protein and Nucleic Acid Assemblies. Annu. Rev. Biophys.

Biomol. Struct. 1999, 28, 1–27. 10.1146/annurev.biophys.28.1.1. [PubMed: 10410793]

(6). Raman CV; Krishnan KS A New Type of Secondary Radiation [11]. Nature 1928, 121 (3048),

501–502. 10.1038/121501c0.

Du et al.

Page 13

ACS Chem Biol

. Author manuscript; available in PMC 2023 November 26.

Author Manuscript

(7). Zumbusch A; Holtom GR; Xie XS Three-Dimensional Vibrational Imaging by Coherent

Anti-Stokes Raman Scattering. Phys. Rev. Lett. 1999, 82 (20), 4142–4145. 10.1103/

PhysRevLett.82.4142.

(8). Min W; Freudiger CW; Lu S; Xie XS Coherent Nonlinear Optical Imaging:

Beyond Fluorescence Microscopy. Annu. Rev. Phys. Chem. 2011, 62, 507–530. 10.1146/

annurev.physchem.012809.103512. [PubMed: 21453061]

(9). Ploetz E; Laimgruber S; Berner S; Zinth W; Gilch P Femtosecond Stimulated Raman Microscopy.

Appl. Phys. B Lasers Opt. 2007, 87 (3), 389–393. 10.1007/s00340-007-2630-x.

(10). Freudiger CW; Min W; Saar BG; Lu S; Holtom GR; He C; Tsai JC; Kang JX; Xie S Label-Free

Biomedical Imaging with High Sensitivity by Stimulated Raman Scattering Microscopy. Science

2008, 322 (19), 1857–1861. 10.1126/science.1165758. [PubMed: 19095943]

(11). Ozeki Y; Dake F; Kajiyama S; Fukui K; Itoh K Analysis and Experimental Assessment of

the Sensitivity of Stimulated Raman Scattering Microscopy. Opt. Express 2009, 17 (5), 3651.

10.1364/oe.17.003651. [PubMed: 19259205]

(12). Cheng JX; Xie XS Vibrational Spectroscopic Imaging of Living Systems: An Emerging Platform

for Biology and Medicine. Science 2015, 350 (6264). 10.1126/science.aaa8870. [PubMed:

26472912]

(13). Hu F; Shi L; Min W Biological Imaging of Chemical Bonds by Stimulated Raman Scattering

Microscopy. Nat. Methods 2019, 16 (9), 830–842. 10.1038/s41592-019-0538-0. [PubMed:

31471618]

(14). Saar BG; Freudiger CW; Reichman J; Stanley CM; Holtom GR; Xie XS Video-Rate Molecular

Imaging in Vivo with Stimulated Raman Scattering. Science 2010, 330 (6009), 1368–1370.

10.1126/science.1197236. [PubMed: 21127249]

(15). Camp CH; Lee YJ; Heddleston JM; Hartshorn CM; Walker ARH; Rich JN; Lathia JD; Cicerone

MT High-Speed Coherent Raman Fingerprint Imaging of Biological Tissues. Nat. Photonics

2014, 8 (8), 627–634. 10.1038/nphoton.2014.145. [PubMed: 25621002]

(16). Wakisaka Y; Suzuki Y; Iwata O; Nakashima A; Ito T; Hirose M; Domon R; Sugawara M;

Tsumura N; Watarai H; et al. Probing the Metabolic Heterogeneity of Live Euglena Gracilis

with Stimulated Raman Scattering Microscopy. Nat. Microbiol. 2016, 1 (16124). 10.1038/

nmicrobiol.2016.124.

(17). Bi Y; Yang C; Chen Y; Yan S; Yang G; Wu Y; Zhang G; Wang P Near-Resonance Enhanced

Label-Free Stimulated Raman Scattering Microscopy with Spatial Resolution near 130 Nm.

