Multimodal techniques based on synchrotron radiation X-rays for revealing interactions of metallic nanoparticles with biological matrices

Abstract

With the increasing applications of metallic nanoparticles (MNPs) in biomedicine and daily consumer products, it is crucial to explore the interactions of MNPs with biological matrices (MNPs-bio), determining the medical efficacy and biosafety. The dynamic alterations of MNPs and biological systems, as well as the complexity of the biological components, pose great challenges in clarifying the underlying details behind MNPs-bio interactions. The synchrotron radiation X-ray, as an advanced light source, has been emerging as a powerful and versatile tool to reveal the complicated MNPs-bio interaction with the advantages of label-free, in situ, strong penetration capability, quantitative analysis, high sensitivity and high resolution. It is increasingly critical to further develop multimodal techniques based on synchrotron radiation X-rays since the dynamic and complex MNPs-bio interactions generally involve alterations of composition, chemical states, structure, morphology and functions. Multimodal techniques based on synchrotron radiation X-rays have been established by integrating different synchrotron radiation X-rays techniques or combining the synchrotron radiation X-rays technique with other state-of-the-art analytical techniques, realizing a comprehensive all-aspect analysis of MNPs-bio interactions. Here, we summarize recent progress in multimodal synchrotron radiation X-ray techniques for the cross-scale analysis of MNPs-bio interactions. The perspectives on potential improvements of multimodal synchrotron radiation X-ray techniques in the future are also highlighted in the review.

1. Introduction

Metallic nanoparticles (MNPs) have received tremendous attention due to their unique and tunable features, including excellent optical, magnetic and electronic properties. MNPs open a myriad of pathways in the field of nanotechnology and have been applied in biomedicine, drug delivery, imaging, biosensor, food, agriculture, cosmetics, catalysis and energy, etc. [[1], [2], [3], [4], [5]]. The metallic nanodrugs for disease diagnosis and therapy have been developed based on metallic elements, including Fe, Al, Zn, Cu, Au, Ag, Gd, Pt and Hf [6,7]. Iron oxide-based MNPs have been approved for clinical use as magnetic resonance imaging agents (such as ferumoxytol), while hafnium oxide MNPs (NBTXR3) obtained the EU marketing approval as a first-in-class radioenhancer for tumor radiotherapy [8]. MNPs based on Al, Mn, Zn, Fe, Ca and Pt have been demonstrated as effective immunology regulators by acting as vaccine adjuvants, immunotherapy sensitizers, immune supplements and reprogramming immune cells, etc. [[9], [10], [11], [12]]. Nanobiotechnology has been leveraged to realize targeted delivery, effective and safe metalloimmunotherapies. Some metal or metal oxide NPs, including Au, Ag, TiO₂, ZnO and Al₂O_3, are widely used in daily care products for skincare or antibacterial purposes [13,14]. Besides, MNPs based on Ag, Au, ZnO, CuO and TiO₂ are helpful to improve the quality of food and extend the product shelf life by preventing microbial contamination and increasing mechanical strength of food packaging and water repellency [15].

Whether used as biomedicines or consumer products, MNPs will inevitably encounter and interact with the biological system to exert their therapeutic functions or cause biological outcomes [[16], [17], [18], [19]]. The absorption, distribution, metabolism and excretion (ADME) process related to medical efficacy and nanosafety involves the complicated biological interactions of MNPs in the organism. Understanding the fundamental interaction mechanisms, including the temporospatial relationships between biological matrices and MNPs, as well as impacts of biological behavior of MNPs on the efficacy and toxicity, assists us in optimizing the design of MNPs nanomedicines and promoting the safe application of MNPs in the life cycle of consumer products. However, the intrinsic complexity and dynamic features of biological matrices, as well as the measurement with low signal-to-noise ratio resulting from the low dosage of MNPs, pose great challenges for the precise and in situ analysis of MNPs-bio interactions. Current bioanalytical techniques, such as electron microscopy (EM), fluorescence microscopy (FM) and radionuclide-based imaging, play an irreplaceable and imperative role in exploring the MNPs-bio interactions and the ADME process of MNPs. EM can acquire the localization of the nanoparticles and structures of cells or tissues with a high spatial resolution of angstroms, and can also discriminate elemental composition with energy-dispersive X-ray spectroscopy (EDS). The weak penetration capability of electrons limits the measurement of bulk or thick specimens; only the ultrathin biological sections can be investigated. With the new development of volume EM technique, three-dimensional (3D) images can be obtained [20]. However, it’s hard to observe the label-free structures, since staining with heavy metals is necessary to enhance image contrast. FM, including super-resolution microscopy and intravital fluorescence microscopy, is a powerful tool to provide temporal, spatial and functional information, extensively applied in the life science field. This technique depends on fluorescence signal and is very suitable for visualizing biological behavior, including the translocation, distribution and clearance of the fluorescent nanoparticles, e.g., quantum dots (QDs) [21]. Fluorophore labeling for the non-fluorescent nanoparticles is necessary, which may change the physicochemical properties of nanoparticles and could cause the release of the fluorophore. Similarly, radionuclide-based imaging is dependent on radionuclide labeling and requires radioactive tracers to visualize nanoparticles. It can analyze the ADME process of nanoparticles but cannot provide the structural and chemical information.

As complementary analytical methods, techniques based on X-rays, especially synchrotron radiation X-rays, can provide plentiful information on structure, morphology, spatial distribution, chemical state and elemental composition. Various X-ray microscopy and spectroscopy analytical techniques, including X-ray computed tomography (CT), X-ray fluorescence (XRF) imaging, coherent diffraction imaging (CDI), X-ray absorption fine structure spectroscopy (XAFS) and Fourier transform infrared (FTIR) micro-spectroscopy, have been developed [[32], [33], [34], [35]]. These techniques are powerful tools to explore the biological behavior and effects of MNPs at molecular, cellular and animal levels due to the high absorption contrast, phase contrast and element-specific absorption/fluorescence signal, possessing advantages of label free, in situ, high resolution, high sensitivity, quantitative analysis, and simple sample preparation (Table 1). Furthermore, multimodal analysis strategies integrating different synchrotron radiation X-ray-based techniques realize the comprehensive all-aspect analysis of interactions between MNPs and biological matrices by providing information on the spatial distribution and biochemical transformation of MNPs, as well as the structural and functional alterations of the biological system (Fig. 1). The simultaneous, sensitive, label-free and in situ measurement of MNPs and biological matrices guarantees the precision of data interpretation and the revealing of deeper underlying relationships.

Table 1.

Comparison between multimodal synchrotron radiation technology and other bio-imaging technology applied in the study of MNPs-bio interactions.

Technique	Spatial resolution	Penetration depth	Labeling
Electron microscopy (EM) [22]	TEM: 0.1–0.2 nm SEM: 0.5–1 nm	TEM: <200 nm SEM(SE): 1–10 nm	Heavy metal staining
Fluorescence microscopy (FM) [23,24]	CLSM: 200–250 nm STORM: 5–20 nm	CLSM: 100–400 µm STORM: 50–100 µm	Fluorophore
Radionuclide-based imaging [25]	SPECT: 1–2 mm PET: 1–2 mm	SPECT: Moderate (Depends on nuclide energy); PET: Deep	Radionuclide
X-ray nano-computed tomography (Nano-CT) [26]	15–50 nm	1–50 µm	No
X-ray fluorescence (XRF) imaging [27,28]	0.1–10 µm	10–100 µm (Depends on X-ray energy)	No
Coherent diffraction imaging (CDI) [29]	Atomic-level	Atom-centimetre	No
X-ray absorption fine structure spectroscopy (XAFS) [30]	µm - mm (Depends on beam focusing)	µm - mm (Depends on X-ray energy)	No
Fourier transform infrared (FTIR) micro-spectroscopy [31]	∼10 µm	2–20 µm	No

Note: SPECT, single photon emission computed tomography; PET, positron emission computed tomography; CLSM, confocal laser scanning microscopy; STORM, stochastic optical reconstruction microscopy.

