Recent advances in background-free Raman scattering for bioanalysis

Journal Pre-proof Recent Advances in Background-Free Raman Scattering for Bioanalysis Xuehui Liu, Xiaoman Liu, Pengfei Rong, Dingbin Liu PII:

S0165-9936(19)30530-8

DOI:

https://doi.org/10.1016/j.trac.2019.115765

Reference:

TRAC 115765

To appear in:

Trends in Analytical Chemistry

Received Date: 14 September 2019 Revised Date:

26 November 2019

Accepted Date: 28 November 2019

Please cite this article as: X. Liu, X. Liu, P. Rong, D. Liu, Recent Advances in Background-Free Raman Scattering for Bioanalysis, Trends in Analytical Chemistry, https://doi.org/10.1016/j.trac.2019.115765. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Elsevier B.V. All rights reserved.

Recent

Advances

in

Background-Free

Raman Scattering for Bioanalysis Xuehui Liu,a,# Xiaoman Liu,a,b,# Pengfei Rong, b,* Dingbin Liua,* a

College of Chemistry, Research Center for Analytical Sciences, State

Key Laboratory of Medicinal Chemical Biology, and Tianjin Key Laboratory of Molecular Recognition and Biosensing, Nankai University, Tianjin 300071, China. b

Department of Radiology, The Third Xiangya Hospital, Central South

University, Changsha, Hunan 410013, China. #

The authors contributed equally to this work.

*Corresponding authors: Dingbin Liu, E-mail: [email protected]. Pengfei Rong, E-mail: [email protected]

1

Highlights · Background-free Raman reporter possesses a vibration in the cellular Raman-silent region. · Background-free probes were designed to detect and probe biomarkers with high signal-to-background ratios by surface-enhanced Raman scattering. · Biomolecules were labeled with background-free vibrational reporters for visualizing their metabolism by stimulated Raman scattering.

Abstract With the characteristics of intrinsically free from photobleaching, blinking, and self-quenching, Raman microscopy has become a powerful platform in detecting molecular vibrations for diverse applications. In particular, Raman spectroscopy has proven very useful for bioanalysis but suffers from several challenges such as poor sensitivity, spectral overlapping, and background interference. In this review, we would like to introduce two kinds of high-fidelity imaging strategies, including background-free surface-enhanced Raman scattering (SERS) and stimulated Raman scattering (SRS) that are capable of significantly enhancing detection sensitivity and spectral resolution while minimizing background

interference

simultaneously.

Recent

advances

in

background-free SERS and SRS and their applications in biological detection and cellular imaging are summarized and discussed.

Keywords: SERS; SRS; bioanalysis; Raman imaging; nanoprobes Abbreviations 3-O-propargyl-d-glucose (3-OPG), 3-[4-(phenylethynyl) benzylthio] 2

propanoic

acid

(PEB),

4-thiophenylacetylene

4-mercaptobenzonitrile

(TPA),

5-ethynyl

5-(1,7-octadinyl)uridine

(MBN),

uridine

triphosphate

(EU), (ODUTP),

5’-ethynyl-2’-deoxyuridine (EdU), 17-octadecenoic acid (17-ODYA), Aggregation-induced emission (AIE), Alkyne-labeled choline (propargyl choline), Au nanoparticles (AuNPs), Au core-Ag shell nanoparticle (Au@Ag

NP),

Au-core

and

dopamine/Ag-shell

(ACDS),

Ag

nanoparticles (AgNPs), Carbon-deuterium (C-D), Cholesteryl esters (CE), Cyclic arginine-glycine aspartic acid (cRGD), Deuterium oxide (D2O), Deuterium-labeled amino acids (D-AA), Deuterium-labeled essential amino acid (leucine-d10), Deuterium-labeled fatty acids (D-FAs), Deuterium-labeled glucose (D7-Glc), Deuterium-labeled palmitate acid (d31-PA), D2O probing with stimulated Raman scattering (DO-SRS), (E)-2-[4-(ethynylbenzylidene)

amino]

ethane-1-thiol

(EBAE),

Enhancement Factor (EF), Epidermal growth factor receptor (EGFR), electromagnetic

(EM),

Epithelial-mesenchymal

transition

(EMT),

Estrogen receptor (ER), Folic acids (FAs), Finite-element method (FEM), Graphene-isolated-Au-nanocrystals (Hpg),

Hyperspectral

(GIANs),

stimulated

Raman

Homopropargylglycine scattering

(hsSRS),

Interference-free SERS (IF-SERS), Locked nucleic acid (LNA), Luteinizing hormone-releasing hormone (LHRH), 4-mercaptobenzonitrile (MBN), Peracetylated N-(4-pentynoyl) mannosamine (Ac4ManNAl), 3

Phenylboronic acid (PBA), Polyallylamine (PAH), Polyethylene glycol (PEG), Polydopamine (PDA), Poly-L-lysine (PLL), Progesterone receptor (PR), Prussian blue (PB), Pyrophosphate (PPi), Raman reporters (RRs),

Reactive

oxygen

species

(ROS),

Sialic

acid

(SA),

Signal-to-background ratio (SBR), Surface plasmon resonance (SPR), Stimulated Raman gain (SRG), Stimulated Raman loss (SRL), Stimulated Raman scattering (SRS), Surface-enhanced Raman scattering (SERS), Surface-enhanced resonance Raman scattering (SERRS), Single-stranded DNA (ssDNA), Terbinafine hydrochloride (TH), Triacylglycerols (TAG), Trimethyl (phenylethynyl) silane (TPS), W-alkynyl palmitic acid (Alk-16).

