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Cell membrane–targeted bioimaging is a prerequisite for studying the roles of membrane-associated biomolecules in various physiological and pathological processes. However, long-term in situ bioimaging on the cell membrane with conventional fluorescent probes leads to diffusion into cells from the membrane surface. Therefore, we herein proposed a de novo strategy to construct an antidiffusion probe by integrating a fluorochrome characterized by strong hydrophobicity and low lipophilicity, with an enzyme substrate to meet this challenge. This precipitating fluorochrome HYPQ was designed by conjugating the traditionally strong hydrophobic solid-state fluorochrome 6-chloro–2-(2-hydroxyphenyl) quinazolin-4(3H)-one (HPQ) with a 2-(2-methyl–4H-chromen–4-ylidene) malononitrile group to obtain closer stacking to lower lipophilicity and elongate emission to the far-red to near-infrared wavelength. As proof-of-concept, the membrane-associated enzyme γ-glutamyltranspeptidase (GGT) was selected as a model enzyme to design the antidiffusion probe HYPQG. Then, benefiting from the precipitating and stable signal properties of HYPQ, in situ imaging of GGT on the membrane was successfully realized. Moreover, after HYPQG was activated by GGT, the fluorescence signal on the cell membrane remained unchanged, with incubation time even extending to 6 h, which is significant for in situ monitoring of enzymatic activity. In vivo testing subsequently showed that the tumor region could be accurately defined by this probe after longterm in situ imaging of tumor-bearing mice. The excellent performance of HYPQ indicates that it may be an ideal alternative for constructing universal antidiffusion fluorescent probes, potentially providing an efficient tool for accurate imaging-guided surgery in the future.

Carbon dots (CDs) have attracted significant interest as one of the most emerging photoluminescence (PL) nanomaterials. However, the realization of CDs with dominant near‐infrared (NIR) absorption/emission peaks in aqueous solution remains a great challenge. Herein, CDs with both main NIR absorption bands at 720 nm and NIR emission bands at 745 nm in an aqueous solution are fabricated for the first time by fusing large conjugated perylene derivatives under solvothermal treatment. With post‐surface engineering, the polyethyleneimine modified CDs (PEI‐CDs) exhibit enhanced PL quantum yields (PLQY) up to 8.3% and 18.8% in bovine serum albumin aqueous and DMF solutions, which is the highest PLQY of CDs in NIR region under NIR excitation. Density functional theory calculations support the strategy of fusing large conjugated perylene derivatives to achieve NIR emissions from CDs. Compared to the commercial NIR dye Indocyanine green, PEI‐CDs exhibit excellent photostability and much lower cost. Furthermore, the obtained PEI‐CDs illustrate the advantages of remarkable two‐photon NIR angiography and in vivo NIR fluorescence bioimaging. This work demonstrates a promising strategy of fusing large conjugated molecules for preparing CDs with strong NIR absorption/emission to promote their bioimaging applications. Carbon dots with both main near‐infrared (NIR) absorption and emission bands in aqueous solution are fabricated through solvothermal fusion of large conjugated perylene derivatives. After post‐surface modification with polyethyleneimine, high photoluminescence quantum yields in NIR region up to 8.3% and 18.8% are obtained in their bovine serum albumin aqueous and DMF solutions, respectively.

Carbon dots (CDs) or carbonized polymer dots (CPDs) are an emerging class of optical materials that have exceptional applications in optoelectronic devices, catalysis, detection, and bioimaging. Although cell studies of CPDs have produced impressive results, in vivo imaging requires available CPDs to fluoresce in the near‐infrared‐II (NIR‐II) window (1000−1700 nm). Here, a two‐step bottom‐up strategy is developed to synthesize NIR‐CPDs that provide bright emissions in both NIR‐I and NIR‐II transparent imaging windows. The designed strategy includes a hydrothermal reaction to form a stable carbon core with aldehyde groups, followed by the Knoevenagel reaction to tether the molecular emission centers. This procedure is labor‐saving, cost‐efficient, and produces a high yield. The NIR‐CPDs enable high‐performance NIR‐II angiography and real‐time imaging of the disease degree of colitis noninvasively. This technology may therefore provide a next‐generation synthesis strategy for CPDs with rational molecular engineering that can accurately tune the absorption/emission properties of NIR‐emissive CPDs. Carbonized polymer dots (CPDs) are remarkable optical materials with low preparation cost, high biocompatibility, and excellent optical properties. The emission wavelengths of current CPDs are limited in the visible to NIR‐I range, impeding their applications in the transparent NIR‐II window. A new series of NIR‐CPDs is reported through a two‐step hydrothermal/Knoevenagel reaction with adjustable absorption/emission wavelength for NIR‐II bioimaging.

