2D NMR Research Articles

Discovery of New Cyclic Lipodepsipeptide Orfamide N via Partnership with Middle School Students from the Boys and Girls Club.

We established a university-community partnership with the Boys and Girls Clubs of Chicago (BGCC)-named Chicago Antibiotic Discovery Lab-to involve middle school students in antibiotic discovery research. In the course of working with a cohort of students from the BGCC, one student isolated a Pseudomonas idahonensis bacterium from a goose feces sample that produced a new cyclic lipodepsipeptide, which was characterized as orfamide N. Orfamide N is composed of ten mixed D/L-amino acids and a (Z)-3R-hydroxyhexadec-9-enoic acid residue. The planar structure of orfamide N was elucidated by one-dimensional (1D) and two-dimensional nuclear magnetic resonance (2D NMR), electrospray ionization-mass spectrometry (ESI-MS), and ozone-induced dissociation mass spectrometry (OzID-MS). The absolute configuration was determined by advanced Marfey's analysis, phylogenetic analysis of C-domains within the orfamide N biosynthetic gene cluster, and chiral high-performance liquid chromatography (HPLC) analysis of the hydrolyzed and reduced lipid tail. Orfamide N was cytotoxic against human melanoma and human ovarian cancer cells with IC50 values of 11.06 and 10.50 μM, respectively. Overall, we demonstrated it is possible to integrate educational outreach with high-end natural product discovery while strengthening the relationship between the university and the community it serves.

In vitro spectroscopic analysis of the chemical interaction between calcium hypochlorite and chlorhexidine.

This study aimed to identify the chemical composition resulting from the chemical interaction between calcium hypochlorite [Ca(OCl)2] and chlorhexidine (CHX) using different 1H and 13C nuclear magnetic resonance spectrometry (NMR), correlated two-dimensional spectroscopy (2D COSY), heteronuclear single quantum coherence (HSQC), and Fourier Transform Infrared Spectroscopy (FTIR) experiments. The 5.25% Ca(OCl)2 was mixed with 2% CHX in a 1:1 ratio, obtaining an orange-brown precipitate that was filtered, washed in ultrapure water, dried and characterized by 1H and 13C NMR, 2D COSY, HSQC and FTIR. One-dimensional and two-dimensional NMR spectra demonstrated the chemical changes around the protons and carbon atoms of the initial CHX and after reacted with Ca(OCl)2. These techniques and FTIR show that the interaction generated two by-products from the degradation of CHX, none of which were parachloroaniline (PCA) despite both products obtained being substituted benzene compounds. Using NMR and FTIR data together with a previously proposed catalytic cleavage degradation mechanism for CHX, the products appear to be parachlorophenylurea (PCU) and parachlorophenylguanidyl-1,6-diguanidyl-hexane (PCGH). Therefore, this in vitro study demonstrated that the precipitate formed from the chemical interaction between Ca(OCl)₂ and CHX results in two by-products, PCU and PCGH, neither of which is PCA. Due to the absence of cytotoxicity studies on the products generated from the combination of Ca(OCl)₂ and CHX, an intermediate rinse with an inert solution is recommended to remove these compounds from the root canals.

Epigenetic modulation via the C-terminal tail of H2A.Z

H2A.Z-nucleosomes are present in both euchromatin and heterochromatin and it has proven difficult to interpret their disparate roles in the context of their stability features. Using an in situ assay of nucleosome stability and DT40 cells expressing engineered forms of the histone variant we show that native H2A.Z, but not C-terminally truncated H2A.Z (H2A.Z∆C), is released from nucleosomes of peripheral heterochromatin at unusually high salt concentrations. H2A.Z and H3K9me3 landscapes are reorganized in H2A.Z∆C-nuclei and overall sensitivity of chromatin to nucleases is increased. These tail-dependent differences are recapitulated upon treatment of HeLa nuclei with the H2A.Z-tail-peptide (C9), with MNase sensitivity being increased genome-wide. Fluorescence correlation spectroscopy revealed C9 binding to reconstituted nucleosomes. When introduced into live cells, C9 elicited chromatin reorganization, overall nucleosome destabilization and changes in gene expression. Thus, H2A.Z-nucleosomes influence global chromatin architecture in a tail-dependent manner, what can be modulated by introducing the tail-peptide into live cells.

Rapidly Screening the Correlation between the Rotational Mobility and the Hydrogen Bonding Strength of Confined Water.