Light Sci. Appl. 2018, 7 (1), 1–10. 10.1038/s41377-018-0082-1. [PubMed: 30839587]

(18). Qian C; Miao K; Lin LE; Chen X; Du J; Wei L Super-Resolution Label-Free Volumetric

Vibrational Imaging. Nat. Commun. 2021, 12 (1), 1–10. 10.1038/s41467-021-23951-x. [PubMed:

33397941]

(19). Shi L; Klimas A; Gallagher B; Cheng Z; Fu F; Wijesekara P; Miao Y; Ren X; Zhao Y;

Min W Super-Resolution Vibrational Imaging Using Expansion Stimulated Raman Scattering

Microscopy. bioRxiv 2021, 2021.12.22.473713. 10.1101/2021.12.22.473713.

(20). Zipfel WR; Williams RM; Webb WW Nonlinear Magic: Multiphoton Microscopy in the

Biosciences. Nat. Biotechnol. 2003, 21 (11), 1369–1377. 10.1038/nbt899. [PubMed: 14595365]

(21). Jonkman J; Brown CM; Wright GD; Anderson KI; North AJ Tutorial: Guidance for Quantitative

Confocal Microscopy. Nat. Protoc. 2020, 15 (5), 1585–1611. 10.1038/s41596-020-0313-9.

[PubMed: 32235926]

(22). Miller DR; Jarrett JW; Hassan AM; Dunn AK Deep Tissue Imaging with

Multiphoton Fluorescence Microscopy. Curr. Opin. Biomed. Eng. 2017, 4, 32–39. 10.1016/

j.cobme.2017.09.004. [PubMed: 29335679]

(23). Hill AH; Manifold B; Fu D Tissue Imaging Depth Limit of Stimulated Raman Scattering

Microscopy. Biomed. Opt. Express 2020, 11 (2), 762. 10.1364/boe.382396. [PubMed: 32133223]

(24). Fu D; Holtom G; Freudiger C; Zhang X; Xie XS Hyperspectral Imaging with Stimulated Raman

Scattering by Chirped Femtosecond Lasers. J. Phys. Chem. B 2013, 117 (16), 4634–4640.

10.1021/jp308938t. [PubMed: 23256635]

(25). Zhang D; Wang P; Slipchenko MN; Ben-Amotz D; Weiner AM; Cheng JX Quantitative

Vibrational Imaging by Hyperspectral Stimulated Raman Scattering Microscopy and Multivariate

Du et al.

Page 14

ACS Chem Biol

. Author manuscript; available in PMC 2023 November 26.

Author Manuscript

Curve Resolution Analysis. Anal. Chem. 2013, 85 (1), 98–106. 10.1021/ac3019119. [PubMed:

23198914]

(26). Figueroa B; Hu R; Rayner SG; Zheng Y; Fu D Real-Time Microscale Temperature Imaging

by Stimulated Raman Scattering. J. Phys. Chem. Lett. 2020, 11 (17), 7083–7089. 10.1021/

acs.jpclett.0c02029. [PubMed: 32786960]

(27). Yue S; Li J; Lee S-Y; Lee HJ; Shao T; Song B; Cheng L; Masterson TA; Liu X; Ratliff TL; et

al. Cholesteryl Ester Accumulation Induced by PTEN Loss and PI3KAKT Activation Underlies

Human Prostate Cancer Aggressiveness. Cell Metab. 2014, 19, 393–406. [PubMed: 24606897]

(28). Li J; Condello S; Thomes-Pepin J; Ma X; Xia Y; Hurley TD; Matei D; Cheng JX Lipid

Desaturation Is a Metabolic Marker and Therapeutic Target of Ovarian Cancer Stem Cells. Cell

Stem Cell 2017, 20 (3), 303–314.e5. 10.1016/j.stem.2016.11.004. [PubMed: 28041894]

(29). Du J; Su Y; Qian C; Yuan D; Miao K; Lee D; Ng AHC; Wijker RS; Ribas A; Levine RD;

et al. Raman-Guided Subcellular Pharmaco-Metabolomics for Metastatic Melanoma Cells. Nat.

Commun. 2020, 11 (1), 4830. 10.1038/s41467-020-18376-x. [PubMed: 32973134]

(30). Chen T; Yavuz A; Wang MC Dissecting Lipid Droplet Biology with Coherent Raman Scattering

Microscopy. J. Cell Sci. 2022, 135 (5). 10.1242/jcs.252353.

