Recent developments in ionization techniques for single-cell mass spectrometry.

The variation among individual cells plays a significant role in many biological functions. Single-cell analysis is advantageous for gaining insight into intricate biochemical mechanisms rarely accessible when studying tissues as a whole. However, measurement on a unicellular scale is still challenging due to unicellular complex composition, minute substance quantities, and considerable differences in compound concentrations. Mass spectrometry has recently gained extensive attention in unicellular analytical fields due to its exceptional sensitivity, throughput, and compound identification abilities. At present, single-cell mass spectrometry primarily concentrates on the enhancement of ionization methods. The principal ionization approaches encompass nanoelectrospray ionization (nano-ESI), laser desorption ionization (LDI), secondary ion mass spectrometry (SIMS), and inductively coupled plasma (ICP). This article summarizes the most recent advancements in ionization techniques and explores their potential directions within the field of single-cell mass spectrometry.

Differences in multielement concentrations in rice (Oryza sativa L.) between longevity and non-longevity areas in China and their relations with lifespan indicators.

Geographical distribution pattern of centenarians in China runs counter to the global trend, and centenarians are mainly distributed in low-latitude South China but rarely in high-latitude North China. To investigate the effect of the elemental content of rice on the differences between the longevity areas and the non-longevity areas in China, the multielement concentrations in 84 rice samples collected from 28 counties/cities were measured, of which 37 belonged to the longevity areas in South China (SLs), 36 belonged to non-longevity areas in South China (SNLs), and 11 belonged to non-longevity areas in North China (NNLs). The concentrations of 29 different elements (Ca, K, Mg, Na, P, S, Se, As, Ba, Cr, Cu, Fe, Mn, Mo, Ni, Sr, Ti, Zn Cd, Co, Li, Pb, Tl, Cs, Ga, In, Rb, U and Bi) in rice samples were determined by ICP-OES, ICP-MS and HG-AFS. Notably, the concentrations of macroelements in SLs rice samples (3682.92 mg/kg) were significantly higher than those of SNLs (3046.62 mg/kg) and NNLs (2590.38 mg/kg) (p < 0.05). The concentrations of essential microelements in rice samples of SLs (38.87 mg/kg) and NNLs (41.79 mg/kg) were not significantly different, but both significantly higher than those of SNLs (31.91 mg/kg) (p < 0.05). Significant differences were also found in the concentrations of K, Mg, Na, P, S, Cu, Mn, Mo, Ni, Pb, Tl, In and Cs in rice among SLs, SNLs and NNLs (p < 0.05). The rice samples of SLs had the characteristic of higher concentrations of K, Mg, P, Mn and Ni. Thus, rice intake in SLs provided greater proportions of the dietary mineral requirements than those in SNLs and NNLs. Besides, the concentrations of K, Mg, P, Ni and Ti in rice, especially K, were demonstrated to be significantly positively correlated with lifespan indicators (p < 0.05), while the concentrations of Ga, Cd, In and U in rice were negatively correlated with lifespan indicators. Our findings are expected to provide a basis for reasonable dietary mineral element intakes and promote understanding of the relationships between natural environment and regional longevity in China.

In Situ Identification and Spatial Mapping of Microplastic Standards in Paramecia by Secondary-Ion Mass Spectrometry Imaging.

Microplastics (MPs) are universally present in the ecosystem and pose great threats to the environment and living organisms. Research studies have shown that small MPs (<50 µm in diameter) are especially toxic and account for more than half of all MPs collected in the Atlantic Ocean. Nevertheless, current methods for the detection and analysis of MPs are incapable of achieving rapid and in situ analysis of small MPs in the biota to ultimately enable the study of their biological effects. In this work, we report a method that allows rapid in situ identification and spatial mapping of small MPs directly from paramecia with high accuracy by acquiring chemical composition information using secondary-ion mass spectrometry (SIMS) imaging. Specifically, six types of common MPs (polymethyl methacrylate, polyvinyl chloride, polypropylene, polyethylene terephthalate, polyglycidyl methacrylate, and polyamide 6) with a diameter of 1-50 µm were simultaneously imaged with high chemical specificity at a spatial resolution of 700 nm. In situ spatial mapping of a group of MPs ingested by paramecia was performed using SIMS fragments specific to the plastic composition with no sample pretreatment, revealing the aggregation of MPs in paramecia after ingestion. Compared with existing methods, one additional advantage of the developed method is that the MPs and the organism can be analyzed in the same experimental workflow to record their fingerprint spectra, acquiring biochemical information to evaluate MP fate, toxicity, and the MP-biota interaction.