Since its inception in the early 80s, inductively coupled plasma–mass spectrometry has developed to the method of choice for the analysis of elements in complex biological systems. High sensitivity paired with isotopic selectivity and a vast dynamic range endorsed ICP-MS for the inquiry of metals in the context of biomedical questions. In a stand-alone configuration, it has optimal qualities for the biomonitoring of major, trace and toxicologically relevant elements and may further be employed for the characterisation of disrupted metabolic pathways in the context of diverse pathologies. The on-line coupling to laser ablation (LA) and chromatography expanded the scope and application range of ICP-MS and set benchmarks for accurate and quantitative speciation analysis and element bioimaging. Furthermore, isotopic analysis provided new avenues to reveal an altered metabolism, for the application of tracers and for calibration approaches. In the last two decades, the scope of ICP-MS was further expanded and inspired by the introduction of new instrumentation and methodologies including novel and improved hardware as well as immunochemical methods. These additions caused a paradigm shift for the biomedical application of ICP-MS and its impact in the medical sciences and enabled the analysis of individual cells, their microenvironment, nanomaterials considered for medical applications, analysis of biomolecules and the design of novel bioassays. These new facets are gradually recognised in the medical communities and several clinical trials are underway. Altogether, ICP-MS emerged as an extremely versatile technique with a vast potential to provide novel insights and complementary perspectives and to push the limits in the medical disciplines. This review will introduce the different facets of ICP-MS and will be divided into two parts. The first part will cover instrumental basics, technological advances, and fundamental considerations as well as traditional and current applications of ICP-MS and its hyphenated techniques in the context of biomonitoring, bioimaging and elemental speciation. The second part will build on this fundament and describe more recent directions with an emphasis on nanomedicine, immunochemistry, mass cytometry and novel bioassays.

Photoluminescent materials (PLNs) are photoluminescent materials that can absorb external excitation light, store it, and slowly release it in the form of light in the dark to achieve long-term luminescence. Developing near-infrared (NIR) PLNs is critical to improving long-afterglow luminescent materials. Because they excite in vitro, NIR-PLNs have the potential to avoid interference from in vivo autofluorescence in biomedical applications. These materials are promising for biosensing and bioimaging applications by exploiting the near-infrared biological window. First, we discuss the biomedical applications of PLNs in the first near-infrared window (NIR-I, 700–900 nm), which have been widely developed and specifically introduce biosensors and imaging reagents. However, the light in this area still suffers from significant light scattering and tissue autofluorescence, which will affect the imaging quality. Over time, fluorescence imaging technology in the second near-infrared window (NIR-II, 1000–1700 nm) has also begun to develop rapidly. NIR-II fluorescence imaging has the advantages of low light scattering loss, high tissue penetration depth, high imaging resolution, and high signal-to-noise ratio, and it shows broad application prospects in biological analysis and medical diagnosis. This critical review collected and sorted articles from the past 5 years and introduced their respective fluorescence imaging technologies and backgrounds based on the definitions of NIR-I and NIR-II. We also analyzed the current advantages and dilemmas that remain to be solved. Herein, we also suggested specific approaches NIR-PLNs can use to improve the quality and be more applicable in cancer research.