(31). Shen Y; Zhao Z; Zhang L; Shi L; Shahriar S; Chan RB; Di Paolo G; Min W Metabolic Activity

Induces Membrane Phase Separation in Endoplasmic Reticulum. Proc. Natl. Acad. Sci. 2017,

114 (51), 13394–13399. 10.1073/pnas.1712555114. [PubMed: 29196526]

(32). Wei L; Hu F; Shen Y; Chen Z; Yu Y; Lin CC; Wang MC; Min W Live-Cell Imaging of

Alkyne-Tagged Small Biomolecules by Stimulated Raman Scattering. Nat. Methods 2014, 11 (4),

410–412. 10.1038/nmeth.2878. [PubMed: 24584195]

(33). Chiu WS; Belsey NA; Garrett NL; Moger J; Delgado-Charro MB; Guy RH Molecular Diffusion

in the Human Nail Measured by Stimulated Raman Scattering Microscopy. Proc. Natl. Acad. Sci.

U. S. A. 2015, 112 (25), 7725–7730. 10.1073/pnas.1503791112. [PubMed: 26056283]

(34). Fu D; Zhou J; Zhu WS; Manley PW; Wang YK; Hood T; Wylie A; Xie XS Imaging

the Intracellular Distribution of Tyrosine Kinase Inhibitors in Living Cells with Quantitative

Hyperspectral Stimulated Raman Scattering. Nat. Chem. 2014, 6 (7), 614–622. 10.1038/

nchem.1961. [PubMed: 24950332]

(35). Huang KC; Li J; Zhang C; Tan Y; Cheng JX Multiplex Stimulated Raman Scattering Imaging

Cytometry Reveals Lipid-Rich Protrusions in Cancer Cells under Stress Condition. iScience

2020, 23 (3), 100953. 10.1016/j.isci.2020.100953. [PubMed: 32179477]

(36). Nitta N; Iino T; Isozaki A; Yamagishi M; Kitahama Y; Sakuma S; Suzuki Y; Tezuka H; Oikawa

M; Arai F; et al. Raman Image-Activated Cell Sorting. Nat. Commun. 2020, 11 (1), 1–16.

10.1038/s41467-020-17285-3. [PubMed: 31911652]

(37). Ji M; Orringer DA; Freudiger CW; Ramkissoon S; Liu X; Lau D; Golby AJ; Norton I; Hayashi

M; Agar NYR; et al. Rapid, Label-Free Detection of Brain Tumors with Stimulated Raman

Scattering Microscopy. Sci. Transl. Med. 2013, 5 (201). 10.1126/scitranslmed.3005954.

(38). Hollon TC; Pandian B; Adapa AR; Urias E; Save AV; Khalsa SSS; Eichberg DG; D’Amico

RS; Farooq ZU; Lewis S; et al. Near Real-Time Intraoperative Brain Tumor Diagnosis Using

Stimulated Raman Histology and Deep Neural Networks. Nat. Med. 2020, 26 (1), 52–58.

10.1038/s41591-019-0715-9. [PubMed: 31907460]

(39). Ji M; Arbel M; Zhang L; Freudiger CW; Hou SS; Lin D; Yang X; Bacskai BJ; Sunney Xie

X Label-Free Imaging of Amyloid Plaques in Alzheimer’s Disease with Stimulated Raman

Scattering Microscopy. Sci. Adv. 2018, 4 (11), 1–9. 10.1126/sciadv.aat7715.

(40). Lin LE; Miao K; Qian C; Wei L High Spatial-Resolution Imaging of Label-Free in Vivo Protein

Aggregates by VISTA. Analyst 2021, 146 (13), 4135–4145. 10.1039/d1an00060h. [PubMed:

33949430]

(41). Berto P; Andresen ER; Rigneault H Background-Free Stimulated Raman Spectroscopy and

Microscopy. Phys. Rev. Lett. 2014, 112 (5), 1–5. 10.1103/PhysRevLett.112.053905.

(42). Casacio CA; Madsen LS; Terrasson A; Waleed M; Barnscheidt K; Hage B; Taylor MA; Bowen

WP Quantum-Enhanced Nonlinear Microscopy. Nature 2021, 594 (7862), 201–206. 10.1038/

s41586-021-03528-w. [PubMed: 34108694]

Du et al.