1. INTRODUCTION Optical microscopy, in particular, fluorescence microscopy, is a powerful tool to study living systems at the microscopic level. The development of confocal fluorescence microscopy, total internal fluorescence microscopy, and recently super-resolution fluorescence microscopy has deepened our understanding of biological systems with unprecedented and explicit details. However, there remains a fundamental limitation for fluorescence imaging of small biomolecules (such as nucleosides, amino acids, fatty acids, choline, glucose, cholesterol, and small-molecule

drugs)

in

living

systems,

because

most

small

biomolecules are intrinsically nonfluorescent. In general, visualization of the biomolecules depends on fluorescent tags such as organic dyes, 4

fluorescent proteins, or quantum dots. However, these tags are often too large relative to the size of a small biomolecule, severely compromising the

native

biochemical

or

biophysical

properties

of

these

fluorophore-labeled small biomolecules inside live cells. Additionally, fluorescent imaging is always facing the problems of photobleaching and self-quenching. Raman spectroscopy offers the spatially resolved chemical information of a molecule without the need of chemical labeling[1]. With the advantages of free from photobleaching, blinking, and self-quenching[2], Raman spectroscopy has been widely used as a platform for determining chemical components, molecular structure and conformation, and molecular interactions. Furthermore, due to the intrinsically weak Raman signal of water, this technique is particularly suitable for the detection and imaging of biomolecules in living systems, which is useful for the accurate determination of cancer biomarkers for diagnosis and treatment[3]. However, the obtained Raman spectra require complicated and laborious spectral unmixing[4]. Moreover, it is difficult to distinguish the Raman signals of target molecules from those of endogenous biomolecules, as they may share the same chemical bonds. Nevertheless, spontaneous Raman scattering is an intrinsically weak process because of the notoriously small cross-sections of biomolecules, which are often overwhelmed by cell autofluorescence[5, 6]. All in all, the issues including slow acquisition rates[7], poor resolution[8], a lack of sensitivity, and vulnerability to sample autofluorescence have hampered further development of spontaneous Raman scattering. As a result, spontaneous Raman microscopy is an unsatisfactory bioanalysis method[9]. Thus, there is a high demand for developing Raman probes with high sensitivity and low background interference simultaneously to overcome the limited 5

molecular specificity in complex biological environments[10]. Surface-enhanced Raman scattering (SERS) and stimulated Raman scattering (SRS) have raised to address the issues mentioned above. SERS and SRS could provide much higher sensitivity and lower signal-to-background ratio (SBR). The SBR, defined as the amount of desired signal relative to the level of background signal, is the key determinant to realize the measurement of low-abundance targets of interest in single cells[11]. It has recently been demonstrated that the SBR can be significantly improved if the detection signals appear in the cellular Raman-silent vibrational region (1800-2800 cm-1), where the endogenous intracellular biomolecules do not generate any Raman signals[12-15]. Traditional Raman reporters possess multiple peaks in the fingerprint region (1000-1700 cm-1), which typically overlap with each other and cause crosstalk. Therefore, background-free reporter, a Raman reporter possessing a vibration in the cellular Raman-silent region, springs up whose Raman signals would not be overlapped by the signals derived from endogenous biological substances. Several chemical groups, including alkyne, azide, nitrile, and deuterium have been utilized as background-free Raman reporters[6,

16]

. These Raman reporters present

strong and sharp single peaks in the cellular Raman-silent region. The carbon-deuterium (C-D) bonds have a lower stretching frequency at 2000-2400 cm−1[17]. Alkyne or carbon-carbon triple bonds exhibit the Raman signal at ∼2120 cm−1[18]. Moreover, these narrow, distinguishable, and single peaks are completely resolved without complicated spectral unmixing when conducting multiplexed imaging[4]. The interference of C-H, which is ubiquitous in nearly all biomolecules including proteins, nucleosides, triglycerides, and cholesterol, can be efficiently eliminated in the cellular-silent vibrational region by the background-free Raman reporters[19, 20]. 6

Vibrational imaging by Raman contrast is a rapidly growing field[9]. In Raman imaging, chemical modification of the target molecule by tagging a background-free Raman reporter can further enhance the detection sensitivity and specificity[5, reporters

are

small,

19-21]

. On the other hand, these Raman

structurally

stable,

and

unsusceptible

to

photobleaching, enabling continuous live-cell imaging[22]. Currently, small bioorthogonal imaging reporters such as stable-isotope labeling (C-D,

13

C), triple bonds (alkyne, cyano) and their combined technology

have opened a new window for correctly tracking small biomolecules like DNA, proteins, lipids, sugars, and drugs[7, 9, 18, 23]. In this review, SERS and SRS, as powerful background-free analytical tools in biology, are discussed. We provide an overview of recent advances in SERS and SRS microscopy with a particular focus on developments

relevant

to

the

background-free

strategies.

The

characterization, as well as the progress of background-free Raman scattering strategies of SERS and SRS, are summarized in sections 2 and 3, respectively. Here, we highlight Raman tagging strategies that exploit the ‘silent region’ in the Raman spectra of cells, and these have allowed the development of spectroscopically bioorthogonal reporters that maximize signal contrast. The progress and perspective are summarized in section 4.

2. SERS The observation and confirmation of SERS phenomena on a roughened Ag metal surface in the 1970s have significantly sparked growing interest in the examination of the optical properties of nanostructured metal surfaces (e.g. gold nanoparticles, AuNPs). The detection limit of SERS 7

can be down to the single-molecule level when the analytes are properly immobilized in the hot spot of plasmonic nanoparticles. The hot spots can gather the incident light around the nanostructures and greatly enhance the electromagnetic field on the metal surfaces. There are two primary theoretical mechanisms of enhancement, long-range electromagnetic (EM) enhancement

and

short-range

chemical

enhancement.[24]

EM

enhancement plays a major role in the enhancement phenomenon of SERS. Figure 1 shows the four major enhancement effects of SERS. The incident laser can cause collective oscillations of the surface electrons on rough metals, which are defined as surface plasmon resonance (SPR) (Figure 1A). When the frequency of the incident laser is resonant with the plasmon, the EM field will be redistributed and enhanced at a specific position around the nanoparticle, because the metal nanoparticle stimulated by the laser will radiate a dipolar field to coherent with the exciting electric field (Figure 1C). Thus, the surface electric field will be greatly enhanced, and the Raman scattering of the surface molecules will be increased. Importantly, this enhancement happens only when molecules locate on or are very close to the metal surface (Figure 1B). Besides, there are extremely small radii of curvature in the tip of the nanoparticles, with a stronger local surface electromagnetic field compared with circular nanoparticles, which is defined as a “sharp tip effect”,[24] because the charge is limited in a very small area due to the 8

continuity of the electric field force (Figure 1D). Moreover, the smaller the tip of the nanostructure, the greater the surface electric field intensity. Some electromagnetic enhancement happens in the gap of the nanoparticle aggregation (Figure 1E). Compared with the edge, the intensity of the electric field is enhanced by E`/E= (2D+d)/d times. Furthermore, nanoshell surfaces can also provide average integrated SERS enhancements similar to nanosphere dimers (Figure 1F). Besides, the SPR wavelength of the bimetallic nanoparticles, like Au core and Ag shell, can be continuously tuned in a wide range, which will be more easily to match with the given laser excitation wavelength and to achieve the strongest SERS enhancement[24]. The characteristics of different kinds of SERS hot spots are shown in Table 1.