Fig. 1.

Multimodal techniques based on synchrotron radiation facilities for comprehensive analysis of cross-scale MNPs-bio interactions. MS, mass spectrometry. Figures from CT to EM clockwise are adapted with permission from references [94,50,91,87,50,83,94,100].

Herein, different from the synchrotron radiation techniques reported in previously published review papers [[34], [35], [36]], we emphasized the integration of multiple synchrotron radiation X-rays techniques or a combination of the synchrotron radiation X-rays technique with other state-of-the-art analytical techniques, such as X-ray CT imaging, X-ray fluorescence microscopy (XFM), X-ray ptychography, XAFS, FTIR, fluorescence microscopy, EM, and mass spectrometry. We summarize the recent progress in multimodal synchrotron radiation X-ray techniques for the cross-scale analysis of MNPs in biological matrices. These multimodal techniques can be divided into two main categories: integration of multiple synchrotron radiation X-ray techniques and combination of synchrotron radiation X-ray techniques with other state-of-the-art analytical methods. The basic principles, the research status and outlook of the related techniques are included in this review. Following the introduction part, the development and basic theory of several selected synchrotron radiation X-ray techniques are described in the second part. Subsequently, typical application examples of multimodal synchrotron radiation X-ray techniques are also provided to assist researchers in understanding and making better use of the approaches in their studies. The perspectives on potential improvements of multimodal synchrotron radiation X-ray techniques in the future are finally highlighted.

2. Basic theory of synchrotron radiation X-ray analytical techniques

Synchrotron radiation X-rays are electromagnetic radiations emitted in the motion direction of charged particles when they move at speeds close to the speed of light and deflect their trajectories in magnetic fields. Compared to the common X-rays, synchrotron radiation X-rays possess numerous merits of high brightness, coherence, collimation, strong penetration capability and broad energy range. Since synchrotron radiation X-rays were observed in 1947, the development of the synchrotron light source has witnessed four generations all over the world. Synchrotron radiation X-rays, as one of the advanced light sources, act as powerful and ubiquitous research tools in almost all branches of science, such as life science, materials science, chemistry, physics, archaeology, and geology [[37], [38], [39], [40]].

Synchrotron radiation X-rays interact with matter and lead to phenomena of absorption, fluorescence, scattering, diffraction, refraction, and reflection. These distinct signals of metal elements with X-rays benefit the label-free and in situ exploration of interactions between MNPs and biological matrices. Information on spatial distribution, chemical environment, morphology and internal structure can be obtained by detecting the signal. Based on these basics, diverse synchrotron radiation X-ray techniques have been developed to meet the demands of scientists in a wide range of fields. In general, synchrotron radiation X-rays-based technologies can be divided into microscopy imaging and spectroscopy measurements, such as X-ray tomographic imaging based on absorption or phase contrast, X-ray fluorescence imaging, ptychographic imaging based on diffraction signal, XAFS and FTIR spectroscopy [41]. Microscopy imaging, including TXM, STXM, XFM and ptychography, possessing the features of label-free, in situ and high-resolution, can provide information on spatial distribution and elemental analysis of MNPs, as well as the structural change and elemental composition of biological matrices. Spectroscopic techniques, such as XAFS, FTIR and SAXS, can realize the in situ characterization of chemical states, composition and structure of MNPs and endogenous biomolecules. Details of several selected techniques are described in the following part (Table 2).

Table 2.

List of current synchrotron radiation (SR)-based techniques commonly used to study the interactions of MNPs with biological matrices.

Techniques	Signal detected	Resolution	2D/3D	MNPs’ information	Biological response
TXM-HXT	Absorption/Phase	30 nm	3D	Spatial distribution	Structural change
TXM-SXT	Absorption/Phase	30 nm	3D
STXM	Absorption	30 nm	3D	Spatial distribution/ Elemental mapping
XFM	Element characteristic X-ray emission	>100 nm	3D	Elemental mapping and quantification	Elemental composition
Ptychography	Diffraction	Atomic-level	3D	Intracellular localization	Structural change
XAFS	Absorption and fluorescence	0.1 eV (XANES) 3–10 eV (EXAFS)	2D	Chemical states and local coordination	Coordination environment and active site of biomolecules
FTIR	Excitation of molecular vibrations by X-rays	Several microns	3D	Compositions and structures of surface ligands on MNPs	Compositions and structures of endogenous biomolecules
SAXS	Scattering	100 nm	3D	Size distribution, shape, morphology and the assembly of MNPs	Structural change

Note: TXM-HXT, full-field transmission X-ray microscopy-hard X-ray tomography; TXM-SXT, full-field transmission X-ray microscopy-soft X-ray tomography; STXM, scanning transmission X-ray microscopy; XFM, X-ray fluorescence microscopy; XANES, X-ray absorption near-edge structure; EXAFS, extended X-ray absorption fine structure; FTIR, Fourier transform infrared micro-spectroscopy; SAXS, small-angle X-ray scattering.

2.1. Synchrotron radiation X-ray imaging techniques

2.1.1. X-ray tomographic imaging

Absorption-contrast and phase-contrast tomography are two representative imaging modes of X-ray tomographic imaging, which is also named X-ray CT. Absorption-contrast CT images are acquired by collecting several transmission radiographic projections at different angles of a specimen with components of distinct X-ray attenuation coefficients. In comparison, phase-contrast CT imaging is superior for the sample with weak absorption contrast (e.g., soft matter/tissue) because the imaging mode uses the effects of X-ray diffraction, interference, or refraction, rather than absorption phenomena [41]. With X-ray CT imaging, a nondestructive technique, internal structures of samples can be investigated with a resolution of microns or nanometers, which are micro-CT and nano-CT, respectively (Fig. 2a). High resolution of X-ray CT imaging can be achieved down to nanometers by TXM and STXM. TXM uses a focused X-ray beam created by condenser optics to irradiate a sample, then refocused onto a detector with an objective zone plate lens [42]. In contrast, STXM employs an X-ray beam focused on a zone plate to scan a sample point by point [43]. The spatial resolution of TXM and STXM is determined by the outermost zone width of the zone plate and the focused size of the X-ray, respectively, which can achieve down to ∼20 nm. The CT images using the TXM technique are acquired by collecting the absorption or phase signal of metals and biological specimens, providing inner architecture and morphology information. 3D CT imaging with the STXM technique also can investigate elemental and chemical distribution by detecting absorption, diffraction or fluorescence signals [34]. 3D CT imaging with TXM and STXM can be achieved with both soft and hard X-rays. Among them, cryo-soft X-ray tomography (cryo-SXT) can reveal the 3D spatial distribution of MNPs and their effects on cellular ultrastructure [44,45]. With the advancement of X-ray CT imaging, especially nano-CT, the interaction of MNPs with subcellular architectures can be investigated label-free and in situ [35]. An advanced hierarchical phase-contrast tomography (HiP-CT) based on hard X-rays of the European Synchrotron Radiation Facility (ESRF) has been developed to realize the imaging of intact human organs from organ to cellular scale in three dimensions by hierarchically scanning and zooming locally in the interested areas (Fig. 2b) [46,47]. Additionally, artificial intelligence plays a vital role in assisting synchrotron radiation X-ray CT imaging. Deep learning has been combined with synchrotron radiation hard X-ray nano-CT to visualize the self-assembly of iron oxide nanoparticles in intact single cells (Fig. 2c) [48].

Fig. 2.