Automated Deuterium Relaxation-Ordered SpectroscopY in solution (ADROSYS), an automated two-dimensional deuterium NMR methodology, discriminates between D2O populations (as well as deuterium-labeled alcohol groups) whose properties differ as a result of being confined inside nanoscale volumes. In this contribution, a proof-of-principle demonstration on reverse micelles (RMs) yields the insight that as the length scale of the confinement decreases from several nanometers down to less than a nanometer, the position of the signal peak migrates through the two-dimensional (2D) spectrum, tracing out a distinctive path in the 2D space (of relaxation time vs chemical shift). The signals typically follow a relatively gentle linear path for water confined on the scale of several nanometers, before curving once the surfactants confine the water molecules to length scales smaller than 1-2 nm. The qualitative shape of this path, especially in the regime of strong confinement, can change with different choices of surfactants, i.e., a different choice of chemistry at the edges of the confining environment. An important facet of this research was to demonstrate the relatively wide applicability of these techniques by showing that both: (1) Standard modern NMR instrumentation is capable of deploying an automated measurement, even though the choice of a deuterium nucleus is nonstandard and frequently requires companion proton spectra in order to reference the chemical shifts; and (2) well-established (though underutilized) modern techniques can process the resulting signal even though it involves the somewhat unusual combination of chemical shifts along one dimension and a distribution of relaxation times along another dimension. In addition to demonstrating that this technique can be deployed across many samples of interest, detailed facts pertaining to the broadening or shifting of resulting signals upon inclusion of various guest molecules are also discussed.

Mapping Natural Sugars Metabolism in Acute Myeloid Leukaemia Using 2D Nuclear Magnetic Resonance Spectroscopy.

Metabolism plays a central role in cancer progression. Rewiring glucose metabolism is essential for fulfilling the high energy and biosynthetic demands as well as for the development of drug resistance. Nevertheless, the role of other diet-abundant natural sugars is not fully understood. In this study, we performed a comprehensive 2D NMR spectroscopy tracer-based assay with a panel of 13C-labelled sugars (glucose, fructose, galactose, mannose and xylose). We assigned over 100 NMR signals from metabolites derived from each sugar and mapped them to metabolic pathways, uncovering two novel findings. First, we demonstrated that mannose has a semi-identical metabolic profile to that of glucose with similar label incorporation patterns. Second, next to the known role of fructose in driving one-carbon metabolism, we explained the equally important contribution of galactose to this pathway. Interestingly, we demonstrated that cells growing with either fructose or galactose became less sensitive to certain one-carbon metabolism inhibitors such as 5-Flurouracil and SHIN1. In summary, this study presents the differential metabolism of natural sugars, demonstrating that mannose has a comparable profile to that of glucose. Conversely, galactose and fructose contribute to a greater extent to one-carbon metabolism, which makes them important modulators for inhibitors targeting this pathway. To our knowledge, this is the first NMR study to comprehensively investigate the metabolism of key natural sugars in AML and cancer.

Physivitrins I–R, lanostane triterpenoids with anti-inflammatory activities from the fungus Physisporinus vitreus

Chemical investigation on the rice fermentation of the fungus Physisporinus vitreus led to the isolation of ten previously undescribed lanostane triterpenoids, physivitrins I–R, and three known analogues. The new structures were elucidated on the basis of extensive spectroscopic methods, including 1D & 2D NMR, HRESIMS, UV and ECD. Physivitrins I and P exhibited significant inhibitory activities against NO production in LPS-activated RAW267.4 macrophages with IC50 values of 8.2 and 11.5 μM, respectively. The comprehensive data indicated that P. vitreus is rich in lanostane triterpenes and has potential anti-inflammatory application prospects.

Discovery of Chemical Constituents with Anti-Atopic Dermatitis Properties from Aster koraiensis.