Tuning the pKa of Carboxyfluorescein with Arginine-Rich Cell-Penetrating Peptides for Intracellular pH Imaging.

5-Carboxylfluorescein (FAM) is a conventional pH-responsive fluorophore widely used in fluorescence labeling and imaging. Because of its nonfluorescent structure under acidic conditions, FAM has long been limited to pH determination in a neutral-basic environment. Here, we modified the optical properties of FAM with cationic arginine-rich cell-penetrating peptides (CPPs), tuning the pKa value of FAM to adapt well to pH measurement under diverse pH conditions. With increasing length of polyarginine, the pKa value of FAM was tuned from 6.20 ± 0.06 to 5.17 ± 0.05. The key mechanism for pKa variations was attributed to intramolecular electrostatic attraction and the positive charge of cationic CPPs tend to stabilize the fluorescent dianionic form of FAM. Apart from tunable pKa, arginine-rich CPPs also improved the water solubility, membrane permeability, and organelle-specific localization of FAM. Two conjugated probes FAM-R12 and FAM-(Fxr)3 were selected to monitor intracellular pH fluctuations. Compared to FAM-(Fxr)3, highly positively charged FAM-R12 was more effective in lower pH condition and realized targeted visualization of lysosomal pH changes. The arginine-rich CPP-based strategy offers a promising approach to obtain optimized fluorescent pH probes with adjustable pKa values for organelle-specific pH measurement.

A cell-penetrating ratiometric probe for simultaneous measurement of lysosomal and cytosolic pH change.

A new ratiometric fluorescent probe based on cell-penetrating peptides (CPPs) was constructed for whole-cell pH mapping and simultaneous measurement of pH changes in the cytoplasm and lysosomes. The arginine-rich CPP, R12K worked as linker, carrier and part of the fluorophore. Benefiting from R12K, the fluorescent probe is completely water soluble, membrane permeable and well biocompatible. It shows high selectivity, sensitivity and reversibility to pH fluctuations. The ratio of fluorescence intensities F519/F582 increased from 0.2 to 9.2 over the pH range from 3.3 to 8.1. Intracellular pH mapping was successfully realized owing to the wide distribution of the probe in live cells (even in nucleus). Moreover, cytosolic and lysosomal pH change caused by the stimuli can be simultaneously detected. Compared to other ratiometric pH probes, RhB-R12K-FITC can provide more precise information about H+ redistribution between different cellular compartments.

Chemical Visualization of Sweat Pores in Fingerprints Using GO-Enhanced TOF-SIMS.

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) has been used in imaging of small molecules (<500 Da) in fingerprints, such as gunshot residues and illicit drugs. However, identifying and mapping relatively high mass molecules are quite difficult owing to insufficient ion yield of their molecular ions. In this report, graphene oxide (GO)-enhanced TOF-SIMS was used to detect and image relatively high mass molecules such as poison, alkaloids (>600 Da) and controlled drugs, and antibiotics (>700 Da) in fingerprints. Detail features of fingerprints such as the number and distribution of sweat pores in a ridge and even the delicate morphology of one pore were clearly revealed in SIMS images of relatively high mass molecules. The detail features combining with identified chemical composition were sufficient to establish a human identity and link the suspect to a crime scene. The wide detectable mass range and high spatial resolution make GO-enhanced TOF-SIMS a promising tool in accurate and fast analysis of fingerprints, especially in fragmental fingerprint analysis.

Cell-Penetrating Peptide Spirolactam Derivative as a Reversible Fluorescent pH Probe for Live Cell Imaging.

A colorless and nonfluorescent spirolactam derivative, RhB-R12K, was synthesized by amide condensation between the carboxyl group of rhodamine B (RhB) and the amino group of cell-penetrating peptide (CPP). The fluorescence intensity of RhB-R12K sharply increased as the pH value decreased from 8.0 to 4.9, demonstrating sensitive and reversible response to intracellular pH distribution. This CPP probe was completely water soluble, had low cytotoxicity, was membrane permeable, and was suitable for pH measurement in various organelles by choosing organelle-specific CPP sequences. Interestingly, CPPs acted not only as carriers but also as indispensable parts of fluorophores here. The presence of active groups on the peptides potentially allows for modification with additional dyes to construct multifunctional and ratiometric probes for cell imaging.

Supramolecular assembly-induced yellow emission of 9,10-distyrylanthracene bridged bis(pillar[5]arene)s.

9,10-Distyrylanthracene has been introduced to bridge two pillarenes to form a dimeric host, which can assemble into a linear supramolecular polymer upon cooperatively binding to a neutral guest linker, exhibiting yellow fluorescence emission in solution and solid states.