Synchrotron radiation X-ray tomographic imaging. (a) Nano-CT and micro-CT imaging are used for MNPs-bio interactions with the in situ, label-free, noninvasive and highly sensitive advantages. Images (1)-(7) are adapted with permission from references [47,[49], [50], [51], [52], [53], [54], [55]]. (b) Hierarchical phase-contrast tomography (HiP-CT) developed for intact human organ imaging from organ to cellular scale in three dimensions. Adapted with permission from ref. [47]. (c) Left: workflow for deep learning-assisted automatic image segmentation and quantitative analysis; right: organelles segmentation. Adapted with permission from ref. [48].

2.1.2. XRF imaging

XRF is the emission of characteristic X-rays of elements after irradiation with primary X-rays (Fig. 3a). XFM, based on the XRF signal, is a powerful technique to determine the chemical composition and elemental distribution of objects by employing the fingerprint radiation of the elements. Multiple elements can be investigated simultaneously and sensitively, which is beneficial for in situ and quantitative study of MNPs and the biological elements in biological matrices (Fig. 3b). The multielement Z-tag X-ray fluorescence (MEZ-XRF) technology, combined with SABER Z-tag metal amplification, is adapted for metals for 13 markers to achieve either ultrafast imaging speeds or detection of low-abundance markers. This will enable new avenues for multiomic tissue analyses in health and disease [49]. The spot size of the X-ray is tuned for the imaging of samples with different volumes (e.g., cells and tissues). For example, the spatial distributions of several MNPs (MoS₂, TiO₂, Au@Gd, Cu, Co, etc.) in C. elegans, plants, different mammalian cells and tissues have been successfully investigated with XFM, providing new insights into the ADME process of MNPs (Fig. 3c-d) [50,[56], [57], [58], [59]]. Since the biological processes are closely related to the inherent elements in living organisms (e.g., Ca, K, Mg, P), the impacts of MNPs on the biological structures and functions could be analyzed by detecting other endogenous biological elements simultaneously. Additionally, by combining XFM with in situ XAFS, we can analyze the chemical states of the interested element at specific positions of the biological specimen. Moreover, 3D XFM for spatial distribution of different elements or chemical species could be obtained by rotating samples and collecting the XRF signal at each rotated angle.

Fig. 3.

Synchrotron radiation X-ray fluorescence microscopy. (a) Basic theory of XRF. (b) Schematic illustration of synchrotron XFM. Adapted with permission from ref. [34]. (c) Distribution of Au and Gd in tumors to demonstrate the enhanced permeation of Au@Gd nanocomposites into tumors by laser irradiation. Adapted with permission from ref. [58]. (d) Spatial sub-organ localization of MoS₂ NPs in the splenic red pulp. Adapted with permission from ref. [50].

2.1.3. X-ray ptychography

In the fourth generation of synchrotron radiation sources, the significantly improved coherence of the light source is very beneficial to promoting the development of CDI technology [60]. Unlike the TXM and STXM, CDI uses coherent light to illuminate the sample and collects the far-field scattering signal with a detector. The diffraction signal produced by the interaction between the X-ray and the sample carries information related to the electron density and lattice structure. CDI can reconstruct the electron density and structure information according to the corresponding relationship between the scattered signal and the Fourier transform, combined with iterative phase recovery algorithm and solving the inverse problem [61] (Fig. 4a). The image resolution does not depend on the size of the focused spot, thus overcoming the resolution limitation of X-ray optical components. The actual resolution is ultimately determined by the coherent X-ray flux, wavelength and sample stability, and atomic-level resolution can be achieved.

Fig. 4.

Basic principle of X-ray ptychography and applications in imaging cell and tissue structures with ultrahigh resolution. (a) Schematic illustration of CDI. (b) Schematic illustration of X-ray ptychography based on overlap correlation. (c) Ptychography image and 3D volume rendering [63]. Violet: Au@citrate NPs; Pink: different types of lysosomes; Green: vacuoles. (d) Alterations in sub-axonal architecture of Parkinson’s diseased human brains using label-free cryo-PXCT [64]. 2D orthoslices and 3D volume rendering of a Parkinson’s diseased human brain. Aqua: myelinated axons; Purple: swellings along the axons; Orange: nuclei of non-neuromelanin-containing cells; Pink: neuromelanin-containing organelles; Red: blood vessel; Dark red: blood cell.

Modern X-ray ptychography can be considered a marriage of CDI and STXM. As shown in Fig. 4b, a series of coherent diffraction patterns are collected by scanning a sample with the focused X-ray beam. There are overlapping regions in point-by-point scanning, and the additional redundancy caused by this oversampling is beneficial for image reconstruction [62]. Compared with STXM technology, ptychography has higher spatial resolution and a larger field of view. In addition, based on two-dimensional oversampling scanning and sample rotation, X-ray ptychography and CT technology can be fused to achieve 3D imaging, i.e., ptychographic X-ray computed tomography (PXCT).

Jiang et al. demonstrated high-resolution and large-field imaging of intact and unstained cancer cells using X-ray ptychography and equally sloped tomography (EST). They investigated the transport and distribution of Au@citrate NPs in cancer cells [63] (Fig. 4c). Single intracellular Au@citrate NPs were observed, and the amount of Au@citrate NPs in aggregations could be accurately quantified. In addition, the morphological changes of lysosomes containing Au@citrate NPs could be observed in high contrast mass density images. The application of PXCT to explore the MNPs fate in intact cancer cells at nanoscale resolution may greatly benefit interdisciplinary research in materials science, nanomedicine and nanotoxicology.

Cryo-PXCT in the several keV energy range is an emerging technology providing phase contrast with high sensitivity, allowing label-free and non-destructive 3D imaging of large continuous tissue volumes, currently spanning up to 4 × 10⁵ µm³ [64]. Tran et al. used cryo-PXCT to image unstained, chemically fixed, frozen-hydrated human brain tissue and display myelinated axons and subcellular features [64] (Fig. 4d). Cryo-PXCT sets an unprecedented foundation for studies addressing axonal integrity and disease-related changes in unstained brain tissues.

2.2. Synchrotron radiation X-ray spectroscopic techniques

X-ray spectroscopic techniques can be used to study the local chemical structures of elements by measuring signal spectra generated from the interactions between electromagnetic radiation and matters, such as absorption, scattering, diffraction and fluorescence (Fig. 5a). XAFS, also known as X-ray absorption spectroscopy (XAS), is the absorption spectra generated when the incident X-rays eject the core-level electrons (K, L, M shell) to the continuum (Fig. 5b). XAFS reflects the energy-dependent oscillations resulting from neighboring atoms at energies near and above the absorption edge, comprising XANES and EXAFS. XAFS has the advantages of element-specificity and high sensitivity (limit of detection down to ppm level), determining the chemical states and local coordination of elements. MNPs and biological matrices undergo dynamic, complex and diverse changes after the interaction of MNPs with the surrounding microenvironment. Various biomolecules, such as proteins, oxidants and reductants, are the main factors involved in transforming MNPs [19]. The stability, dissolution, degradation and enzymation of nanomaterials resulting from active biomolecules in biofluid have been demonstrated with XAFS. Furthermore, in situ XAFS can be realized by combining synchrotron radiation X-ray imaging and XAFS. For example, XAFS integrated with XFM or STXM was employed to investigate the in situ biodistribution and degradation of Se-based nanomaterials (SeNPs and CdSe@ZnS QDs) [66,67] and silver nanoparticles [68], suggesting the decomposition of nanostructures in the sub-tissue structures. STXM together with XANES was used to study the heterogeneous chemical forms in a single haze particulate, indicating the ferrous and ferric distribution in single particles and providing strong evidence for catalytic activity of these particles [69].

Fig. 5.

Introduction of synchrotron radiation X-ray spectroscopic techniques. Schematic illustration of (a) interactions between electromagnetic radiation and matter and (b) X-ray absorption spectroscopy. (c) Experimental setup for XRR and GIXOS. Figures b and c are adapted with permission from ref. [34,65].