Page 15

ACS Chem Biol

. Author manuscript; available in PMC 2023 November 26.

Author Manuscript

(43). Lin H; Lee HJ; Tague N; Lugagne JB; Zong C; Deng F; Shin J; Tian L; Wong W; Dunlop MJ;

et al. Microsecond Fingerprint Stimulated Raman Spectroscopic Imaging by Ultrafast Tuning

and Spatial-Spectral Learning. Nat. Commun. 2021, 12 (1), 1–12. 10.1038/s41467-021-23202-z.

[PubMed: 33397941]

(44). Kobayashi-Kirschvink KJ; Gaddam S; James-Sorenson T; Grody E; Ounadjela JR; Ge B;

Zhang K; Kang JW; Xavier R; So PTC; et al. Raman2RNA: Live-Cell Label-Free Prediction of

Single-Cell RNA Expression Profiles by Raman Microscopy. bioRxiv 2021, 2021.11.30.470655.

10.1101/2021.11.30.470655.

(45). Manifold B; Men S; Hu R; Fu D A Versatile Deep Learning Architecture for Classification

and Label-Free Prediction of Hyperspectral Images. Nat. Mach. Intell. 2021, 3 (4), 306–315.

10.1038/s42256-021-00309-y. [PubMed: 34676358]

(46). Wei L; Hu F; Chen Z; Shen Y; Zhang L; Min W Live-Cell Bioorthogonal Chemical Imaging:

Stimulated Raman Scattering Microscopy of Vibrational Probes. Acc. Chem. Res. 2016, 49 (8),

1494–1502. 10.1021/acs.accounts.6b00210. [PubMed: 27486796]

(47). Wei L; Chen Z; Shi L; Long R; Anzalone AV; Zhang L; Hu F; Yuste R; Cornish VW; Min W

Super-Multiplex Vibrational Imaging. Nature 2017. 10.1038/nature22051.

(48). Miao Y; Qian N; Shi L; Hu F; Min W 9-Cyanopyronin Probe Palette for Super-Multiplexed

Vibrational Imaging. Nat. Commun. 2021, 12, 4518. 10.1038/s41467-021-24855-6. [PubMed:

34312393]

(49). Shi L; Wei M; Miao Y; Qian N; Shi L; Singer RA; Benninger RKP; Min W Highly-Multiplexed

Volumetric Mapping with Raman Dye Imaging and Tissue Clearing. Nat. Biotechnol. 2021.

10.1038/s41587-021-01041-z.

(50). Fujioka H; Shou J; Kojima R; Urano Y; Ozeki Y; Kamiya M Multicolor Activatable Raman

Probes for Simultaneous Detection of Plural Enzyme Activities. J. Am. Chem. Soc. 2020, 142

(49), 20701–20707. 10.1021/jacs.0c09200. [PubMed: 33225696]

(51). Ao J; Fang X; Miao X; Ling J; Kang H; Park S; Wu C; Ji M Switchable Stimulated Raman

Scattering Microscopy with Photochromic Vibrational Probes. Nat. Commun. 2021, 12 (1), 1–8.

10.1038/s41467-021-23407-2. [PubMed: 33397941]

(52). Shou J; Ozeki Y Photoswitchable Stimulated Raman Scattering Spectroscopy and Microscopy.

Opt. Lett. 2021, 46 (9), 2176. 10.1364/ol.418240. [PubMed: 33929447]

(53). Lee D; Qian C; Wang H; Li L; Miao K; Du J; Shcherbakova DM; Verkhusha VV; Wang LV; Wei

L Toward Photoswitchable Electronic Pre-Resonance Stimulated Raman Probes. J. Chem. Phys.

2021, 154 (135102), 1–10. 10.1063/5.0043791.

(54). Du J; Wei L Multicolor Photoactivatable Raman Probes for Subcellular Imaging and Tracking by

Cyclopropenone Caging. J. Am. Chem. Soc. 2021. 10.1021/jacs.1c09689.