9

Figure 1. (A) Localized surface plasmon resonance (LSPR). [25] (B) Diagram of SERS. Schematic illustration of localized surface plasmon resonance (LSPR) effects under the incident light by finite-element method (FEM) simulating spatial |E|4 distribution for (C) Au nanosphere, (D) Au nanostar, (E) Au core/Au satellite particle, and (F) Au/Ag nanoshell.[26]

Table 1. Characteristics of different kinds of SERS hot spots SERS tags Surface plasma resonance (SPR) effect Sharp effect

Enhancement Factor (EF) range

SPR range

Sensing properties

Au nanospheres

102-103 [27]

λmax ~560 nm [28]

enhancement factors vary with analyte concentration [27].

Au nanorods

103-107 [29]

λmax ~735 nm [30]

very high filed enhancement on both ends [31].

tip

10

The coupling effect between nanoparticles

Au nanostars

104 [32]

λmax ~600 nm λmax ~800 nm[28]

Silver nanocubes Au nanotriangles Nanoparticles aggregation

5.5×104 [33]

~ 800 nm[34]

6×104 [36]

λmax ~800 nm[28]

Gold nanoshells Au@Ag bimetallic nanospheres

106-1010 [29]

having exceptional sensing capabilities due to its extreme sharpness [32]. very high electromagnetic enhancement in the corners [35]. SERS hot spots on the edges [36].

λmax ~660 nm[28]

resonance Raman and electronparamagnetic resonance spectroscopy measurements [37].

108-109 [38]

657–957 nm [39]

0.94×107 [40]

400–520 nm[41]

near-infrared-resonant nanoshells [38] . The analytical enhancement factor increased obviously as the diameter of Au@Ag nanospheres went up[40].

Over the past 40 years, SERS has found important applications in chemistry, material sciences, analytical sciences, surface sciences, biomedical research, etc.[42] SERS inherits the rich chemical fingerprint information

on

Raman

spectroscopy

and

gains

sensitivity

by

plasmon-enhanced excitation and scattering. In particular, most Raman peaks have a narrow width suitable for multiplex analysis, and the measurements can be conducted under ambient and aqueous conditions. These merits make SERS promising for studying complex biological systems. Especially, the SERS substrate can be optimized for the near-infrared laser to avoid the native autofluorescence from the biological samples and to minimize the photodamage of visible laser to living cells. SERS has been widely used for biological imaging through combining nanotechnology with molecular spectroscopy, holding the 11

advantages of high sensitivity, high spectral resolution, reliable signals free from quenching or photobleaching, non-invasive measurements with minimum damage to biological samples, as well as little interference from autofluorescence and water. As a result, the past few decades have witnessed impressive developments in SERS and its bioanalytical applications. Recent developments in the synthesis of SERS active materials and techniques have led to impressive progress not only in molecular detection but also in the chemical mapping of single molecules and molecular imaging in living systems. Table 2 lists representative background-free Raman molecules commonly used in current literature. Table 2. Representative SERS active molecules used as background-free reporters Types

SERS active molecules

Detected objects

Probe’s design

Properties

Refs #

1. Alkyne

5-ethynyl-2’-de oxyuridine(Ed U)

active synthesis

DNA

the alkyne tag can be employed for click-free molecular imaging

the Raman signal at 2123 cm-1 localized in the nucleus is derived from EdU.

[18]

4-ethynylbenze nethiol

AS1411), cRGD, anti-CD44

showed discernible SERS peaks at 2105, 2159, and 2212 cm-1

[36]

phenylboronic acid

sialic acid

(E)-2-[4-(ethyn ylbenzylidene) amino]ethane-1 -thiol

polydopamine

three 4-ethynylbenzenet hiol-derivatives modified Au@AgNPs AuNP dimers; an alkyne-bridged plasmonic dimer probe Au-core and dopamine/Ag-shell

12

the 2210 cm-1 channel [3] presented clear signals without background noise. the [32] AuNPs@EBAE@PD A nanoparticles showed a peak at 2100

cm−1 trimethyl HER2, ER, PR, AuNPs at 1982 cm−1, 2121 [40] (phenylethynyl) and EGFR cm−1, 2216 cm−1 and silane 2240 cm−1 bind to the surfaces 4-(phenylethyn progesterone signals at 2230 cm-1 [33] yl) aniline receptor of AuNPs , then tags were further conjugated with

progesterone receptor antibodies

2. Nitrile

phenylthiocyan ate

epidermal growth factor receptor

bind to the surfaces

signals at 2120 cm-1

[33]

of AuNPs , then tags were further conjugated with

epidermal growth factor receptor antibodies

4-mercaptoben zonitrile

3. Alkyneand nitrile-

alkyneand nitrile-terminat ed molecules

Pyrophosphate; monitor ROS in mitochondria

AuNPs; Au core-Ag shell nanoparticle

miRNA-21 (miR-21) miRNA-155 (miR-155)

a

and

target-mediated

nanoparticle dimerization strategy

to

build

electromagnetic hot spots in living cells

its nitrile moiety presents a strong and sharp single peak (2220 cm-1) in the cellular Raman-silent region -1 (1800-2800 cm ). Coupling target-programmed nanoparticle dimerization with the background-free Raman reporters

[28], [30]

[4]

In this section, we would like to review the recent progress regarding background-free SERS strategies in direct intracellular analysis, investigation of biological processes and cellular functions, as well as SERS imaging of cell-surface species.