Background Imaging data contains a substantial amount of information which can be difficult to evaluate by eye. With the expansion of high throughput microscopy methodologies producing increasingly large datasets, automated and objective analysis of the resulting images is essential to effectively extract biological information from this data. CellProfiler is a free, open source image analysis program which enables researchers to generate modular pipelines with which to process microscopy images into interpretable measurements. Results Herein we describe CellProfiler 4, a new version of this software with expanded functionality. Based on user feedback, we have made several user interface refinements to improve the usability of the software. We introduced new modules to expand the capabilities of the software. We also evaluated performance and made targeted optimizations to reduce the time and cost associated with running common large-scale analysis pipelines. Conclusions CellProfiler 4 provides significantly improved performance in complex workflows compared to previous versions. This release will ensure that researchers will have continued access to CellProfiler’s powerful computational tools in the coming years.

Rolling circle replication (RCR), including rolling circle amplification (RCA) and rolling circle transcription (RCT), is an isothermal enzymatic reaction. Because of its high amplification efficiency, RCR is a powerful biosensing tool for detecting biomolecules. In recent years, RCR has also been extended to the field of bioimaging to better understand biological pathways. Furthermore, RCR provides a simple technique to design and generate DNA/RNA structures with unique advantages in delivering drugs and enhanced targeting ability. In this review, we introduce the fundamentals of RCR and describe the most recent advances in RCR-based detection methods and delivery vehicles for biosensing, bioimaging, and biomedicine. Finally, some challenges and further opportunities of RCR-based biotechnology are discussed. Rolling circle replication (RCR) is an isothermal nucleic acid amplification approach that holds great potential in biotechnology.RCR can serve as a powerful biosensing platform for the detection of various biomolecules in bioanalysis and diagnosis.In bioimaging, RCR can provide a unique perspective in comprehensive understanding of biological processes and effective diagnosis of diseases.Programmable, biodegradable RCR-based assemblies have been used as versatile advanced drug carriers for targeted theranostics.

Hyperspectral imaging (HSI) is a technique that allows for the simultaneous acquisition of both spatial and spectral information. While HSI has been known for years in the field of remote sensing, for instance in geology, cultural heritage, or food industries, it recently emerged in the fields of nano- and micromaterials as well as bioimaging and -sensing. Herein, the attractiveness of HSI arises from the suitability for generating knowledge about environment-specific optical properties, such as photoluminescence of optical probes in a biological sample or at a single-crystal/particle level, to be leveraged into better understanding of structure-property relationships and nano-bio interactions, respectively. Moreover, given its excellent spectral resolution, HSI is highly suitable for optical multiplexing in multiple dimensions, as sought after for, e.g., high throughput biological imaging by simultaneous tracking of multiple targets. Overall, HSI is an emerging technique that has the potential to transform analytical approaches from biomedicine to advanced materials research. This Trends Article provides insight into the potential of HSI, highlighting selected examples from well-established fields including environmental monitoring and food quality control to set the stage for the discussion of emerging opportunities at the micro- and nanoscale. Herein, special focus is set on photoluminescent micro- and nanoprobes for health and spectral conversion applications.

Surface-enhanced Raman scattering (SERS) has emerged as a valuable technique for molecular identification. Due to the characteristics of high sensitivity, excellent signal specificity, and photobleaching resistance, SERS has been widely used in the fields of environmental monitoring, food safety, and disease diagnosis. By attaching the organic molecules to the surface of plasmonic nanoparticles, the obtained SERS tags show high-performance multiplexing capability for biosensing. The past decade has witnessed the progress of SERS tags for liquid biopsy, bioimaging, and theranostics applications. This review focuses on the advances of SERS tags in biomedical fields. We first introduce the building blocks of SERS tags, followed by the summarization of recent progress in SERS tags employed for detecting biomarkers, such as DNA, miRNA, and protein in biological fluids, as well as imaging from cell, bacteria, tissue to tumors. Further, we illustrate the appealing applications of SERS tags for delineating tumor margins and cancer diagnosis. In the end, perspectives of SERS tags projecting into the possible obstacles are deliberately proposed in future clinical translation.