Atopic dermatitis is an inflammatory dermatological disease characterized by persistent scratching and recurrent eczema. Due to the influence of environmental variables on the cause of this disease, there remains an ongoing interest in the development of therapeutic interventions. Previous studies have shown that various plants of the genus Aster and its derived phytochemicals possess efficacy in treating inflammatory-mediated diseases, including atopic dermatitis. Therefore, the present study investigated a potential compound with anti-atopic dermatitis properties derived from Aster koraiensis leaves, specifically targeting HaCaT keratinocyte cells. First, we isolated eleven compounds with three unknown compounds, including two polyacetylenes (1 and 3) and a benzoic acid derivative (4). The chemical structures of the isolates were elucidated by 1D and 2D NMR, specific rotation, acid hydrolysis, and quantum chemical calculations. Next, we treated an A. koraiensis extract and all isolates to HaCaT keratinocyte, followed by stimulation with TNF-α/IFN-γ. Among bioactive compounds, astersaponin J (7) exhibited a significant reduction in the levels of inflammatory cytokines associated with atopic dermatitis at a concentration of 2.5 μM. These findings suggest that chemicals obtained from an A. koraiensis 95% ethanol extract and derived compounds are potential therapeutics to help reduce the immunological response driven by atopic dermatitis.

Synthesis of Sulfonamide-Based Schiff Bases as Potent Anticancer Agents: Spectral Analyses, Biological Activity, Molecular Docking, ADME, DFT, and Pharmacophore Modelling Studies.

The current study focuses on the synthesis and characterization of six benzenesulfonamide-based Schiff base derivatives (7-12) with various electron-withdrawing and electron-donating substituents (-F, -CI, -Br, -CH3, and -OCH3) and the assessment of their antiproliferative activities against human lung (A549) and liver (HepG2) cancer cell lines using in vitro and in silico approaches. The structures of the synthesized compounds (7-12) were elucidated by elemental analysis and FT-IR, 1D (1H, 13C, APT, and DEPT-135), and 2D (HMQC and HMBC) NMR spectroscopies. The cytotoxic activities of the targeted compounds were determined at various concentrations against these cancer cell lines for 72 h, using the MTT method. The targeted molecules (7-12) demonstrated remarkable antiproliferative activities, with IC50 values ranging from 6.032-9.533 μM against the A549 cell line and 5.244-9.629 μM against the HepG2 cell line. These compounds showed activities at lower or very similar concentrations to cisplatin against the A549 cell line and at much lower concentrations than cisplatin against the HepG2 cell line. Among them, compounds 10 and 12 were found to be more effective against A549 and HepG2 cells, respectively, than cisplatin. These compounds were analyzed by interacting with the 1BNA, 4HJO, and 4ASD crystal structures in molecular docking studies.

Ultrasound-assisted low-temperature extraction of polysaccharides from Lavandula angustifolia Mill.: optimization, structure characterization, and anti-inflammatory activity

Lavandula angustifolia Mill. is a traditional Chinese medicinal herb with high economic and pharmacological value. In this study, we optimized the conditions for low-temperature ultrasound-assisted extraction of L. angustifolia Mill. polysaccharides using response surface methodology (RSM), and two homopolysaccharides LAPW1 (33.6 kDa) and LAPS1 (95.9 kDa) were obtained after isolation and purification by DEAE-650 M and Superdex™ 200 chromatography. The primary structure of LAPS1 was determined using “partial acid hydrolysis, methylation, two-dimensional (2D NMR) spectroscopy” as the core method. The results revealed that LAPS1 is a heteropolysaccharide whose main chain consists of [-1)-β-Galp-(6→1)-α-Araf-(5→]3-1-α-Araf-(3→1)-α-Araf-(5→[1)-β-Galp-(6→1)-α-Araf-(5→]3-1-α-Araf-(5→1)-α-Araf-(5→[-1)-β-Galp-(6→1)-α-Araf-(5→]3. In vitro experiments revealed that LAPW1 and LAPS1 significantly reduced the production of the inflammatory cytokines Interleukin-1β (IL-1β), Interleukin-6 (IL-6), and Tumor Necrosis Factor (TNF), as well as the expression of Nitric Oxide Synthase 2 (NOS2) and release of nitric oxide (.NO) free radical in the inflammatory model established by lipopolysaccharide (LPS) stimulated RAW264.7. Besides, the zebrafish inflammatory model, stimulated by CuSO4, was employed to assess the impact of polysaccharides on neutrophil migration, indicating a notable decrease in zebrafish neutrophils and confirming their potential anti-inflammatory activity. These results indicate that polysaccharides from L. angustifolia Mill. be used in the development of functional foods and pharmaceutical products.