2.1. Dimeric nanostructure-based SERS imaging 13

The electromagnetic hot spots could be generated by inducing AuNPs dimerization, and a much higher electromagnetic field can be detected in the gap of the dimer than that on the surface of AuNP monomers (Figure 2A). Once the background-free Raman reporters are sandwiched into the gapped hot spot, the Raman intensity of the reporters can be significantly enhanced (Figure 2B). Several strategies have been introduced to initiate the generation of AuNP dimerization, which has been employed to probe targets of interest inside cells. We reported a background-free SERS probe for highly sensitive and selective detection of pyrophosphate (PPi) in aqueous solutions and imaging PPi in living cells with nearly zero background interference[43] (Figure 2C). 4-mercaptobenzonitrile (MBN) was employed as a background-free reporter showing a nitrile vibration at 2232 cm−1 in the cellular Raman-silent region

[44]

(1800-2800 cm-1). The

surfaces of AuNPs were asymmetrically functionalized with PEG and the mixture of MBN and DPASH-Zn2+ complex, respectively. The in situ formation of hot spots was based on the specific coordination of DPASH-Zn2+ with PPi by a molecular ratio of 2:1, resulting in AuNPs dimerization and thus markedly enhancing the Raman intensity of MBN. The results demonstrated that the Raman signal of exogenous nitrile (CN) could be completely resolved from the background noise. The distribution of PPi in living cells was mapped at the CN channel with high accuracy (Figure 2D). Further, we proposed a similar target-mediated nanoparticle dimerization strategy to build electromagnetic hot spots in living cells[4], which enabled background-free multiplexed imaging of two intracellular oncogenic markers microRNAs (miRNAs namely, miR-21 and miR-155) in living cancer cells. Alkyne- and nitrile-terminated molecules were applied as multiplex Raman reporters because of their strong and sharp single peaks in the cellular Raman-silent region[18, 20]. The hot spots of the dimeric nanostructures were originated from the binding events between 14

target miRNAs and the locked nucleic acid (LNA) sequences on the asymmetrically-modified SERS probes, where the newly generated electromagnetic hot spots provided significantly enhanced Raman scattering (Figure 2E). The results indicated that this approach could be applied to detect and differentiate miR-21 and miR-155 in a single cell simultaneously, opening an exciting avenue for multiplexed genetic profiling at the single live-cell level (Figure 2F). The dramatic enhancement of Raman scattering in the dimeric nanoparticles encouraged us to design background-free SERS tags. To achieve this, we synthesized a set of alkyne-bearing dyes, which were resided into the gaps of the plasmonic dimeric nanoprobes for the preparation of high-performance SERS tags[3] (Figure 2G). The probes were modified with phenylboronic acid (PBA) for profiling the expression of sialic acid (SA) on cancer cell surfaces and for further revealing the clinical relevance of SA expression with various metastasis levels (Figure 2H).

15

Figure 2. Dimeric nanostructure-based SERS probes for biological detection and imaging. (A) Near-field distributions of the normalized electric field |E|/|Einc| at the excitation laser’s wavelength (633 nm) for AuNP monomer (left) and dimer (right). (B) SERS spectra of the AuNP probes before (black line) and after (red line) addition of PPi. (C) Schematic illustration of AuNP Dimerization through PPi-mediated metal coordination. (D) Bright-field (BF) and SERS mapping images of single living cells acquired in 1580 and 2220 cm

-1

channel and their merged image.

Scale bar: 5 µm. SERS spectra in point 1 and point 2[43]. (E) Schematic representation of the miRNA-programmed nanoparticle dimerization. (F) BF, Raman mapping at 1580 cm-1 (blue channel) and 2101 cm-1 (red channel), merged images, and statistic Raman intensity for the MCF-7 cells treated with a) probe 1, b) probe 2, and c) the mixture of probe 1 and probe 2 at 37 °C for 12 h. The scale bar in all the BF images is 10 µm[4]. (G) Schematic illustration of alkyne-bridged AuNP dimers conjugated with PBA for specific profiling of SA on the cell 16

membrane. (H) Profiling SA expression in clinical tissues with various degrees of metastasis. The Raman mapping images were acquired in 1580 (green) and 2210 cm-1 (red) channels: a) benign breast specimen and b), c) two different malignant breast cancer specimens with II to III degree of metastasis and d) that of III degree. The specimens were analyzed using confocal Raman imaging with a laser excitation of 785 nm (30 mW). Scale bars: 20 µm[3].

2.2. Core-shell structure-based SERS imaging The background-free Raman reporters can also be embedded in the core-shell layered nanoparticles to obtain enhanced Raman signals. Shen et al.[45] designed an Au core-Ag shell nanoparticle (Au@Ag NP) which contains the cellular Raman-silent reporter MBNs. As the Ag shell was etched by reactive oxygen species (ROS), the SERS intensity of MBN decreased accordingly, allowing this nanoprobe to be used in ROS monitoring in mitochondria during the photothermal therapy. Furthermore, Prussian blue (PB) has been demonstrated as an ideal Raman reporter because it is a cyanide (CN)-bridged coordination polymer showing a strong and sharp single vibrational peak at 2156 cm-1 in the cellular Raman-silent region. PB can be assembled onto AuNPs to provide surface-enhanced resonance Raman scattering (SERRS) probes with high SBR (Figure 3A). We have demonstrated the performance of the PB-based SERRS probes (Au@PB NPs) for high-sensitivity immunoassay (Figure 3B) and cancer cell imaging (Figure 3C)[46]. The Au@PB NPs were initially wrapped with poly-L-lysine (PLL) to provide positively charged amine moieties combining with folic acids (FAs) for cell targeting as FA receptors were overexpressed in diverse cancer cells. The result presented the cell distribution of the background-free Raman reporters in the HeLa cell, where a specific Raman peak of CN at 2156 cm-1 was noticeably exhibited in the Raman-silent region. Otherwise, Chen et al.[47] synthesized a Raman reporter (E)-2-[4-(ethynylbenzylidene) 17

amino] ethane-1-thiol (EBAE) with terminal alkyne. The EBAE molecule equipped with S- and C-termini could directly bond to AuNPs and dopamine/silver, forming Au-core and dopamine/Ag-shell (ACDS), respectively. The bimetallic ACDS exhibited excellent live-cell imaging due to the strong electromagnetic field between AuNPs and Ag nanoparticles (AgNPs) induced by EBAE and the distinct Raman signal from EBAE in the cellular Raman-silent region with nearly zero background interference. To improve the chemical stability and brightness of the core-shell nanoprobes, we reported an interference-free SERS (IF-SERS) probe using MBN as the background-free Raman reporter[11] for single-cell imaging (Figure 3D). MBN reporters were embedded in the core-shell layered AuNPs to obtain enhanced Raman signals. The IF-SERS probes were functionalized with EpCAM antibodies to profile the expression of EpCAMs in single cancer cells. The results suggested that the IF-SERS probes were potent tools for profiling biomarkers in complex biological systems with high SBR. This background-free imaging technique can also be applied to probe most other biomarkers of interest by simply replacing the ligands, which improves biosensing with high SBR in complex environments. Hence, it provides new opportunities in both basic biological studies and clinical applications for early diagnosis and assessment of tumor malignancy and metastatic degrees during cancer treatment.

