Rapid Staining Research Articles

Feline sporotrichosis: occurrence in the Metropolitan Region of the municipality of Recife and anatomical distribution of lesions on cats’ body

Sporotrichosis is a fungal infection of worldwide importance caused by a saprophyte fungus of the genus Sporothrix. The objective of this study was to report the occurrence of feline sporotrichosis in the Metropolitan Region of the city of Recife and the anatomical distribution of lesions on the bodies of cats. Stray cats (n=100) with suggestive sporotrichosis lesions were examined. Material from the lesion was collected from all patients using an imprint and/or sterile swab for cytology. The material was placed on a glass slide and stained using rapid panoptic staining. Data on the localization of each skin lesion were recorded, and cigar-shaped yeast structures were searched at the microscopical examination. A heat map graphically represented the anatomical distribution of each lesion. In 70% (70/100) of samples were visualized fungal structure compatible with Sporothrix spp. There was a predominance of positive males (?2=6.691; p=0.0097). Skin lesions related to Sporothrix spp. infections were unevenly distributed throughout the animal's body. Based on the interpolation of data, the chance for detecting lesions was higher on the nose, anterior paw, and ears than on other body sites (H=40.0043; p=0.0000). Because of the high positivity in these cats, measures of health vigilance are imperative to reduce the risk of animal and human infection.

Thioflavin-T: application as a neuronal body and nucleolar stain and the blue light photo enhancement effect

Thioflavin-T (THT) is a common and indispensable tool for the study of amyloid pathologies and protein aggregation, both in vitro and for histological samples. In this study we expand the use of THT beyond its canonical usage for staining amyloid plaques and demonstrate its novel use as an easy and rapid stain comparable to fluorescent Nissl staining, allowing for clear discernment of neuronal cell bodies and also nucleoli in fixed tissue and live cells. We believe that this is of value for any lab that studies central nervous system (CNS) tissues. Furthermore, we show that THT could potentially be used as a an alternative to the use of fluorescent reporters or other more costly RNA binding compounds in the study of nucleolar dynamics owing to its ability to clearly stain nucleoli in live cells. We also discovered the previously unreported effect of blue light exposure on the photo enhancement of THT excited by a 488 nm laser in stained tissue sample and how to avoid complications arising from this effect. Finally, we provide a simple protocol that can be easily adjusted either for using THT as a neuronal cell body and nucleoli stain, compatible with antibody based staining methods tested up to 4 fluorophores, or alternatively by using an additional washing step the protocol may be used for amyloid plaque detection in fixed brain tissue.

A fluorescent probe for rapid staining and real-time detection of autophagy in single cells by lighting-up G-quadruplexes

Autophagy is a crucial physiological mechanism in eukaryotes for maintaining metabolic balance, and its dysregulation is closely linked to human neurodegenerative diseases, tumors, and others. Real-time tracking of autophagy in live cells is essential for elucidating pathogenesis and developing new therapeutics. Here, a supramolecular assembly, SCY-3, has been designed to monitor autophagy by selectively lighting-up the G-quadruplex structures entering the lysosomes. SCY-3 exhibits low cytotoxicity, long optical detection wavelength, and excellent photostability, making it ideal for prolonged tracking in single cells and living organisms. Notably, SCY-3 can rapidly penetrate lysosomes, offering significant advantages in fast staining. These attributes position SCY-3 as a promising universal tool for autophagy tracking with broad applicability.

Bed bugs, Cimex lectularius: Undercover agents in forensic investigations.

Insects have long played a role in forensic investigations and can be used to estimate minimum time since death, corpse translocation, and link an individual to a crime scene. Bed bugs (Cimex lectularius) are wingless ectoparasitic insects of potential forensic utility, given that all mobile life stages feed on vertebrate blood. Successful profiling of autosomal short tandem repeats (STRs) from human DNA isolated from bed bugs has been previously reported. This proof-of-concept study looked to expand this work and determine any possible limitations of using bed bugs for both rapid stain identification (RSID™) for human blood and Y-STR profiling. To achieve this, bed bugs were fed either human male only or human pooled (female:male) blood for 30 min and subsequently collected at 12-h intervals up to 108 h post-blood meal (PBM). RSID™ blood testing was successful from the bed bug carcass remaining after DNA isolation, regardless of blood meal type and time of collection PBM. Complete Y-STR profiles were generated from bed bugs <60 h PBM. As the time PBM increased, DNA quantity decreased, while the degradation index increased. Collection of bed bugs at a crime scene could provide a valuable source of human blood for Y STR profiling and be used to link an individual to a crime scene or for potential male suspect exclusion. Future studies should look to replicate the results of this proof-of-concept study with larger numbers of bed bugs, more diverse blood donors, and additional STR profiling kits.

Fast Green FCF Improves Depiction of Extracellular Matrix in Ex Vivo Fluorescence Confocal Microscopy.