FTIR spectroscopy determines the structure of molecular chemistry based on the excitation of molecular vibrations by X-rays, and is a common analytical method for organic molecules. Synchrotron radiation X-ray source endows conventional FTIR with many advantages: high signal-to-noise ratio, fast measurement, broad wavelength range (from near IR to far IR). FTIR imaging can also be performed by collecting the spectra of each point of the specimen, giving a two-dimensional or three-dimensional mapping of the chemical composition with a spatial resolution down to several microns. FTIR spectroscopy has been applied to investigate the compositions and structures of surface ligands on MNPs and endogenous molecules, such as nucleic acids, lipids and proteins, at the cell or tissue levels. Due to the advantages of non-destructive methods, the multimodal measurements can be performed on the same sample and the same area of interest. The interaction of MNPs with biological matrices can be realized by combining FTIR with other X-ray techniques, such as CT, XFM and XAFS, to obtain the elemental and biomolecular distribution, as well as the morphology and structures of specimens.

X-ray scattering techniques, such as SAXS and X-ray liquid surface scattering, have been utilized to characterize the structure of NPs and the interactions of NPs with bio-interfaces. SAXS can provide detailed information on the size distribution, shape, morphology and assembly of NPs in liquid suspensions, as well as the structures of cell samples [70,71]. The characterization of NPs with X-ray scattering techniques in cells and tissues remains challenging. However, integration of SAXS with other X-ray techniques (e.g., XFM, CT imaging) benefits the analysis of NPs in vitro and in vivo [72]. Synchrotron radiation X-ray liquid surface scattering techniques, including X-ray reflectivity (XRR), grazing incidence X-ray off-specular scattering (GIXOS) and grazing incidence X-ray diffraction (GIXRD), are key tools to study the interfacial interactions of nanoparticles with amphiphilic molecules (e.g., bio-membranes) at air-water interfaces (Fig. 5c). Electron density profile along the direction perpendicular to the interface is detected to obtain the interfacial structure information, including the thickness, the molecular arrangement and the structure. For example, XRR and GIXOS were applied to characterize the interface structure between biological membranes and gold nanorods (AuNRs) with different surface modifications [65]. The amount and the orientation of AuNRs and the thickness of the membrane were measured by detecting the electron density profile of the phospholipid monolayer, revealing the molecular mechanism behind the impacts of surface ligands on nanoparticles on the membrane integrity.

3. Applications of multimodal synchrotron radiation X-ray analytical techniques in the study of MNPs-bio interactions

The dynamic interactions of MNPs with biological matrices generally involve alterations of composition, chemical states, structure, morphology and functions, which are always interrelated. The researchers pay much attention to the heterogeneous chemical and physical properties, e.g., valence state, coordination bonds, electron density, crystallinity and chemical composition, beyond the structure and morphology, motivating the employment of multimodal synchrotron radiation X-ray analytical techniques. Synchrotron radiation X-ray imaging and spectroscopic techniques have been integrated to provide comprehensive and complementary data in the study of MNPs-bio interactions. For example, XFM combined with XANES is a powerful way to analyze the elemental composition of MNPs and biological specimens, as well as the chemical states of MNPs. Multimodal imaging with XFM and CT is two complementary modalities to investigate 3D spatial distributions of elements and the morphology of biological specimens with native absorption contrast with hard X-rays and soft X-rays, respectively. The elemental mapping of MNPs and the structures of biological samples can be obtained by combining XFM and ptychography. Moreover, synchrotron radiation X-ray techniques have also been combined with other state-of-the-art analytical techniques (e.g., FM, EM and MS) to provide complementary information on biological functions, composition, chemical states, structure and morphology (Table 3). The selection of the modalities is highly determined by specific scientific research. Here we discuss the related scientific cases to make researchers understand the selection principles of multimodal synchrotron radiation X-ray analytical techniques.

Table 3.

Information on nano-biological interactions provided by multimodal synchrotron radiation X-ray techniques.

Multimodal techniques	MNPs’ information	Biological information	MNPs examples	References
XFM & XAFS	Spatial distribution and chemical transformation of MNPs	Endogenous element distribution and valence changes	CdSe@ZnS QDs; MoS2 NPs; ZnO NPs	[50,67,[73], [74], [75], [76], [77], [78]]
Cryo-SXT & XAFS		3D structural change	Pt(IV) prodrug; Ca-based NPs	[79,80]
CT & XFM	The spatial and elemental distribution of MNPs	3D structural change; Endogenous element distribution and valence changes	Iridium half-sandwich complex; Uranium	[81,82]
Ptychography & XFM		3D ultrastructural change; Endogenous element distribution and valence changes	Iron oxide NPs	[[83], [84], [85], [86]]
FTIR & XFM		Quantify the alterations of endogenous biomolecules	Au-PEG NPs TiO2 NPs	[87,88]
LC-MS & XFM & MSI	Identification and quantification of biomolecules adsorbed by MNPs	Spatial localization of biomolecules in biological tissues at cellular and subcellular scales	Ag NPs	[50,91]
CT & FM	Intracellular localization of MNPs and fluorescently labeled molecules	3D structural change; Cellular functions	Iron oxide NPs; Mn-based NPs	[[92], [93], [94],51]
CT & EM	Subcellular localization	3D ultrastructural change in large intact specimens	Fluorescent nanodiamonds	[99,100]

3.1. Observing spatial distribution and chemical states of MNPs in biological matrices

Based on the absorption, fluorescence and phase signal, the spatial distribution of MNPs in cells or organs can be observed. Synchrotron radiation X-ray CT imaging and XFM are useful to visualize the spatial localization of MNPs. When combined with XAFS, they can realize the analysis of the distribution and chemical species of MNPs in cells or organs. Synchrotron XFM is a powerful and versatile imaging method for elemental mapping with high sensitivity and resolution, but without the ability to distinguish different chemical forms of one element. XAFS is a sensitive fingerprint that can non-destructively determine chemical species of elements in complex biological samples [32]. When combining XFM with XAFS, we can obtain the composition, chemical states and transformed forms of the MNPs interacting with biological matrices (biofluids, cells, tissues), even the information on in situ spatial distribution of different chemical forms. For instance, the biodistribution and biotransformation of MoS₂, Ag, ZnO, CeO₂ nanomaterials, Fe-based magnetic nanoparticles and Cd-based quantum dots (QDs) in cells, Caenorhabditis elegans or biological tissues have been determined by the multimodal analysis with XFM and XANES. At the cellular level, subcellular distribution and transformation of AgNPs in hepatocyte spheroids were revealed by XFM imaging of Ag and detecting K-edge XANES of Ag [73]. The hepatocyte internalized AgNPs partially in the endolysosomes in an exposure time-dependent manner, while the Ag(I) complexes were present in all cell compartments, such as the cytoplasm and nuclei. After 7-day exposure, the observed Ag signal in bile canaliculi indicated the excretion pathway. The AgNPs transformed into Ag sulphide or Ag–organothiol complexes in hepatocytes. The biomineralization of magnetite or greigite nanoparticles in magnetosome organelles of magnetotactic bacteria (MTB) was explored to understand the biosynthesis of magnetic NPs and the contribution of MTB in iron cycling [74]. The Fe Kα XRF signal was observed along the distribution and orientation of magnetosomes. The composition of magnetic NPs included Fe₃O₄ (60%), Fe₂O₃ (33%) and FeO(OH) (7%), revealed by the whole-cell XANES. In situ XANES was utilized to demonstrate the intracellular heterogeneity with the content of the ferric compounds via principal components analysis of cluster center maps and the XANES spectra.