(55). Hong S; Chen T; Zhu Y; Li A; Huang Y; Chen X Live-Cell Stimulated Raman Scattering Imaging

of Alkyne-Tagged Biomolecules. Angew. Chemie - Int. Ed. 2014, 53 (23), 5827–5831. 10.1002/

anie.201400328.

(56). Lee HJ; Zhang W; Zhang D; Yang Y; Liu B; Barker EL; Buhman KK; Slipchenko LV; Dai M;

Cheng JX Assessing Cholesterol Storage in Live Cells and C. Elegans by Stimulated Raman

Scattering Imaging of Phenyl-Diyne Cholesterol. Sci. Rep. 2015, 5, 1–10. 10.1038/srep07930.

(57). de Moliner F; Knox K; Gordon D; Lee M; Tipping WJ; Geddis A; Reinders A; Ward JM;

Oparka K; Vendrell M A Palette of Minimally Tagged Sucrose Analogues for Real-Time Raman

Imaging of Intracellular Plant Metabolism. Angew. Chemie - Int. Ed. 2021, 60 (14), 7637–7642.

10.1002/anie.202016802.

(58). Seidel J; Miao Y; Porterfield W; Cai W; Zhu X; Kim SJ; Hu F; Bhattarai-Kline S; Min W;

Zhang W Structure-Activity-Distribution Relationship Study of Anti-Cancer Antimycin-Type

Depsipeptides. Chem. Commun. 2019, 55 (63), 9379–9382. 10.1039/c9cc03051d.

(59). Gaschler MM; Hu F; Feng H; Linkermann A; Min W; Stockwell BR Determination of the

Subcellular Localization and Mechanism of Action of Ferrostatins in Suppressing Ferroptosis.

ACS Chem. Biol. 2018, 13 (4), 1013–1020. 10.1021/acschembio.8b00199. [PubMed: 29512999]

(60). Koike K; Bando K; Ando J; Yamakoshi H; Terayama N; Dodo K; Smith NI; Sodeoka M;

Fujita K Quantitative Drug Dynamics Visualized by Alkyne-Tagged Plasmonic-Enhanced Raman

Du et al.

Page 16

ACS Chem Biol

. Author manuscript; available in PMC 2023 November 26.

Author Manuscript

Microscopy. ACS Nano 2020, 14 (11), 15032–15041. 10.1021/acsnano.0c05010. [PubMed:

33079538]

(61). Chen Z; Paley DW; Wei L; Weisman AL; Friesner RA; Nuckolls C; Min W Multicolor Live-Cell

Chemical Imaging by Isotopically Edited Alkyne Vibrational Palette. J. Am. Chem. Soc. 2014,

136 (22), 8027–8033. 10.1021/ja502706q. [PubMed: 24849912]

(62). Waegele MM; Culik RM; Gai F Site-Specific Spectroscopic Reporters of the Local Electric

Field, Hydration, Structure, and Dynamics of Biomolecules. J. Phys. Chem. Lett. 2011, 2 (20),

2598–2609. 10.1021/jz201161b. [PubMed: 22003429]

(63). Bakthavatsalam S; Dodo K; Sodeoka M A Decade of Alkyne-Tag Raman Imaging (ATRI):

Applications in Biological Systems. RSC Chem. Biol. 2021, 2, 1415–1429. 10.1039/d1cb00116g.

[PubMed: 34704046]

(64). Yamakoshi H; Dodo K; Palonpon A; Ando J; Fujita K; Kawata S; Sodeoka M Alkyne-Tag Raman

Imaging for Visualization of Mobile Small Molecules in Live Cells. J. Am. Chem. Soc. 2012,

134 (51), 20681–20689. 10.1021/ja308529n. [PubMed: 23198907]

(65). Hu F; Chen Z; Zhang L; Shen Y; Wei L; Min W Vibrational Imaging of Glucose Uptake Activity

in Live Cells and Tissues by Stimulated Raman Scattering. Angew. Chemie - Int. Ed. 2015, 54

(34), 9821–9825. 10.1002/anie.201502543.