18

Figure 3. (A) Schematic illustration of Au@PB NPs and their Raman signals in comparison with that of cells. The Au@PB NP probes were employed in immunoassays (B) and single-cell background-free imaging (C)[46]. (D) Schematic illustration of the procedure for the preparation of Au@MBN@AuNPs and the Raman spectra of single MCF-7 cells before (black) and after (red) treating with the as-prepared IF-SERS nanotags. The nanotags exhibit multiple peaks in the fingerprint region (<1800 cm-1) and a strong peak (2232 cm-1) in the cellular Raman-silent region (1800-2800 cm-1). Mapping images were acquired in 1570 cm-1 channel (strong background noise) and 2232 cm-1 channel (negligible background noise)[11].

2.3. Multicolor SERS imaging SERS reporters are quite suitable for multiplex detection owing to their extremely narrow peak width (down to 1-2 nm). The background-free SERS reporters without overlapping peaks for multiplex imaging could be applied to profile multiple biomarkers expressed in cancer cells and tissues. We introduced a one-pot approach[48] to preparing SERS reporters through the direct coordination of alkyne and nitrile groups with AuNPs based on σ-π[49] or π-π interactions[50] for multiplex targeting and multicolor identification of cancer biomarkers with nonoverlapping Raman signals. In the meantime, a library of Raman reporters containing 19

the exogenous alkyne and nitrile groups were designed as both anchors and background-free reporters (Figure 4A). The SERS tags bearing reporters 4-ethynyl-biphenyl, phenylthiocyanate, and 4-(phenylethynyl) aniline were further conjugated with different antibodies against estrogen receptor

(ER),

epidermal

growth

factor

receptor

(EGFR)

and

progesterone receptor (PR) respectively for multicolor imaging. The signals at 2000, 2120, and 2230 cm-1 reflected the expression of ER, EGFR, and PR, respectively, of three different biological targets in cancer cells and human breast cancer tissues. Chen et al.[51] proposed a highly sensitive Raman probe equipped with an alkyne-modulated SERS palette, which possessed super sensitivity and minimal optical interferences for optical interference-free multiplex cellular imaging and multi-target assay. Considering the chemical structure of the thiol-containing small molecule, the 4-ethynylbenzenethiol was applied as the parent structures of potential alkynes of SERS-palette. The Raman shifts of alkynyl could be tuned by linking different substituent groups at the benzene ring or terminal alkynes. The alkyne-reporters were successfully encapsulated in the polyallylamine (PAH, MW = 17 000) shell through the Ag-S bond between

the

reporters

and

4-ethynylbenzenethiol-derivatives

the

NPs

modified

surface.

The

Au@AgNPs

three showed

discernible SERS peaks at 2105, 2159, and 2212 cm-1 with excitation at 532 nm. In addition, FA, luteinizing hormone-releasing hormone (LHRH), or CALNNR8, a polypeptide chain containing multiple arginines, could be conjugated with the abundant-NH2 groups of PAHylation by EDC reaction to help the nanoparticles enter cells quickly,[52] (Figure 4B). Wang et al.[53] demonstrated a multiplex cocktail of bioorthogonal SERS nanoprobes containing three bioorthogonal Raman reporters with alkyne, azide, or nitrile groups with specific Raman shift peaks at 2205, 2120, and 2230 cm-1, respectively. Then, the surface of nanoprobes was 20

modified by silica and polyethylene glycol (PEG) and conjugated with three specific targeting ligands: oligonucleotide aptamer (AS1411), cyclic arginine-glycine aspartic acid (cRGD) peptide, and homing cell adhesion molecule antibody (anti-CD44) separately for simultaneous targeting and diagnosis of multiple breast cancer phenotypes. There is another method for multiplexed Raman imaging. Zou et al.[54] designed a kind of isotopic cellular

Raman-silent

SERS

graphene-isolated-Au-nanocrystals

nanoprobe (GIANs).

Intact

defined graphene

as was

deposited on AuNPs, providing a protective shell, and showing a 2D-band (2706 cm-1) at cellular Raman-silent region. Moreover, multiplexed GIAN SERS tags with Raman signals at the cellular Raman-silent region could be achieved by varying the fraction of isotopic compositions of 12C and 13C, and they had synthesized five GIAN multiplexed tags conjugated with three specific phospholipid-polyethylene glycol-linked aptamers for targeted cancer cell imaging. The chemical stability and signal reproducibility of SERS tags depend heavily on the interactions between the SERS substrate and the molecular structure of Raman reporters incorporated. Recently, we reported a simple, rapid, and universal platform methodology[34] for the one-pot preparation of Raman nanoprobes without the constraints of Raman dye chemical structures. In this way, Raman reporters with or without an amine or thiol group could be incorporated in situ during dopamine polymerization onto the nanoparticle surface (Figure 4C). For example, the background-free Raman reporter, trimethyl (phenylethynyl) silane (TPS) equipped with alkyne vibration without an anchoring group, would be detectable through the in situ incorporation method. Owing to the advantages of in-situ formation of polydopamine (PDA), the Raman signal of DMSO-d6 from the deuterium (C-D), which was undetectable by conventional Raman detection, would be enhanced and detectable through this one-pot 21

method. This method enabled the easy preparation of a large library of imaging probes and offered new opportunities for multiplex biological detection and diagnosis (Figure 4D). The background-free SERS reporters without overlapping peaks for multiplex imaging could be applied to profile multiple biomarkers expressed in cancer cells and tissues[48]. Thus, four SERS reporters were chosen to conjugate with the targeting ligands via Michael addition through the exposed dopamine quinone to simultaneously probe HER2, ER, PR, and EGFR at 1982 cm−1, 2121 cm−1, 2216 cm−1, and 2240 cm−1 respectively (Figure 4E). Three cancer biopsies and clinical FFPE tissue specimens were evaluated for the cancer biomarkers.

22

Figure 4. (A) The molecular structures and SERS spectra of the three background-free Raman reporters[48]. (B) Three-color SERS imaging using SERS probes of alkyne SERS palette in the same live HeLa cells[51]. (C) Schematic illustration of the steps in the conventional preparation of SERS tags. The reporters are coated onto metallic nanoparticles via an anchoring group such as thiol or amine. Another protective layer, including thiolated polyethylene glycol, silica, or bovine serum albumin, is added next. Schematic illustration of the one-pot synthesis of SERS tags. The reporters are embedded in situ during PDA polymerization in a weak basic solution (pH 8.5). (D) Single Raman bands of no. 4, 19, 68, 18, 35, and 38-coded SERS probes in the biological Raman-silent region. (E) Raman mapping images of four pseudo-color (red, green, magenta, and blue) channels corresponding to the no. 4, 68, 35, and 38-encoded SERS probes targeting HER2, ER, PR, and EGFR, respectively. Scale bar = 50 µm[55].