The metabolism of Cd-based QDs in small model organisms (Caenorhabditis elegans, microorganisms) was analyzed in situ by combining XFM and XANES at the beamline BL15U1 of Shanghai Synchrotron Radiation Facilities (SSRF) and 4W1B beamline of Beijing Synchrotron Radiation Facility (BSRF) [67,76]. CdSe@ZnS QDs were mainly observed in the alimentary system and underwent metabolism in the digestive microenvironment after exposure. The changes of Se XANES in the retral part of the intestine indicated the oxidation of QDs and the collapse of the core/shell structure in the digestive environment (Fig. 6a). Furthermore, researchers developed the fluorescence imaging XANES via the synergy of XFM and XANES to visualize the coordination environments of metals in biological specimens. They have realized the imaging of iron-protein coordination complexes in Caenorhabditis elegans [77].

Fig. 6.

Synchrotron XFM combined with XAFS to observe the spatial distribution and transformation of MNPs in biological matrices. (a) XFM of CdSe@ZnS QDs in C. elegans after exposure for 24 h and the in situ XAFS of the indicated position of the C. elegans shown in XFM images [67]. (b) The spatial distribution of MoS₂ NPs in liver and the biotransformation process revealed by micro-XFM and XAFS [50]. (c) Elemental mapping of the pepper plant exposed to ZnO_pH NPs and ZnO NPs and the PCA plots of the Zn XANES spectra [75].

The sequestration of MoS₂ nanomedicines in hepatic Kupffer cells and splenic red pulp macrophages, as well as the degradation in the liver and spleen, were demonstrated by mapping the Mo element and investigating the chemical forms of Mo in tissues [50]. The findings explained the underlying mechanism of bioavailability of MoS₂ nanomedicines in the liver by incorporating into the biosynthesis of molybdenum cofactors in molybdenum enzymes and clarified the in vivo transport–transformation–bioavailability process of two-dimensional transition metal dichalcogenide nanomaterials (Fig. 6b). Similarly, the biodistribution and chemical biotransformation of CdSe@ZnS core/shell QDs in vivo were quantified analyzed by XRF and XANES measurement [78]. Moreover, correlative cryo-XFM and XANES were adopted to investigate the translocation and biotransformation of ZnO NPs in plants, revealing the crucial enhanced effects of surface modification with Zinc phosphate [75]. In this research, XRF mapping and XANES were performed at the same beamline of the synchrotron radiation facilities (ID21 beamline of the European Synchrotron Radiation Facilities (ESRF)). Bare ZnO NPs and ZnO modified with a Zn₃(PO₄)₂ shell (ZnO_pH NPs) could be absorbed by leaves and transported to phloem and epidermal tissues after foliar application to pepper plants. Zn₃(PO₄)₂ shell coating enhanced the leaf uptake, loading and persistence in exposed leaf and plant stem, as well as the mobility of Zn nutrients. Principal component analysis (PCA) plots of XANES spectra of Zn indicated that the Zn speciation was dependent on spatial localization and the exposure time. After 2 h exposure, Zn-based NPs were still present in the exposed leaves, while Zn elements present in upper epidermis cells, palisade, or spongy mesophyll were mainly associated with the carboxyl (-COOH) and phosphate (-PO₄) groups. ZnO_pH NPs exposure induced more Zn speciation bonds to thiol-like groups in upper epidermis cells and palisade than ZnO NPs exposure. After 1 week of exposure, there were no NPs remained in the exposed leaves. The association of Zn with carboxyl, phosphate and thiol groups was variable depending on the distribution in the plant organs (Fig. 6c).

So far, XFM integrated with XAFS has been widely applied to explore the elemental distribution, composition and chemical states of MNPs both at cellular and tissue levels. Sometimes, these two techniques are adopted with other analytical methods, such as STXM absorption-contrast imaging, electron microscopy, fluorescence imaging or absorption-contrast/ phase-contrast CT imaging to provide more comprehensive understanding, such as morphology, structure and function, beyond elemental and chemical information.

In addition, CT imaging is also a powerful method to obtain the 3D spatial localization of MNPs in biological matrices. The combination of cryo-SXT and XAFS technology can play a crucial role in the biochemical transformation and spatial localization of MNPs or metal ions, providing unique insights into subsequent biological effects. Pt(IV) prodrug trans, trans, trans-[Pt(pyridine)₂(N₃)₂(OH)₂] (Pt1) and its coumarin derivative trans, trans, trans-[Pt(pyridine)₂ (N₃)₂(OH)(coumarin-3-carboxylate)] (Pt2) is a promising photoactivated chemotherapeutic agent. These complexes are inert in the dark, but release Pt(II) substances and free radicals upon visible light irradiation, thereby producing photocytotoxicity to cancer cells. Sadler et al. used synchrotron technology to study the intracellular behavior of these prodrugs and, for the first time, to visualize changes in cell morphology and Pt localization with and without light irradiation [79]. Cryo-SXT showed that photoactivation of Pt2 caused significant cellular damage, including plasma membrane blebbing, cytoplasmic vacuolization, and reduction in lipid droplet number. In addition, XANES measurements showed that the prodrug was only partially reduced after irradiation, emphasizing that phototoxicity in cancer cells may involve not only Pt (II) photoproducts but also photoexcited Pt (IV) substances (Fig. 7a). Combined cryo-SXT and XANES were used to investigate the anticancer mechanism of MNPs in cancer cells in their near-native state. Application of such techniques provided complementary information at subcellular resolution on the biological response of cancer cells to MNPs treatment and the cellular distribution and oxidation state of the MNPs and their products.

Fig. 7.

The chemical morphology and structural changes of intracellular NMPs were studied by cryo-SXT and XANES. (a) The single-cell chemistry of photoactivatable platinum anticancer complexes was investigated by cryo-SXT and XANES [79]. (b) Speciation and ultrastructure of cellular calcium during coccolith formation [80]. (1) Time-resolved evolution of the XANES spectra (black) of forming calcite and of the calculated fit (orange) using linear combinations of three reference standards (coccolith calcite, free calcium ions and amorphous CaCO₃). (2) Ca K-edge XANES spectra. Calcium species include 10 mM CaCl₂, amorphous CaPO₄, amorphous CaCO₃, calcite and C cells. (3) Soft X-ray tomographic slice of coccolith (blue arrow) and calcium-rich body (red arrow). 3D segmentation of the calcium-rich bodies (red) and intracellular coccoliths (blue).

Coccoliths are calcitic particles produced inside the cells of unicellular marine algae known as coccolithophores. Scheffel et al. combined cryo-SXT and XANES techniques to characterize a highly concentrated, previously unrecognized intracellular calcium pool and to study its corresponding ultrastructural environment [80]. Co-localized with calcium are high concentrations of phosphorus and small amounts of other cations, such as Mg²⁺. The calcium stored appears to be dynamic, and at some stage this compartment is in direct contact with the vesicle that produces coccyliths, suggesting an active role in coccylithic formation (Fig. 7b). The combined cryo-SXT and XANES techniques provide important insights into calcium accumulation in calcifying organism.

3.2. Investigating the elemental distribution and structural alterations in the MNPs-bio interaction system

3D structural information of cells and living organisms can be obtained by X-ray tomographic imaging. Especially, cryo-SXT is superior for investigating the 3D spatial distribution of MNPs and the cellular ultrastructure. XFM can quantitatively track the distribution of multiple elements in cells, living organisms or biological tissues without chemical fluorophore labeling, and the detection limit is within the ppm range. Both endogenous and exogenous elements can be mapped simultaneously, providing important biochemical information on MNPs-bio interactions. The elemental distribution and structural alterations in the MNPs-bio interaction system, including the elemental mapping, chemical composition, ultrastructure, and molecular structure, can be obtained by combining XFM with CT imaging, ptychographic imaging, FTIR or mass spectrometry (MS).

