Applications of Metallic Nanoparticles in Bio-imaging and Molecular Spectroscopy

Introduction

Metallic nanoparticles have been attractive for imaging and spectroscopic analysis of biological samples because of their unique optical properties induced by localized plasmon resonance.¹ Metallic nanoparticles with a variety of different physical parameters such as size, shape, metal composition, and assembly (Figure 1)² can be synthesized for use in a wide range of biological applications. In particular, gold nanoparticles (AuNPs) are widely used as optical contrast agents for bioimaging since they possess several advantages such as strong light scattering properties at visible wavelengths without photobleaching, high chemical stability, and low toxicity for use in biological samples. Researchers have demonstrated high spatiotemporal resolution optical imaging of AuNP-labeled biomolecules and intracellular organelles.^3–5 In addition to their use as a contrast agent, metallic nanoparticles also work as probes for surface-enhanced Raman scattering (SERS) spectroscopy to analyze biological molecules and cells.⁶ With SERS spectroscopy, information of the molecular species near the metal surface can be obtained with high sensitivity and high spatial confinement. This enables analysis of biological functions and phenomena, such as organelle transportation,⁷ drug uptake,⁸ and cell division.⁹ Herein, we review recent progress on metallic nanoparticle applications for bio-imaging and molecular spectroscopy of biological systems.

Tracking Biomolecules at Angstrom Precision and Microsecond Time Resolution

When labeled with AuNPs, the motions of biomolecules and intracellular organelles can be investigated by tracking the bright spot of the AuNP scattering image (Figure 2A). The localization precision of the optical image is inversely proportional to the square root of the photon number.¹⁰ AuNPs provide a high scattering signal without photobleaching, allowing for nanometer-scale localization even at microsecond time resolution,^3,11 and AuNPs enable the analysis of the fast dynamics of biomolecules such as lipids and proteins.^4,12 To understand the working mechanism of these tiny and complex biological molecules in detail, further improvements in localization precision are essential. Recently, researchers reported the development of an annular illumination total internal reflection dark-field microscope to illuminate AuNPs at high laser intensity.¹³ The authors achieved a localization precision of 1.3 Å and 5.4 Å with 40 nm AuNPs at 1 ms and 33 μs time resolution (Figure 2B). This system revealed the fast stepping motions of kinesin-1 in detail, including a transition pathway of the motor domain from bound to unbound state on microtubule measured at a 10 μs time resolution (Figure 2C). Stepping motions of a processive chimeric dynein were also observed with a 30 nm AuNP at 100 μs time resolution, revealing 8 nm forward and backward steps and 5 nm side steps, consistent with the pitch of binding domains on the microtubule.¹⁴ The use of small AuNPs as optical probes is crucial to minimize possible steric hindrances on the target biomolecules. Recently, the development of highly sensitive interferometric scattering microscopy achieved localized precision at a few nanometers with 20 nm AuNPs at microsecond time resolution.4 Detection using smaller diameter AuNPs, such as 10 nm,¹⁵ or even label-free direct detection of proteins,^16,17 has also been performed, expanding the range of measurable biomolecule types and phenomena.

Multicolor Imaging of Biomolecules with Ag, AgAu Alloy, and AuNPs

AuNP-based biomolecular tracking to date has been limited to monochromatic imaging. However, new methods have emerged that enable analysis of multiple biomolecules at high spatiotemporal resolution using additional color channels.