Rapid microscopic analysis of tissue is an essential diagnostic tool in oncological surgery. The gold standard for intraoperative histological tissue evaluation is frozen sections. However, frozen sections are prone to a variety of artefacts and require skilled staff and specialized lab equipment. A potential method for rapid intraoperative tissue evaluation that does not require fixation, freezing, or sectioning of the tissue is ex vivo fluorescence confocal microscopy (FCM). The visualization of the structurally important extracellular matrix (ECM) in conventional ex vivo FCM lags behind the standards of conventional histology. The objective of this study was to find a stain that would improve the depiction of the ECM to resemble FFPE H&E sections as closely as possible. Eleven different tissue stains were tested on 122 tissue samples submitted to the Department of Pathology at the Medical University of Vienna. This study was conducted on the RS-G4 Upright (Caliber I.D. Rochester, NY, USA, distributed in Europe by MAVIG GmbH, Munich, Germany). Fast Green FCF (FGFCF) in combination with acridine orange as a nuclear stain improved the visibility of the structural details of the ECM. Morphological details in FCM were equivalent or even superior to frozen sections in most analyzed categories. The addition of FGFCF to the conventional staining protocol improves the assessment of the ECM and analysis of fibrosis. The rapid staining protocol is compatible with an application in intraoperative microscopy.

Optimization of spermatozoa analysis in mice: A comprehensive protocol

Sperm quality is critical to predict reproductive alterations caused by immunological factors or toxicant agents. Yet, no detailed protocol has been published focusing on analyses of sperm parameters in mice. Our aim was to evaluate the most efficient diluent for mice sperm analyses and to optimize the sperm morphology classification, through the comparison of different staining methods. The diluents assessed were PBS (baseline), HTF, DMEM, 1 % BSA in PBS and 9 % skimmed powdered milk diluted in PBS. Spermatozoa were evaluated for vitality, motility, and morphology, smears were stained with Papanicolaou, HE, Giemsa, and Rapid staining. Sperm vitality and total motility reached better scores in milk based and DMEM diluents. HE raised up as an effective option since its combination with any of the diluents we tested, resulted in a fair staining, which was appropriated to evaluate mice spermatozoa. Finally, based on WHO manual, we have updated the current morphological classification for mice sperm, since we have detailed the head defects as well as included midpiece and tail defects on it. Taken together, we presented a useful, low cost, and reliable method to assess sperm morphology that could be employed worldwide by laboratories dedicated to study reproductive biology on mice model.

A Dual-Modal, Label-Free Raman Imaging Method for Rapid Virtual Staining of Large-Area Breast Cancer Tissue Sections.

As one of the most common cancers, accurate, rapid, and simple histopathological diagnosis is very important for breast cancer. Raman imaging is a powerful technique for label-free analysis of tissue composition and histopathology, but it suffers from slow speed when applied to large-area tissue sections. In this study, we propose a dual-modal Raman imaging method that combines Raman mapping data with microscopy bright-field images to achieve virtual staining of breast cancer tissue sections. We validate our method on various breast tissue sections with different morphologies and biomarker expressions and compare it with the golden standard of histopathological methods. The results demonstrate that our method can effectively distinguish various types and components of tissues, and provide staining images comparable to stained tissue sections. Moreover, our method can improve imaging speed by up to 65 times compared to general spontaneous Raman imaging methods. It is simple, fast, and suitable for clinical applications.

P-041 Impact of cigarette smoking on level of sperm reactive oxygen species in normozoospermic men