Combining X-ray tomography and XFM techniques can obtain 3D elemental and structural information within cells and living organisms in quasi-native states. Iridium half-sandwich complex [Ir(η⁵:κ¹-C₅Me₄ CH₂py) (2-phenylpyrine)] PF₆ is highly cytotoxic: 15- to 250-fold more potent than clinically used cisplatin in several cancer cell lines. Pizarro et al. developed a 3D cryo-X-ray imaging method that allows for specialized localization and quantification of iridium in whole hydrated cells at nanometric resolution [81]. With the help of cryo-SXT, which provides cellular ultrastructure at 50 nm resolution, and cryogenic hard X-ray fluorescence tomography (cryo-XRF), which provides elemental sensitivity in a 70-nm step range, iridium anticancer agents were found to be specifically localized in mitochondria (Fig. 8a-b). Cryo-SXT and cryo-XRF correlation imaging methods can provide unique information about the intracellular fate of metallic drugs without chemically fixing, labeling, or mechanically manipulating the cells. Byrnes et al. used micro- and nano-X-ray techniques to investigate the relationship between the spatial distribution of uranium (U) in tissues and the adverse gastrointestinal effects of exposure to uranium nanoparticles (UNPs) in the aquatic model organism Daphnia magna [82]. X-ray CT images of intact daphnids exposed to sublethal concentrations of UNPs showed adverse morphological changes to the midgut and the hepatic ceca. The µ-XRF element map showed that U was colocalized with morphological changes, and U accumulated in the lumen and epithelial tissues. With the use of high-resolution nano-XRF, 400 to 1000 nm UNPs could be identified within cells throughout the midgut and hepatic cecum, which is consistent with tissue damage (Fig. 8c). The results emphasized that the destruction of intestinal function was an important mode of acute U toxicity to D. magna, and the midgut epithelial cells and hepatic cecum were the key target organs.

Fig. 8.

Correlation imaging technology is used to study the interaction of NMPs with cells and living organisms. (a) Schematic representation of correlative 3D structural and elemental information in whole cell context [81]. (b) Intracellular localization and quantification of a potent iridium anticancer compound by correlative 3D cryogenic X-ray imaging. The iridium anticancer compound was localized in the mitochondria [81]. (c) Synchrotron XRF and µ-CT techniques were used to investigate the relationship between uranium (U) distributions and adverse effects on the digestive tract of Daphnia magna following UNPs exposure [82].

Though XFM can visualize trace element distribution in multi-micron-thick samples, independent of fluorophore labeling, X-ray fluorescence is not very sensitive to visualize cellular structures and organic molecules composed of low-Z elements. Ptychography, a scanning CDI method, offers a path to the imaging of weakly fluorescing biological ultrastructure beyond focusing-optic resolution. Simultaneous XFM and pytchography imaging of frozen hydrated specimens guarantees excellent structural and chemical preservation.

Deng et al. reported the development of 3D correlative microscopy through a combination of cryogenic X-ray ptychography and X-ray fluorescence microscopy [83]. High-quality 3D maps of the ultrastructure and intracellular element distribution of unlabeled whole algal cells could be obtained by utilizing a tomographic reconstruction algorithm (named GENFIRE) (Fig. 9a). This will open the door to studying a myriad of biological problems where high-resolution elemental identification and complex internal structure information are required in 3D.

Fig. 9.

Simultaneous structural and elemental nano-imaging of biological sample by XFM and ptychography. (a) Correlative 3D XFM and ptychographic tomography of frozen-hydrated green algae [83]. (b) The structural and chemical characterization of complex biological tissues [84]. X-ray ptychography was used to construct spatial phase contrast and electron density images of each identified feature. LB: lewy bodies; SOD1: superoxide dismutase 1; NM: neuromelanin. XFM revealed elemental maps of LB, SOD1 aggregate and neuromelanin in the PD brain. (c) Correlative XFM and ptychography imaging of nanocontainer-treated macrophages [86]. (1) Ptychographic imaging of macrophage cells. Black arrows indicate the internalization of two agglomerates of nanocontainers in the cell. (2) Fe areal mass map was obtained through XRF superimposed on the ptychographic phase. (3) Histogram of the estimated antibiotic load per agglomerate.

Metallic elements play crucial roles in normal neuronal biochemical processes, but their dyshomeostasis is implicated in Parkinson's disease (PD) aetiology. Genoud et al. examined the neuropathological microfeatures of Lewy bodies (LB), superoxide dismutase 1 (SOD1), and neuromelanin (NM) in human post-mortem Parkinson's disease brain tissue [84]. Results suggested that LB and SOD1 aggregates had distinct elemental fingerprints but were structurally similar, whereas NM exhibited a different elemental composition and a unique disordered structure (Fig. 9b). Characterizing the elemental and structural composition of these microfeatures with nanoscale resolution can provide new insights into pathological pathways within the PD brain.

Metallic compounds are being used as novel drug delivery systems by addressing the metabolic properties of bacterial agents [85] or as more efficient medical imaging markers to track tissues of interest. Stachnik et al. [86] quantified the iron distribution in macrophages treated with iron oxide nanocontainers targeting Mycobacterium tuberculosis. These nanocontainers were actively internalized into macrophages and released antibiotics in the vicinity of Mycobacterium tuberculosis. From the iron area mass map, the distribution of antibiotic load per agglomerate and an average areal concentration of nanocontainers in the agglomerates were obtained (Fig. 9c). The iron distribution maps and structural images of nanocontainer-treated macrophages were obtained by simultaneous XFM and ptychography, which can quantify the amount of antibiotic delivered to the cells. This combined approach has significant impact on the intracellular localization of NMPs, helps elucidate the role of metals in cell therapy and development, and further studies diseases with dysregulated metal homeostasis, including neurodegenerative diseases.

MNPs-bio interactions always affect biological processes by interacting with endogenous biomolecules, such as proteins, lipids and nucleic acids. FTIR can be employed to investigate and quantify the alterations of endogenous biomolecules affected by the administration of MNPs. Besides, the in vivo biological behavior of the surface ligands modified on metallic nanocores (such as polymers, small organic molecules and biomacromolecules) can be obtained by measuring the characteristic FTIR spectra. For instance, synchrotron-based XFM and FTIR spectroscopy were utilized to explore the in vivo biodistribution, transport and biological impacts of PEGylated Au NPs (Fig. 10a) [87]. The sub-organ distribution of Au and PEG was imaged with XRF and FTIR, respectively, suggesting similar patterns of these two parts and the stability of PEG surface ligands at 24 h post single intravenous (i.v.) injection of Au-PEG NPs. Simultaneously, FTIR imaging revealed that alterations of proteins, lipids and nucleic acids in the liver and kidney presented similar patterns with PEG, indicating that the changes were strongly dependent on the Au-PEG NPs accumulation. Besides, synchrotron radiation micro-CT imaging was used to clarify the transport mechanism of Au-PEG NPs in the liver by observing the hepatic vascularity in three-dimension. The increased diameters of the hepatic vein after i.v. administration of Au-PEG NPs (1 h, 4 h, 7 d) implied that the induced hepatic blood vessel dilatation could increase the vascular permeability.

Fig. 10.

Synchrotron XFM combined with FTIR to investigate the biological behavior of MNPs and organic molecules. (a) XFM images of the mouse kidney after i.v. injection of Au-PEG NPs and FTIR images of mouse kidney showing distribution of lipids, proteins, nucleic acids and PEG polymers. RC: renal cortex. RM: renal medulla. Adapted with permission from ref. [87]. (b) The translocation of tattoo pigments from skin to lymph nodes and alterations of proteins and lipids in skin and lymph nodes. SC: stratum corneum and epidermis; D: dermis; DP: dermis with particles; P1, P2: particle-containing regions; C1, C2: control regions without particles. Adapted with permission from ref. [88].