(R)-3,3,6-Trimethyl-3,3a,4,5-tetrahydro-1H-cyclopenta[c]furan

(R)-3,3,6-trimethyl-3,3a,4,5-tetrahydro-1H-cyclopenta[c]furan was obtained by the acid cyclization of the alcohol (S)-(2-methyl-5-(prop-1-en-2-yl)cyclopent-1-en-1-yl)methanol, which, in turn, is obtained from (R)-limonene. The structure of the new compound was established using NMR spectroscopy (1H, 13C, COSY, HSQC, and HMBC spectra) and high-resolution mass spectrometry. The chemical shifts of the proton and carbon atoms were calculated at the DFT level with a high correlation between the calculated and experimental values.

Water-Soluble Curcumin Derivatives Including Aza-Crown Ether Macrocycles as Enhancers of their Cytotoxic Activity.

The synthesis of three novel curcumin derivative compounds, featuring aza-crown ether macrocycles of various sizes (aza-12-crown-4, aza-15-crown-5, and aza-18-crown-6), is described. The incorporation of these aza-crown macrocycles significantly enhances their water solubility, positioning them as groundbreaking instances of curcumin derivatives that are fully soluble in aqueous environments. These curcumin ligands (L1, L2, and L3) were then reacted with zinc acetate to afford the coordination metal complexes (L1-Zn, L2-Zn, and L3-Zn). Comprehensive characterization of all compounds was achieved using various analytical techniques, including 1D and 2D NMR spectroscopy, ATR-FTIR spectroscopy, mass spectrometry (ESI+), elemental analysis and UV-Vis spectroscopy. The in vitro cytotoxic activity of both, ligands and complexes were evaluated on three human cancer cell lines (U-251, MCF-7, and SK-LU-1). Compared to conventional curcumin, these compounds demonstrated improved antiproliferative potential. Additionally, a wound healing assay was conducted to assess their antimigration properties. The obtained results suggest that these modifications to the curcumin structure represent a promising approach for developing therapeutic agents with enhanced cytotoxic properties.

Distinction of the Microcrystalline Celluloses from Eurycoma longifolia and Mannihot esculenta by Two-dimensional Correlation and Second Derivative IR Spectroscopy

Distinction of the Microcrystalline Celluloses from Eurycoma longifolia and Mannihot esculenta by Two-dimensional Correlation and Second Derivative IR Spectroscopy

Melt Polycondensation Strategy to Access Unexplored l-Amino Acid and Sugar Copolymers.

Biodegradable polymers from bioresources are highly in demand for the development of sustainable polymer platforms for commodity plastics and in the biomedical field. Here, an elegant one-pot synthetic strategy is developed, for the first time, to access unexplored hybrid polymers from two naturally abundant resources: carbohydrates (sugars) and l-amino acids. A bottleneck in the synthetic strategy is overcome by tailor-making d-mannitol-based six- and five-membered bicyclic acetalized diols, and their structures are confirmed by single-crystal X-ray diffraction and 2D NMR spectroscopy. l-Amino acids are converted into ester-urethane functional monomers, and they are polymerized with sugar-diols under solvent-free melt polycondensation to yield biodegradable poly(ester-urethane)s. Acid-catalyzed deprotection yielded amphiphilic polymers having exclusively alternating residues of sugar and l-amino acid in the polymer backbone. The polymer is self-assembled into 200 ± 10 nm sized nanoparticles that can encapsulate fluorescent dyes, are nontoxic to cells up to 250 μg/mL, and are readily endocytosed for lysosomal enzymatic biodegradation at the cellular level.

Effects of a Serotonergic Psychedelic on the Lipid Bilayer.

Serotonergic psychedelics, known for their hallucinogenic effects, have attracted interest due to their ability to enhance neuronal plasticity and potential therapeutic benefits. Although psychedelic-enhanced neuroplasticity is believed to require activation of 5-hydroxytryptamine (serotonin) 2A receptors (5-HT2ARs), serotonin itself is less effective in promoting such plasticity. Also, the psychoplastogenic effects of these molecules correlate with their lipophilicity, leading to suggestions that they act by influencing the intracellular receptors. However, their lipophilicity also implies that a significant quantity of lipids is accumulated in the lipid bilayer, potentially altering the physical properties of the membrane. Here, we probe whether the serotonergic psychedelic 2,5-dimethoxy-4-iodoamphetamine (DOI) can affect the properties of artificial lipid bilayers and if that can potentially affect processes such as membrane fusion. Solid-state NMR spectroscopy shows that the DOI strongly induces disorder in the lipid acyl chains. Atomic force microscopy shows that it can shrink the ordered domains in a biphasic lipid bilayer and can reduce the force needed to form nanopores in the membrane. Fluorescence correlation spectroscopy shows that DOI can promote vesicle association, and total internal fluorescence microscopy shows that it enhances vesicle fusion to a supported lipid bilayer. While serotonin has also recently been shown to cause similar effects, DOI is more than two orders of magnitude more potent in evoking these. Our results suggest that the receptor-independent effects of serotonergic psychedelics on lipid membranes may contribute to their biological actions, especially those that require significant membrane remodeling, such as neuronal plasticity.