23

Background-free SERS strategy provides insight into the development of a new generation of multiplex imaging, which could be used for in vivo noninvasive and background-free multiplex profiling of tumor biomarker expression. This new imaging technique should facilitate cancer diagnostic procedures, prognosis assessment, and specific treatment based on cancer phenotypes. Also, there are some other detection methods with background-free Raman reporters. Si et al.[56] made use of alloyed Au/Ag nanoparticles as the SERS substrate, which combined the superior properties of both pure Au and pure AgNPs. The alloyed nanoparticles equipped with endonuclease-responsive SERS signaling molecule, the single-stranded DNA (ssDNA) modified with 3-[4-(phenylethynyl)benzylthio]propanoic acid (PEB), and 4-thiophenylacetylene (TPA) employed as the internal standard SERS signal for quantitative detection of the endonuclease. The Raman signals from both the PEB and TPA molecules on the alloyed nanoparticles could be detected and identified. In the presence of endonuclease, the ssDNA was cleaved, releasing PEB molecules and decreasing the SERS signal at 2215 cm-1. As the SERS signal at 1983 cm-1 from TPA remained unchanged, quantitative detection of endonuclease could be achieved, based on the ratiometric peak intensity of I1983/I2215 both in vitro and in living cells. Otherwise, Sumaira Hanif et al.[57] made use of aptamers-functionalized gold-coated nanopipette to capture target, while the AgNPs modified with MBN, and complementary DNA worked as Raman reporter to produce SERS signal at 2223 cm-1. They demonstrated that the SERS signal at 2223 cm-1 was lost when the nanopipette upon nucleolin was captured by the aptamers on nanopipette, which could be helpful in cancer diagnosis and treatment at the cellular level. Overall, SERS with background-free Raman reporter should facilitate 24

cancer diagnostic procedures, prognosis assessment, and specific treatment based on cancer phenotypes.

3. SRS Besides SERS, the newly emerged SRS microscopy is also more sensitive than the spontaneous Raman scattering[58, 59]. In SRS, there are two laser beams at ωp and ωs coinciding with the sample. When the difference frequency, ∆ω=ωp−ωs, defined as the Raman shift, matches a particular molecular vibrational frequency, the intensity of the Stokes beam, IS, experiences a gain, ∆IS (stimulated Raman gain, SRG), and the intensity of the pump beam, IP, experiences a loss, ∆IP (stimulated Raman loss, SRL). In contrast, when the Raman shift does not match any vibrational resonance, SRL and SRG would not occur. The accompanied SRL signal of the transmitted pump beam or the SRG signal of the transmitted Stokes beam can be detected sensitively by a high-frequency modulation scheme through a lock-in amplifier. Thus, high-speed imaging up to the video rate can be achieved, which is 100-1000 faster than spontaneous Raman imaging[10, 60]. The sensitivity enhancement of SRS is based on exploiting the coherent nature of signal generation, which makes use of stimulated excitation to amplify the Raman vibrational excitation by a factor of 107, rendering a quantum leap of sensitivity over spontaneous Raman[10, 61] without enhancing substrates[5]. The SRS signal has a little non-resonant background, well preserved Raman spectra, and straightforward image interpretation[60]. Moreover, the Raman spectral fidelity is preserved, and the intensity of the SRS is proportional to the concentration of the chemical bond, which would provide a linear concentration dependence for quantitative imaging. As an emerging nonlinear vibrational imaging technique, SRS exhibits subcellular resolution straightforward image interpretation in many 25

biological systems[60, 62]. Through combining the advantages of SRS with background-free

reporters,

SRS

microscopy

could

achieve

non-invasiveness, high sensitivity, molecular specificity, and excellent biocompatibility in live-cell imaging[10,

63]

. Hu et al.[64] demonstrated

Raman active polymer dots with high photo-stability under SRS microscopy, which were suitable for live-cell analysis. The Raman active polymer dots equipped with alkyne (2163 cm-1), nitrile (2232 cm-1), and C-D (2293 cm-1) were spectrally orthogonal to each other in live cells with negligible cross-talk, allowing high-fidelity multiplexed imaging without the need of spectral unmixing. The three vibrational background-free reporters had high labeling efficiency for SRS imaging with high signal to noise ratio (S/N>5, at 30 µs time constant) in the cellular Raman-silent region. Finally, each cell type was incubated with one specific Raman-active polymer dot, and then the three cell types were mixed and cultured together. The results could identify the cell types and map out the spatial location of each cell in a co-culture. Recently, a new bioorthogonal SRS strategy has proven powerful for cell imaging by introducing background-free vibrational reporters such as deuterium, nitrile, or alkyne labels to small biomolecules[9, 62]. There are two ways to introduce background-free vibrational reporters: one is labeling biomolecules background-free vibrational reporters directly, and the other is the incorporation of deuterium oxide (D2O) derived deuterium into macromolecules to generate carbon-deuterium (C-D) bonds via metabolism.

3.1. Direct labeling of biomolecules with background-free moieties As biomolecules can be covalently tagged with background-free vibrational reporters, like alkyne (Figure 5A), SRS microscopy can be 26

employed to study the metabolism of glucose uptake[63], choline metabolism[61], cell proliferation[62], membrane synthesis[61], and drug uptake. The investigation into the spatial-temporal dynamics of many metabolites in live cells and animals will provide useful insights into aberrant metabolic processes in multiple diseases. 3.1.1. Lipid imaging Hong et al.[5] made use of w-alkynyl palmitic acid (Alk-16), a palmitic acid analogue functionalized with an alkyne, for tracking palmitoylomes. Wei et al.[61] employed one alkyne-tagged fatty acid, 17-octadecenoic acid (17-ODYA), to depict the formation of numerous lipid droplets, which indicated the transformation into foam cells, a hallmark of early atherosclerosis. Hu et al.[60] reported deuterated (trimethyl-D9)-choline derivatives for in vivo imaging of choline-containing metabolites using SRS microscopy. Deuterium labeling could also image different types of lipid molecules quantitatively, and track their spatiotemporal dynamics in vivo under both physiological and pathological conditions. Fu et al.[7] reported that two classes of neutral lipid molecules, triacylglycerols (TAG) and cholesteryl esters (CE) labeled with deuterium at the single-lipid droplet level, could be distinguished in yeast, C. elegans, mammalian cells, and mouse tissues by hyperspectral stimulated Raman scattering (hsSRS) imaging method. Moreover, the results elucidated the dynamics of different fatty acids molecules during their incorporation and transportation in vivo. 3.1.2. Nuclear imaging The 5’-ethynyl-2’-deoxyuridine (EdU) was employed to image DNA because EdU could be metabolically incorporated into replicating DNA by partly substituting thymidine[65]. SRS imaging of HeLa cells treated with EdU revealed intense alkyne signals at 2120 cm-1 in the nuclei. 27