In another study [88], skin safety of tattoo pigments consisting of inorganic metals and their oxides (e.g., TiO₂ with nano- and micro-size) or polyaromatics was studied with XFM at both micro- and nano-range and micro-FTIR spectroscopy (Fig. 10b). XFM exhibited its strong ability to detect multiple elements simultaneously by imaging all elements included in tattoo inks and other endogenous biological elements such as P, K, Ca, etc. TiO₂ particles and heavy metals in the tattoo inks were translocated from skin to lymph nodes. The accumulation of tattoo particles in skin and lymph nodes induced alterations in the secondary structure of proteins and the amount of lipids. The relations of biological elements (e.g., Fe) to Alzheimer's disease (AD) were explored with FTIR, XFM and XANES [89]. Researchers characterized two types of amyloid plaques (fibrillary and nonfibrillary) in the cortex sections of AD patients in situ by determining the secondary structure of the peptide with micro-FTIR. Nano-XFM and XANES demonstrated that ferrous ions were mainly present in fibrillary plaques, while ferric ions were the major iron species in nonfibrillary plaques.

Mass spectrometry (MS) plays a pivotal role in identifying chemical composition due to its high sensitivity and precise quantification. Inductively coupled plasma mass spectrometry (ICP-MS), laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS), mass spectrometry imaging (MSI), single-particle inductively coupled plasma time-of-flight mass spectrometry (spICP-TOFMS) and mass cytometry (CyToF) have been employed in the field of nanobiotechnology. Inductively coupled plasma mass spectrometry (ICP-MS) is a sensitive and accurate method to quantify trace elements with a limit of detection down to ppt level, which has been applied to detect the pharmacokinetics of MNPs-based drugs and the distribution of MNPs in different tissues ex vivo. Though ICP-MS is a very sensitive and precise method, it is destructive with the digestion procedure in sample preparation, providing the average amount of metal in samples. XFM is a non-destructive imaging method to offer spatial mapping of metal in biological specimens, but the limit of detection is at the ppm level. The quantification with ICP-MS and the imaging with XFM are two complementary methods to explore the biological behavior in cells and tissues [50,52]. Before XFM imaging of the cells or tissues treated with MNPs, ICP-MS could quantify the amount of metals, which helps us predict if the samples are proper for XFM and the settings of the imaging parameters to save beamline time and acquire high-quality images. LA-ICP-MS is a sensitive imaging method for in situ elemental and isotopic mapping, which combines a laser ablation system and the ICP-MS instrument. LA creates fine particles by exciting the sample with a focused laser beam, then the ablated particles are transported to be digested and ionized in the ICP-MS instrument for elemental and isotopic analysis. Though LA-ICP-MS is a destructive method for elemental imaging, sometimes it is a useful alternative to XFM, especially when the elements are not proper for XFM measurement. For instance, the spatial distribution and the degradation of Ag NPs in the brain and spinal cord were analyzed by LA-ICP-MS and XANES to reveal the transneuronal translocation from the gut to the central nervous system [90].

MS is also powerful for organic molecules analysis, except for metal elements measurement. Specifically, in the case of MNPs-bio interactions, MS can detect the biomolecules absorbed on the surface of MNPs and map the biomolecules in cells or tissues. Together with the analysis of MNPs by other synchrotron radiation X-ray methods, all the results can clarify the relations and explain phenomena. Liquid chromatography-mass spectrometry (LC-MS) is one of the key detection methods for proteomics, metabolomics and lipidomics. For example, the protein corona absorbed on MoS₂ nanoparticles has been identified and quantified by LC-MS. XFM was used to observe the spatial distribution of Mo in the liver and spleen of mice [50]. Combined with quantitative analysis of Mo levels in these organs after treatment with MoS₂ coated with different protein coronas, the results demonstrated that the apolipoprotein E corona mediated the sequestration of MoS₂ by liver Kupffer cells and splenic red pulp macrophages. MSI is another label-free and sensitive imaging method for detecting and identifying biomolecules in complex biological matrices, enabling the spatial localization of hundreds of molecules in biological tissues at cellular and subcellular scales. The light sources used for photoionization in Mass spectrometry are laser, vacuum ultraviolet discharge lamp and synchrotron radiation vacuum ultraviolet light. Synchrotron radiation ultraviolet photoionization mass spectrometry (SVUV-PIMS) has emerged as an outstanding technique in biological fields due to the merits of high flux, high energy resolution and adjustable energy, enabling the detection of polar and non-polar molecules with large or small mass including metabolites, lipids and proteins. For example, neutral biomolecules such as creatine, cholesterol and some lipids in mouse brain tissue, as well as the nonpolar constituents in plant tissue, such as catechins in leaf shoots of tea, were visualized by SVUV-PIMS in National Synchrotron Radiation Laboratory, China [91].

3.3. Correlative analysis of structures and biological functions in the MNPs-bio system

X-ray tomography imaging can obtain the 3D structures of cells and subcellular organelles, and has gradually become an important approach to cell imaging. However, the specific biomolecules that perform biological functions and have similar elemental composition within cells cannot be directly observed and distinguished by X-ray tomography imaging. FM can obtain the localization information of fluorescently labeled molecules, but cannot obtain the whole cell and subcellular 3D structure outside the fluorescently labeled molecules. Therefore, the combination of X-ray tomography and FM imaging technology can observe the 3D localization of specific molecules and 3D cellular architectures. Correlated structural and functional imaging is essential to elucidating the pathogenesis of disease, cellular therapeutic mechanisms and toxicological outcomes of MNPs, etc.

Chiappi et al. used SXT and FM to study the interaction of superparamagnetic iron oxide nanoparticles (SPION) with human breast cancer cells [92]. These acidic organelles (lysosomes) were fluorescently labeled, and the SPION in endocytotic vesicles significantly improved the imaging contrast under X-rays. The results showed that the internalized SPION was continuously transferred from the plasma membrane to the region near the nucleus, the number and volume of SPION-containing vesicles increased, and the mitochondria were pushed away from the nucleus (Fig. 11a). Pereiro et al. applied a novel correlative cryogenic 3D imaging approach to determine the intracellular fate of a designed protein–nanomaterial hybrid with antifibrotic properties that showed great potentials in mitigating myocardial fibrosis [93]. For this aim, the fibroblasts were treated by two protein hybrid systems, with and without the engineered Hsp90 inhibitory domain (TPR-Hsp90-AuNC and TPR-AuNC, respectively). Then, the treated fibroblasts were imaged using cryo-SXT and cryo-structured illumination microscopy (SIM) (Fig. 11b). The results confirmed that at the ultrastructural level, TPR-Hsp90-AuNC could inhibit Hsp90 protein in mouse primary fibroblasts while also ensuring its ATPase activity, thereby maintaining a stable intracellular environment.

Fig. 11.

X-ray and fluorescence microscopy correlation approach was used to study the interaction between NMPs and cells. (a) 3D distribution of SPION in breast cancer cells [92]. (1) fluorescently labeled lysosomes (red). (2) SXT image showing lysosomes containing SPION with high X-ray absorption contrast. N: nucleus. (3) 3D segmentation renders images. Red: vesicles containing SPION; Blue: nucleus; Yellow: mitochondria. (b) Cryo-SXT and cryo-SIM were used to investigate the intracellular location and role of antifibrotic protein-nanomaterials hybrids [93]. (1) NIH-3T3 cells. (2) Cardiac primary mouse fibroblasts. Red: mitochondrial; Green: antifibrotic protein-nanomaterial hybrid. (c) HXT combined with the nanoprobe technique was used for 3D imaging of HER2 proteins [94]. (1) Workflow of HXT combined with fluorescence imaging. (2) Confocal fluorescence image of SK-BR-3 cells exposed to Fe@BFK nanoprobes and anti-HER2 antibodies. (3) Left: HXT of cells after targeting HER2 protein with the nanoprobe. Right: 3D segmentation volume of cells after targeting HER2 protein by nanoprobes. (d) The nanovaccine is internalized by DC2.4 cells in vitro [51].