A multicolor high-speed single-particle tracking system was recently developed using silver and silver-gold alloy nanoparticles (AgNPs and AgAuNPs) together with AuNPs (Figure 3A).¹⁸ The peak wavelength of AgNPs in the extinction spectra is more than 100 nm shorter than that of AuNPs, and the AgAuNPs spectra are located between the AgNP and AuNP spectra and can be tuned depending on their composition ratio (Figure 3B).^19,20 To simultaneously observe each metal NP, a total internal reflection multicolor dark-field microscope was constructed with multiple lasers at 404 nm, 473 nm, and 561 nm that match the plasmon resonance wavelength of the AgNPs, AgAuNPs, and AuNPs, respectively. A spectrophotometer was used in the imaging optics to project scattering images at each wavelength on the different portions of a two-dimensional high-speed CMOS camera. With this system, multicolor imaging of phospholipid diffusional motions in a supported membrane (Figure 3C–E) and stepping motions of kinesin-1 along microtubules was achieved at 100 μs time resolution and nanometer-scale localization precision. This system also allows for the capture of the transient dimer formation of the two metal NPs, and a distance-dependent intensity increase in the 649 nm channel (Figure 3F). When two metal NPs are in sufficiently close proximity, plasmon coupling and a resulting redshift of the plasmon resonance wavelength are induced. The distance-dependent spectral shift has been used to create a highly accurate nano-ruler, known as a plasmon ruler, using two AuNPs or AgNPs and millisecond time resolution.¹⁹ Multicolor imaging system extends the scope of the plasmon ruler with microsecond time resolution and multicolor imaging capabilities.

Cell Imaging Using SERS: Drug Uptake and pH Sensing

Metallic nanoparticles are also widely used as SERS probes to amplify the Raman scattering signal from molecules near the metal surface, enabling highly sensitive Raman measurements of biomolecules, such as DNA, RNA, amino acids, proteins, and lipids.^6,21,22 Metallic nanoparticles introduced into cells work as SERS probes that report the molecular and chemical environment in cells near the metal surface.²³ Endocytosis is often used as a method of delivering metallic nanoparticles into cells. Culturing cells in a medium containing metallic nanoparticles allows the uptake by the cells; the nanoparticles eventually accumulate in endosomes and lysosomes through the intracellular transportation function.²³ The uptake of AuNPs into macrophage cells has been observed by slit-scanning Raman microscopy.^24,25 Time-resolved two-dimensional SERS images, captured at 2.5 minutes per frame, revealed the attachment of AuNPs on the cellular outer membranes and the entry into intracellular space. The analysis of SERS spectra obtained during time-lapse observations showed temporal fluctuation of the SERS signal and dependence of SERS spectra on the position of the AuNPs, indicating that the AuNPs report the variation of chemical environments during transportation.

The detection of extrinsic molecules such as drugs, introduced into cells is an important application of SERS. Indeed, SERS detection of anti-tumor and anti-leukemia drugs in cells has revealed much about drug distribution and metabolism in cells.^8,26,27 One of the difficulties of SERS-based drug detection has been its molecular specificity. SERS spectra from cells contain strong background signals from intrinsic molecules, and they often hinder the specific detection of target molecules. We have recently developed a SERS technique for drug imaging with high molecular specificity using an alkyne tag composed of a carbon-carbon triple bond.²⁸ Since the alkyne shows characteristic peaks in the Raman silent region of biomolecules from 1800 to 2600 cm^-1, alkyne-tagged molecules can be selectively detected without overlapping with intrinsic biomolecules.²⁹ Furthermore, the small chemical structure of the alkyne tag allows the drug molecule to essentially maintain its original properties, such as affinity to the drug target. Also, the high affinity of alkyne to metallic nanoparticles improves SERS detection sensitivity of both electromagnetic and chemical enhancement effects.^30–32 As a proof of concept, we used an alkyne-tagged inhibitor for cathepsin B, a drug target of tumor metastasis and localized in lysosomes. AuNPs were introduced into lysosomes in macrophage cells via endocytosis, and the alkyne-tagged inhibitor was subsequently administrated to the cell culture media at a concentration of 20 µM. When we performed time-lapse 3D SERS imaging of drug-treated cells with slit-scanning Raman microscopy, a gradual increase of the intensity of alkyne-SERS peak at 1980 cm^-1 was observed at around 10 minutes after drug administration (Figure 4). The uptake speed of the inhibitor was quantitatively evaluated by counting the number of alkyne-SERS signals. Alkyne-tag SERS microscopy opens new avenues for quantitative analysis of drug dynamics in living cells.