Beyond that, the alkyne staining of nuclei exhibited a positive correlation between the SRS signal intensity and the concentration of EdU[5]. Moreover, the alkyne-tagged uridine analogue 5-ethynyl uridine (EU) was applied to explore RNA transcription and turnover[61]. Anupam A. Sawant et al.[66] developed a new alkyne-modified UTP analog, 5-(1,7-octadinyl)uridine triphosphate (ODUTP), which contains two alkyne labels. The terminal alkyne group of the ODUTP could interact with a variety of biophysical tags (fluorescent, affinity, amino acid and sugar) by using alkyne-azide click reaction, and the reaction would leave the internal disubstituted alkyne group intact, which could be served as a Raman active label at cellular Raman-silent region. In general, ODUTP could be potentially suitable for dual imaging of RNA by using click chemistry and Raman spectroscopy. 3.1.3. Protein imaging SRS microscopy was coupled with deuterium-labeled amino acids for visualization of newly synthesized proteins in live cells without fixing and staining. Wei et al.[9] incorporated the deuterium-labeled essential amino acid (leucine-d10) into nascent proteins on several mammalian cell lines, by which the SRS microscopy provided the spatial maps of the quantitative ratio between new and old proteomes. Moreover, this method could also be used to study de novo protein synthesis during neuronal plasticities.

Furthermore,

homopropargylglycine

(Hpg),

an

alkyne-containing noncanonical amino acid, was used instead of methionine for visualizing the distribution of the newly-synthesized proteins[5]. 3.1.4. Glycan imaging Peracetylated N-(4-pentynoyl) mannosamine (Ac4ManNAl) could be metabolically converted to the corresponding sialic acid and incorporated 28

into the sialylated glycans for visualizing the distribution and activity of glycan. The cells treated with Ac4ManNAl expressed the Raman signal at 2120 cm-1, which reported the cellular distribution of the alkynes that were derived from free ManNAl, metabolic intermediates, and sialylated glycans[5]. 3.1.5. Glucose imaging To visualize glucose uptake activity, Hu et al.[63] designed and synthesized a novel glucose analogue bearing an alkyne vibrational reporter,

named

3-O-propargyl-d-glucose

(3-OPG).

Under

SRS

microscopy, 3-OPG could provide vibrational imaging of glucose uptake activity in living cells and tissues. The results indicated that this method was able to differentiate cancer cells with varied metabolic activities and to reveal heterogeneous glucose uptake patterns in tumor xenograft tissues as well as in neuronal culture and mouse brain tissues. 3.1.6. Imaging of drug uptake process Terbinafine hydrochloride (TH) is an alkyne-bearing antifungal skin drug. The delivery pathway of TH inside mouse ear tissue could be imaged at a depth of about 100 µm by targeting its internal alkyne signal[61]. William et al.[67] demonstrated the simultaneous detection of anisomycin derivative labeled with an alkyne, together with either lipid droplets or EdU. This study confirmed the potential of dual-color SRS microscopy

for

identifying

drug-dependent

changes

in

cellular

composition and cell cycle progression at the single-cell level. In addition to those, there exist a variety of strategies for multicolor chemical imaging of biomolecules in cells, like visualizing the metabolism of glucose, protein, fatty acid, and choline simultaneously. Zhang et al.[62] made use of deuterium-labeled glucose (D7-Glc), deuterium-labeled amino acids (D-AA), deuterium-labeled palmitate acid 29

(d31-PA), and alkyne-labeled choline (propargyl choline) for visualizing the metabolism of glucose, protein, fatty acid, and choline respectively during epithelial-mesenchymal transition (EMT) of the MCF-7 cell (Figure 5B, C). The results showed that the incorporation rates of amino acids, choline, and glucose were all reduced by various levels after EMT, which indicated the need for mesenchymal cells to restrict the biosynthesis of proteins and lipids and to preserve energy for its migration and invasion. Beyond that, Chen et al.[20] developed a three-color vibrational palette of alkyne reporters using a

13

C-based

isotopic editing strategy. The Raman peaks derived from mono-13C (13C ≡ 12C) and bis-13C (13C ≡ 13C) labeled alkyne isotopologues were redshifted and completely resolved from the originally unlabeled (12C≡ 12

C) alkynyl probe (Figure 5D). The three forms of alkynes were

biochemically identical and displayed three mutually resolvable Raman peaks. The simultaneous three-color SRS imaging of DNA, RNA, and lipid metabolism could be obtained by applying three different alkyne-tagged small-molecule metabolic reporters EU-13C≡13C, EdU-13C ≡12C, and 17-ODYA respectively in live mammalian cells (Figure 5E).

30

Figure 5. Direct labeling of biomolecules with background-free moieties for SRS imaging. (A) The metabolic incorporation scheme for a broad spectrum of alkyne-tagged small precursors. a.u., arbitrary units[61]. (B) Spontaneous Raman spectra of MCF-7 cells cultured with metabolic labels. MCF-7 cells cultured in regular medium do not have any Raman peak in 1800 to 2400 cm-1 (gray curve on bottom). Cells cultured in deuterium- or alkyne-labeled metabolites show Raman peaks that are signature of the label (cyan shade). (C) Representative SRS images of MCF-7 cells cultured in mediums with metabolic labels. Left, incorporated metabolites. Middle, cell silent region that is far away from the Raman peak of metabolic labels. Right, SRS images at 1655 cm-1 from amide vibration that represent an intrinsic protein pool. Scale bars, 50 µm[62]. (D) Three-color chemical imaging using isotopically edited alkyne reporters. Left, structures and normalized Raman spectra of RNA probe EU (11), DNA probe EdU (1), and fatty acid probe 17-octadecynoic acid (12). Right, structures and normalized Raman spectra of isotopically edited EU-13C2 (13), EdU-13C (2), and 17-ODYA (12). (E) Three-color SRS imaging of nascent RNA, DNA, and fatty acyl derivatives in live HeLa cells by spectral targeting of different isotopically edited alkyne reporters[20].