Steroids produced by Cladosporium anthropophilum, an endophytic fungus Isolated from Avicennia marina (Forssk.) Vierh and Their Antibacterial Activity

This work was conducted to isolate the steroid compounds from an endophytic fungus Cladosporium anthropophilum and determine their effects against 4 strains of bacteria. The fungi were afforded from a mangrove plant Avicennia marina (Forssk.) Vierh of Acanthaceae family, growing in West Java, Indonesia. The fermentation and isolation of C. anthropophilum yielded 3 ergostane-type steroids including a highly oxidized one, identified as penicisteroid A (1), ergosterol (2), and ergosterol-5,8-peroxide (3), together with 2 stigmastanes, characterized as stigmasterol (4), and stigmasterol-5,8-peroxide (5). The elucidation structure of all isolated steroids was performed by extensive spectroscopic measurements (MS, IR, 1D, and 2D NMR) and supported by a comparison of previously reported spectral data. Subsequently, the potential of the 5 steroids as well as methanol, n-hexane, and ethyl acetate extracts were evaluated against 2 Gram-positive bacteria, Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 and 2 Gram-negative bacteria, Vibrio harveyi ATCC 5339 and Escherichia coli ATCC 25922. The results showed that none of the isolated steroids were active, while the 3 extracts exhibited significant inhibition against all the tested bacteria’s growth and enabled us to propose the synergistic effects between active compounds in these extracts and their antibacterial activity. HIGHLIGHTS An isolated and identified endophytic fungus, Cladosporium anthropophilum, from a mangrove plant Avicennia marina (Forssk.) Vierh yielded five steroids. A chromatography technique and spectroscopic method were utilized for separation processes and structure determination, respectively. An antibacterial assay against two Gram-positive bacteria ( aureus and E. faecalis) and two Gram-negative bacteria (V. harveyi and E. coli) implied the synergistic effects between five isolated steroids and their parent extracts. A further investigation is highly recommended to explore the possibility of these five steroids as a potential source of active compounds. GRAPHICAL ABSTRACT

Enhancing antibacterial efficacy through macrocyclic host complexation of fluoroquinolone antibiotics for overcoming resistance

The use of supramolecular assemblies in pharmaceuticals has garnered significant interest. Recent studies have shown that the activities of antibacterial agents can be enhanced through complexation with cyclic oligomers and metal ions. Notably, these complexes sometimes possess greater therapeutic properties than the parent drugs. To develop microbiologically potent supramolecular drugs, the complexation of macrocyclic hosts with fluoroquinolone (FQ) antibiotics was investigated. FQs are a successful family of antibiotics that target the bacterial enzymes DNA gyrase and DNA topoisomerase IV, leading to bacterial cell death through the inhibition of DNA synthesis. However, antibiotic resistance resulting from the repeated use of FQs over time has limited their effectiveness against resistant pathogens. To overcome this issue, the encapsulation of FQs in polyphenolic macrocycles was investigated. This study highlights resorcinarene, a polyphenolic host with antibacterial properties, and its ability to chemically interact with FQs. The inclusion complexation process was analyzed using NMR and FTIR techniques. The binding constants determined by 1H-NMR titration revealed that levofloxacin forms more stable complexes with resorcinarene than with β-cyclodextrin, which aligned with MD simulations. Assessment of the geometric characteristics of the inclusion complexes using 2D NMR analysis confirmed that different moieties of various FQs can fit into a single host cavity and improve activity against gram-negative bacteria. Overall, these findings suggest that encapsulation in polyphenolic macrocycles is a promising strategy for utilizing FQs against antibiotic-resistant bacteria.

Packaged delivery of CRISPR-Cas9 ribonucleoproteins accelerates genome editing.