(66). Wei L; Yu Y; Shen Y; Wang MC; Min W Vibrational Imaging of Newly Synthesized Proteins

in Live Cells by Stimulated Raman Scattering Microscopy. Proc. Natl. Acad. Sci. U. S. A. 2013,

110 (28), 11226–11231. 10.1073/pnas.1303768110. [PubMed: 23798434]

(67). Saar BG; Contreras-Rojas LR; Xie SX; Guy RH Imaging Drug Delivery to Skin with Coherent

Raman Scattering Microscopy. Mol. Pharm. 2014, 8, 969–975. 10.1007/978-3-642-32109-2_20.

(68). Miao K; Wei L Live-Cell Imaging and Quantification of PolyQ Aggregates by Stimulated Raman

Scattering of Selective Deuterium Labeling. ACS Cent. Sci. 2020, 6 (4), 478–486. 10.1021/

acscentsci.9b01196. [PubMed: 32341997]

(69). Hu F; Brucks SD; Lambert TH; Campos LM; Min W Stimulated Raman Scattering of Polymer

Nanoparticles for Multiplexed Live-Cell Imaging. Chem. Commun. 2017, 53 (46), 6187–6190.

10.1039/c7cc01860f.

(70). Li J; Cheng JX Direct Visualization of de Novo Lipogenesis in Single Living Cells. Sci. Rep.

2014, 4, 1–8. 10.1038/srep06807.

(71). Zhang D; Slipchenko MN; Cheng JX Highly Sensitive Vibrational Imaging by Femtosecond

Pulse Stimulated Raman Loss. J. Phys. Chem. Lett. 2011, 2 (11), 1248–1253. 10.1021/

jz200516n. [PubMed: 21731798]

(72). Hu F; Wei L; Zheng C; Shen Y; Min W Live-Cell Vibrational Imaging of Choline Metabolites

by Stimulated Raman Scattering Coupled with Isotope-Based Metabolic Labeling. Analyst 2014,

139 (10), 2312–2317. 10.1039/c3an02281a. [PubMed: 24555181]

(73). Alfonso-García A; Pfisterer SG; Riezman H; Ikonen E; Potma EO D38-Cholesterol as a Raman

Active Probe for Imaging Intracellular Cholesterol Storage. J. Biomed. Opt. 2015, 21 (6),

061003. 10.1117/1.jbo.21.6.061003.

(74). Shi L; Zheng C; Shen Y; Chen Z; Silveira ES; Zhang L; Wei M; Liu C; de Sena-Tomas C;

Targoff K; et al. Optical Imaging of Metabolic Dynamics in Animals. Nat. Commun. 2018, 9 (1).

10.1038/s41467-018-05401-3.

(75). Lee D; Du J; Yu R; Su Y; Heath JR; Wei L Visualizing Subcellular Enrichment of Glycogen in

Live Cancer Cells by Stimulated Raman Scattering. Anal. Chem. 2020, 92 (19), 13182–13191.

10.1021/acs.analchem.0c02348. [PubMed: 32907318]

(76). Zhu W; Cai EL; Li HZ; Wang P; Shen AG; Popp J; Hu JM Precise Encoding of Triple-Bond

Raman Scattering of Single Polymer Nanoparticles for Multiplexed Imaging Application. Angew.

Chemie - Int. Ed. 2021, 60 (40), 21846–21852. 10.1002/anie.202106136.

(77). Tian S; Li H; Li Z; Tang H; Yin M; Chen Y; Wang S; Gao Y; Yang X; Meng F; et al.

Polydiacetylene-Based Ultrastrong Bioorthogonal Raman Probes for Targeted Live-Cell Raman

Imaging. Nat. Commun. 2020, 11 (1), 1–9. 10.1038/s41467-019-13784-0. [PubMed: 31911652]

(78). Zhao Z; Chen C; Wei S; Xiong H; Hu F; Miao Y; Jin T; Min W Ultra-Bright Raman Dots

for Multiplexed Optical Imaging. Nat. Commun. 2021, 12, 1305. 10.1038/s41467-021-21570-0.

[PubMed: 33637723]

Du et al.

Page 17

ACS Chem Biol

. Author manuscript; available in PMC 2023 November 26.

Author Manuscript