31

This covalent labeling method can also be applied to image many other biomolecules of interest by labeling with background-free vibrational reporters for visualizing the metabolism in vivo. Moreover, the simultaneous imaging of various biomolecules in a single cell is very meaningful for disease diagnosis and surveillance.

3.2. Metabolic labeling of biomolecules with background-free moieties Enzymatic

incorporation

of

D2O

derived

deuterium

into

macromolecules generates carbon-deuterium (C-D) bonds that vibrate in the cell-silent spectral window, which allows tracking of biosynthesis and metabolic rate in tissues by SRS in situ (Figure 6A). D2O is better than deuterium-labeled carbon substrate in monitoring and imaging metabolic activities because it can diffuse into cells freely, probe de novo biosynthesis, but do not perturb native metabolism. For example, Shi et al.[68] proved that D2O was a better probe than deuterium-labeled fatty acids (D-FAs) for detecting lipogenesis in C. elegans. Through comparing the CDL signals generated by D2O and D-FA supplementation, the results showed that 20% D2O treatment produced over a two-fold stronger CDL signal than deuterated palmitic acid at its highest concentration[7, 69]. As bacteria were grown on D2O plates, D2O probes produced a variety of D-labeled fatty acids, lipids, and their metabolic intermediate for real-time background-free SRS imaging. D2O probing with stimulated Raman scattering (DO-SRS) microscopy enabled tracking of in vivo protein synthesis with high efficiency but without tissue bias (Figure 6B). The single peak at 2135 cm-1 represented the newly synthesized D-labeled lipids, while the peaks at 2185 cm-1 and 2210 cm-1 represented 32

the D-labeled proteins and C-D DNA, respectively. Thus, DO-SRS microscopy could achieve in situ visualization of de novo lipogenesis and protein synthesis in animals and tracking lipid metabolism in C. elegans. Particularly, this method succeeded in imaging lipids and proteins metabolism simultaneously, which could be used to distinguish C. elegans during germline development, to identify tumor boundaries, and to detect intratumoral heterogeneity (Figure 6C). The metabolic activities of both protein and lipid could be mapped out simultaneously by DO-SRS. Wei et al.[70] developed a volumetric chemical imaging technique that coupled Raman-tailored tissue-clearing with DO-SRS microscopy to study the metabolic basis of angiogenesis in the tumor, and presented intricate 3D organizations of tumor spheroids, mouse brain tissues, and tumor xenografts. With the help of urea, which was identified as clearing recipe for Raman microscopy[71], the depth of the clearing-enhanced SRS was increased by more than ten times compared with the standard SRS (Figure 6D). Collectively, DO-SRS allows visualization of metabolic dynamics of biomolecules, such as proteins, lipids, and nucleic acids, inside tissues and organisms. Thus, background-free SRS is really useful in both basic biological studies and clinical applications, like tracking metabolic activities in cells and target tissues.

33

Figure 6. Metabolic labeling of biomolecules with background-free moieties for SRS imaging. (A) D2O-derived deuterium can be incorporated into C-D bonds of metabolic precursors for the synthesis of macromolecules through irreversible enzymatic incorporation. (B) A variety of protein-rich organs were collected from adult mice that were administrated with 25% D2O or 2 mg mL-1 of D-AAs in drinking water for 8 days and then imaged for CDP and CHP signals. Scale bar = 20 µm. (C) DO-SRS microscopy identifies tumor boundaries and metabolic heterogeneity at different types of channels[68]. (D) Three-dimensional reconstructions and representative 2D images of a tumor spheroid of MCF-10AHras breast cells with tissue clearing. a) Protein (magenta) and lipid (green) channels are overlaid. b) Protein (magenta) and the d-PA (cyan) channels are overlaid. c) Lipid (green) and d-PA (cyan) channels are overlaid. (Scale bars, 50 µm)[70].

34

4. SUMMARY AND OUTLOOK Live-cell Raman imaging, when coupled with background-free labeling strategies, is emerging as a promising imaging modality applicable to bioanalysis. Advances in the synthesis of Raman reporters and technical instrument improvements are expected to make further gains in detection sensitivity. Although background-free SERS and SRS probes have shown high specificity and sensitivity, Raman imaging is still facing several inherent challenges in terms of scanning rate and penetration depth. Thus, Raman imaging can be combined with other imaging tools with complementary characteristics for studying living systems at multi-mode levels. For example, Li et al.[1] reported a dual-mode probe equipped with both aggregation-induced emission (AIE) characteristic and enhanced alkyne Raman signal, as examined by an FL-SRS microscope system. The integration of fluorescence microscopy with SRS microscopy will potentially promote the development of dual-mode probes, thus benefiting subcellular organelle studies under physiological and pathological conditions. Besides, DO-SRS microscopy can also be combined with fluorescent microscopy to track the metabolic activity of specific cell lineages[5]. The bioorthogonal SRS imaging methodology complements the fluorescence and label-free microscopies to offer a new imaging platform. Further development of imaging platforms by coupling background-free Raman strategies with other imaging modalities could provide significant benefits to study metabolic processes in live systems, allowing a better understanding of life processes and the evolution mechanisms of various diseases. Currently, the imaging instruments for both SERS and SRS depend on relatively complex optical systems, which are generally expensive and complicated for use. This formidable issue must be a big obstacle that limits the wide applications of Raman imaging techniques from the 35

laboratory bench to real-world settings. Although low-cost, portable hand-held Raman spectroscopy has been widely used in various point-of-care fields such as cultural relics identification, food inspection, and criminal investigation, etc., it remains technically challenging to apply this simple hand-held system into rapid biological and biomedical imaging[50]. On the one hand, therefore, we should improve the performance of a hand-held Raman scanner; on the other hand, mass production of Raman probes should be realized with high reproducibility and quality. The combination of ease-of-use Raman imaging platform with high-quality Raman probes could revolutionalize the ways of molecular imaging and facilitate the development of molecular medicine.

Acknowledgments This study was supported by grants from the National Natural Science Foundation of China (21775075), the Fundamental Research Funds for Central Universities (China), and the Thousand Youth Talents Plan of China.

36

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The authors declare no conflict of interests.