Effective genome editing requires a sufficient dose of CRISPR-Cas9 ribonucleoproteins (RNPs) to enter the target cell while minimizing immune responses, off-target editing and cytotoxicity. Clinical use of Cas9 RNPs currently entails electroporation into cells ex vivo , but no systematic comparison of this method to packaged RNP delivery has been made. Here we compared two delivery strategies, electroporation and enveloped delivery vehicles (EDVs), to investigate the Cas9 dosage requirements for genome editing. Using fluorescence correlation spectroscopy (FCS), we determined that >1300 Cas9 RNPs per nucleus are typically required for productive genome editing. EDV-mediated editing was >30-fold more efficient than electroporation, and editing occurs at least two-fold faster for EDV delivery at comparable total Cas9 RNP doses. We hypothesize that differences in efficacy between these methods result in part from the increased duration of RNP nuclear residence resulting from EDV delivery. Our results directly compare RNP delivery strategies, showing that packaged delivery could dramatically reduce the amount of CRISPR-Cas9 RNPs required for experimental or clinical genome editing.

Talaroterpenoids A-F: Six New Seco-Terpenoids from the Marine-Derived Fungus Talaromyces aurantiacus.

Six new highly oxidized seco-terpenoids, including three 3-nor-labdane type diterpenes, talaroterpenoids A-C (1-3), and three meroterpenoids containing an orthoester group, talaroterpenoids D-F (6-8), together with five known compounds (4-5 and 9-11), were isolated from the marine-derived fungus Talaromyces aurantiacus. Their chemical structures were elucidated through 1D, 2D NMR, HRESIMS, J-based configuration analysis (JBCA), computational ECD calculations, and single-crystal X-ray diffraction analysis. Compounds 1 and 2 contain an unusual 6,20-γ-lactone-bridged scaffold. Compounds 10 and 11 presented inhibitory effects on NO release in lipopolysaccharide (LPS)-induced BV-2 cells with IC50 values of 11.47 and 11.32 μM, respectively. Talaroterpenoid C (3) showed moderate antifungal activity against A. alternata and P. theae Steyaert.

Noninvasive optical monitoring of cerebral hemodynamics in a preclinical model of neonatal intraventricular hemorrhage.

Intraventricular hemorrhage (IVH) is a common complication in premature infants and is associated with white matter injury and long-term neurodevelopmental disabilities. Standard diagnostic tools such as cranial ultrasound and MRI are widely used in both preclinical drug development and clinical practice to detect IVH. However, these methods only provide endpoint assessments of blood accumulation and lack real-time information about dynamic changes in ventricular blood flow. This limitation could potentially result in missed opportunities to advance drug candidates that may have protective effects against IVH. In this pilot study, we aimed to develop a noninvasive optical approach using diffuse correlation spectroscopy (DCS) to monitor real-time hemodynamic changes associated with hemorrhagic and sub-hemorrhagic events in a preclinical rabbit model of IVH. DCS measurements were conducted during the experimental induction of IVH, and results were compared with ultrasound and histological analysis to validate findings. Significant changes in hemodynamics were detected in all animals subjected to IVH-inducing procedures, including those that did not show clear positive results on ultrasound. The study revealed progressively elevated coefficients of variation in blood flow, particularly driven by increased oscillations within the 0.05-0.1 Hz frequency band. These hemodynamic changes were more pronounced in animals that developed IVH, as confirmed by ultrasound. Our findings suggest that real-time optical monitoring with DCS can provide critical insights into pathological blood flow changes, offering a more sensitive and informative tool for evaluating potential therapeutics in the context of IVH.

Dynamic X-ray Coherent Diffraction Analysis: Bridging the Time Scales between Imaging and Photon Correlation Spectroscopy.

The advent of diffraction limited sources and developments in detector technology opens up new possibilities for the study of materials in situ and operando. Coherent X-ray diffraction techniques such as coherent X-ray diffractive imaging (CXDI) and X-ray photon correlation spectroscopy (XPCS) are capable for this purpose and provide complementary information, although due to signal-to-noise requirements, their simultaneous demonstration has been limited. Here, we demonstrate a strategy for the simultaneous use of CXDI and XPCS to study in situ the Brownian motion of colloidal gold nanoparticles of 200 nm diameter suspended in a glycerol-water mixture. We visualize the process of agglomeration, examine the spatiotemporal space accessible with the combination of techniques, and demonstrate CXDI with 22 ms temporal resolution.