Different from the combination of SXT and FM, Wang et al. combined HXT with nanoprobe technology to perform 3D imaging of HER2 protein in intact single cells [94]. Firstly, the multifunctional iron oxide (Fe@BFK) nanoprobe with hard X-ray imaging signal, fluorescence signal and specific binding ability to human epidermal growth factor receptor 2 (HER2) protein was synthesized. Then, the samples more suitable for HXT were screened with the help of the fluorescence signal. Finally, after obtaining the confocal fluorescence data, the samples were transferred to the hard X-ray imaging beamline station for CT data acquisition, and the results showed that HER2 protein was mainly localized on the cell membrane and aggregated in a heterogeneous pattern (Fig. 11c). X-ray tomography and fluorescence correlation imaging technology provide a powerful means to study the subcellular localization of NMPs and the distribution of key cellular proteins. In addition, to understand the process of nanovaccines uptake and immune regulations route, Wang et al. imaged FITC-labeled nanovaccines by confocal fluorescence and HXT [51]; as shown in Fig. 11d, MnARK exhibited a time-dependent antigen internalization process in DCs. This validated the possibilities of intracellular antigen delivery by MnARK.

3.4. Multi-scale imaging of MNPs and ultrafine structures in large intact biological specimens

Cryo-soft X-ray microscopy (cryo-SXM) can be used for non-destructive 3D high-resolution imaging of biological samples up to 18 µm thick [95] with spatial resolution better than 15 nm [96]. SXM is particularly suitable for imaging mesoscale structures of cells, from intact single cells (10 µm) to objects larger than molecular machines (50 nm). However, it does not recognize the various molecules in the cell that perform physiological functions, such as proteins and nucleic acids. The resolution of EM can reach the atomic level [97], but the low electron penetration depth leads to the imaging depth of only hundreds of nanometers [98], which makes it hard to achieve whole-cell imaging. EM and SXM have different imaging depths and resolutions. The combination of EM and SXM can obtain the ultra-fine structure information of large samples.

Reineck et al. investigate the fate of fluorescent nanodiamonds (FNDs) in cancer cells using confocal fluorescence microscopy, AFM, SEM, and SXT (Fig. 12a), in which they directly correlated fluorescence, AFM topography, stiffness, and SEM images of FNDs on the cell surface. In addition, SXT revealed that the FNDs were endocytosed and localized in vesicles. Multimodal imaging provides important insights into the nano- and microscale environment of FNDs in cells [99]. Zhang et al. [100] established a unique multi-modal, multi-scale cryo-correlative platform to image Vero cells infected with SARS-CoV-2. This platform combined serial cryo-FIB/SEM volume imaging and cryo-SXT with cell lamellae-based cryo-electron tomography (cryo-ET) and sub-tomogram averaging. This approach provided a comprehensive view of SARS-CoV-2 infection, from the whole cell to individual virus spike molecules, revealing pathways of SARS-CoV-2 assembly and egress and cytopathic effects of SARS-CoV-2 infection (Fig. 12b). The developed correlation imaging method can characterize the fine structure of molecules in intact large cells, which greatly promotes our understanding of processes such as pathogen infection and cell therapy with NMPs.

Fig. 12.

Employing a multimodal imaging approach to investigate the fine structure of molecules in intact large cells. (a) High-resolution correlation of features on the cell surface and cryo-SXT imaging of PC3 cells incubated with FNDs and untreated reference cells [99]. (1) From top to bottom: Confocal fluorescence image; SEM image; AFM topography image; AFM stiffness map; High-resolution SEM image of the area indicated in panel SEM (dotted pink rectangle). (2) Cryo-SXT imaging of PC3 cells incubated with FNDs. The black arrow points to the nuclear membrane and the vesicle containing the FNDs. (3) Cryo-SXT imaging of PC3 cells untreated. The black arrows point to the nuclear envelope, lipid droplets, mitochondria and plasma membrane. (b) Correlative multi-scale cryo-imaging unveils SARS-CoV-2 assembly and egress. (1) A correlative workflow of SARS-CoV-2-infected cells [100]. (2) Cryo-EM/ET revealed portals on DMVs next to assembly sites. Tomographic slice of cryo-FIB lamella depicting SARS-CoV-2 assembly. Cryo-SXT of SARS-CoV-2-infected cells revealed ultrastructural changes.

4. Conclusion and perspectives

Multimodal analysis based on synchrotron radiation X-rays plays an irreplaceable role in delving into the interactions of MNPs with biological matrices. By integrating different state-of-the-art analytical techniques, such as X-ray CT imaging, XFM, X-ray ptychography, XAFS, FTIR, fluorescence microscopy, EM, and mass spectrometry, we can achieve a comprehensive all-aspect analysis of MNPs-bio interactions. The multimodal analytical techniques can not only provide information on spatial distribution, composition, valence state, coordination bonds, electron density and crystallinity of MNPs, but also explore the structure, morphology, chemical composition and biological functions of cells or tissues. The multimodal analysis is beneficial to clarifying the underlying interrelationships and promoting the understanding of the medical efficacy and biosafety of MNPs applied in medicines or daily consumer products. Though the future development of multimodal synchrotron radiation X-ray techniques is still necessary, they act as universal and robust platforms enabling in situ label-free analysis of MNPs-bio interactions.

Multimodal analysis based on synchrotron radiation X-ray techniques has made tremendous inroads in the study of MNPs-bio interactions during recent years. However, future development is still necessary for multimodal data fusion, efficient data collection and analysis. Nowadays, integrating multimodal techniques at one beamline of the synchrotron radiation facilities is the mainstream development trend all over the world. For example, different synchrotron radiation X-ray microscopy techniques (XFM, TXM, STXM, ptychography) are integrated with electron microscopy, fluorescence microscopy and mass spectroscopy to provide structure, morphology, composition, and chemical environment. The online correlation of synchrotron radiation X-ray techniques requires an optimized data acquisition procedure, integrated device control systems and algorithms to improve data collection speed and accuracy. Simultaneous data acquisition in the multimodal analysis could reduce the radiation damage to the specimens, facilitate the data reconstruction and save time for data processing. Moreover, developing intelligent and advanced analysis algorithms, such as machine learning, will help reduce the signal noise and improve data processing precision and the data fusion process. We believe that with the upgrades of optics and improved performance of advanced light sources, multimodal analysis based on synchrotron radiation X-rays will make significant progress in the future.

Declaration of competing interest

The authors declare that they have no conflicts of interest in this work.

Biographies

Mingjing Cao (BRID: 07923.00.01577) is an associate professor at the National Center for Nanoscience and Technology of China. She received double Ph.D. degrees from University of Chinese Academy of Sciences and Aarhus University in 2020 and 2021, respectively. She then worked as a postdoctoral researcher at the National Center for Nanoscience and Technology of China and was selected for “the National Postdoctoral Program for Innovative Talents” in 2021. Dr. Cao is engaged in the basic scientific research in the field of nanobiological effects, focusing on elucidating the underlying mechanism behind biological effects and safety of nanomaterials, in view of interactions between nanomaterials and biological interfaces.

Zheng Dang received his doctor’s degree from University of Science and Technology of China in 2024. He is currently a postdoctoral fellow at National Center for Nanoscience and Technology of China. His research interests mainly focus on synchrotron radiation X-ray imaging technology and biomedical applications, mechanism and anti-tumor application of biological multistage ordered assembly of nanoparticles.

Yaling Wang (BRID: 08537.00.17680) received her Ph.D. in bioinorganic chemistry at the Institute of High Energy Physics, CAS (2012). She focuses on developing nanomedicine for in vivo imaging, cancer therapy, vaccine and single cell analysis via nano−bio and synchrotron radiation technologies. She has authored/co-authored over 90 peer-reviewed papers, including the Nature Protocols, Acc. Chem. Res., ACS Cent. Sci., Angew. Chem. Int. Ed., Nature Nanotechnology. She is the principle investigator or key member of several domestic and international projects, such as China MOST, Program and projects from Natural Science Foundation of China, EU2020 (NanoinformaTIX).