SERS with metallic nanoparticles also works as a sensing method for intracellular environments, such as pH^33–35 and redox potential.³⁶ For this purpose, the nanoparticle surface is functionalized with molecules that show a structural change depending on the surrounding environment. In particular, SERS-based pH sensing of cells has been widely investigated,^33–35 since pH value is related to various biological processes such as proliferation, apoptosis, and ion transport.³⁷ Improvement of SERS sensitivity results in fast pH measurement, providing a better understanding of the biological processes governed by dynamic pH changes. To identify the most sensitive metal nanoparticles for pH sensing, we synthesized 18 types of AuNPs and AgNPs (Figure 1), with different shapes, sizes, metal compositions, and assemblies. By evaluating the SERS activity, it was confirmed that Ag chain, Ag core/satellites, Ag@Au core/ satellites, and Au core/satellites produced the strongest SERS signal, and that this signal is sufficient to perform pH sensing at the single-particle level. With Ag@Au core/satellites as SERS probe, changes in intracellular pH over time during the apoptotic process of HeLa cells were successfully revealed at a temporal resolution of 5 minutes (Figure 5).

Dynamic SERS tracking in a Living Cell

In recent years, particle tracking and SERS spectroscopy have been combined to perform dynamic SERS imaging inside biological cells.^7,25 Since endocytosed AuNPs travel around the intracellular space due to a cellular transportation function, intracellular molecules near the nanoparticle surface can be detected with a high spatiotemporal resolution by tracking the motion of AuNPs and performing SERS spectroscopy at the same time. This allows us to investigate the mechanisms of various biomolecules involved in cellular transportation. For this purpose, a laser-scanning Raman microscope and a dark-field microscope were combined to acquire SERS signal from a specific nanoparticle in a cell continuously.^7,25 Information on particle positions is obtained by dark-field microscopy and used to continuously relocate the focus position of Raman excitation laser on the same nanoparticle. The temporal resolution of this SERS tracking system was 50 ms per spectrum. SERS measurements were performed on macrophage cells with 50 nm AuNPs. During SERS measurement, motion-dependent changes in the SERS spectra were observed, indicating that the AuNPs report local molecular environment during transportation. Furthermore, using a dual-focus dark-field imaging system, the 3D SERS tracking technique has been developed, with which AuNPs that move three-dimensionally can be followed.³⁸ Recently, the 3D SERS tracking system was applied to local pH measurement inside cells (Figure 6A).³⁹ The nanoparticles, functionalized with pH-sensitive molecules, are thought to be transported to endosomes and lysosomes by intracellular trafficking after being taken up into cells by endocytosis. In general, during these trafficking pathways, the pH is gradually reduced from pH 6.3 in early endosomes to pH 5.5 in lysosomes.⁴⁰ Multi-modal imaging with SERS tracking and fluorescence imaging using lysosome-specific dye revealed that the pH value quantified by SERS analysis was low when the nanoparticles exist in lysosomes (Figure 6B and C).

Conclusion

In this manuscript, we discussed the recent progress on the applications of metallic nanoparticles for imaging and spectroscopy of biological systems. For bio-imaging, multicolor, high-speed, and high-precision tracking of target molecules was demonstrated, and dynamic motions of biomolecules were investigated. For spectroscopic analysis with metal nanoparticles, SERS microscopy applications for drug uptake and pH sensing were summarized. Finally, we introduced an approach to combine particle tracking and SERS spectroscopy to analyze the intracellular molecular environment during transportation at high spatiotemporal resolution. These new metallic nanoparticle-based approaches for bio-imaging and molecular spectroscopy will further contribute to a wide range of research fields such as biology, drug development, and medical science in the future.

Acknowledgments

This work was partially supported by the grant of AMED-CREST under Grant JP18gm071000 and the Grants-in-Aid for Scientific Research (grant numbers JP18H01904 to JA) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.