Abstract Study question Does cigarette smoking influence the levels of sperm reactive oxygen species (ROS) in men with normal semen routine parameters? Summary answer Cigarettes impact the sperm ROS of normozoospermic smokers, whose sperm ROS level is elevated when they smoke equal to or more than 10 cigarettes daily. What is known already Increasing evidence indicates that oxidative stress (OS) plays an independent role in the etiology of male infertility, with 30% to 80% of infertile men having elevated ROS levels in semen. Cigarette smoking leads to an imbalance between ROS and antioxidants in the semen of smokers, which may negatively affect semen quality and male fertility. However, few studies concern whether cigarette smoking would influence the sperm ROS levels in normozoospermic men. Study design, size, duration Data of semen analysis from January 2021 to December 2023 were involved. Men with normal semen routine parameters, including sperm concentration, total sperm number, sperm progressive motility, total motility and normal forms of sperm morphology, were included in the study. Sperm ROS levels of normozoospermic men were retrospectively analyzed. Men with varicocele and leucocytospermia were excluded. Participants/materials, setting, methods Sperm count and motility were analyzed by the computer-assisted sperm analysis (CASA) system, while sperm morphology was examined after the Diff-Quik rapid staining procedure. Sperm ROS was measured by flow cytometry using the probe DCFH-DA. Sperm ROS levels of smokers and non-smokers were compared by the Mann-Whitney U test. The Kruskal-Wallis H test was used for the comparison of sperm ROS levels among the control group and smoking subgroups. Main results and the role of chance A total of 2946 subjects were involved in the present study. The included subjects were divided into smokers (n = 730) and non-smokers (n = 2216) according to the clinical records. And the included smokers were subsequently divided into three subgroups according to the daily cigarettes taken, which were group A (1∼9 cigarettes/day, n = 184), group B (1∼19 cigarettes/day, n = 315), and group C (&amp;gt;20 cigarettes/day, n = 231). No statistically significant differences in age or BMI were found between smokers and non-smokers with normozoospermia. The sperm ROS level of the smoker was higher than that of the non-smoker [14.53% (14.84%) vs. 13.07% (12.09%), p = 0.001]. Compared to the control group (non-smoker), the sperm ROS levels of group B [13.07% (12.09%) vs. 15.44% (14.28%), p = 0.002] and group C [13.07% (12.09%) vs. 14.80% (14.31%)., p = 0.042] were significantly elevated. ROS levels in the control group and group A were comparable [13.07% (12.09%) vs. 13.46% (15.74%)., p = 0.391]. Limitations, reasons for caution It is a single-center study and the above results were from a retrospective analysis. Wider implications of the findings The above results indicate that cigarette smoking, although it would not deteriorate semen routine parameters, may increase the ROS level in sperm. Elevated ROS in sperm may lead to an imbalance of redox in sperm, which may contribute to male infertility. Trial registration number not applicable

Rapid multiplex immunohistochemistry for characterizing tumor-immune microenvironment

Intratumoral immune profiles are related to prognosis and therapeutic efficacy, and could result in personalized treatments based on biomarkers. To develop a multiplex, quantitative, and rapid tissue evaluation method based on the clinically established standard immunohistochemistry (IHC), a 6-marker rapid multiplex IHC was developed based on our previously reported 14-marker multiplex IHC by reducing the number of labels and accelerating the staining procedure. First, fewer labels were required to identify the same immunological features linked to prognosis in 14-marker multiplex IHC analyses. The six selected markers showed a significant correlation with the 14 markers in the immune classification. Next, a rapid staining protocol was developed by optimizing the reaction temperature, chromogen, and washing time, allowing the completion of 6-marker analysis in 5 h and 49 min, as opposed to the several days required for conventional multiplex IHC. Validation of benign tonsil and head and neck cancer tissues revealed a significant correlation between rapid and conventional 6-makrer multiplex IHC in terms of staining intensities, densities of T cells, macrophages, lymphoid/myeloid immune cell ratios, and spatial profiles of intratumoral immune infiltrates. This method may enable quantitative assessment of the tumor-immune microenvironment on a clinically feasible time scale, which promotes the development of tissue biomarker-guided therapeutic strategies.

Fluorescent carbon nanodot-based rapid cell nucleus staining and application for diagnosis.

Cell nucleus status decides the activities of corresponding cells, making its rapid and effective staining important for revealing the actual condition of biological environment in life science and related fields. In this study, fast staining of cell nucleus is realized by fluorescent carbon nanodots (CDs). The staining mechanism is due to the positively charged CD surface-induced cell membrane penetration, which facilitates the CD-nucleus binding via electrostatic attraction. The size of cell nucleus is easily measured with fluorescence imaging technique. In addition, the CD-based cell nucleus stain is applied for discriminating the normal and cancer cells by determining the cell-to-nucleus ratio with fluorescence images.

Comparison of staining quality between rapid and routine hematoxylin and eosin staining of frozen breast tissue sections: an observational study.

To compare the staining quality between rapid hematoxylin and eosin (H&E) staining and routine H&E staining of frozen breast tissue sections. In this cross-sectional observational study, 120 frozen breast tissue sections were randomly assigned to rapid or routine H&E staining (n = 60 per group). Rapid H&E staining used a 7:1 mixture of modified Gill's hematoxylin and alcohol-soluble 1% eosin Y. The staining quality of each section was evaluated and scored. A score of >7 was considered excellent, a score of 6 to 7 good, and a score of ≤5 poor. The staining time for rapid staining was approximately 3 minutes, whereas that of routine staining was approximately 12 minutes. There were no significant differences in the staining quality scores or proportions of sections in each grade between the two staining methods. The proportions of sections that were classified as excellent or good were 96.7% and 98.3% for rapid and routine staining, respectively. In frozen breast tissue sections, rapid H&E staining may provide staining quality that is comparable to that of routine staining, while markedly reducing the staining time.

User-Friendly Multifunctional Red-Emissive Carbon Dots for Rapid Cell Nucleus Staining via Targeting Nuclear Proteins.

Cytoarchitectural staining is of great importance in disease diagnosis and cell biology research. This study developed user-friendly multifunctional red-emissive carbon dots (R-CDs) for rapid cell nucleus staining via targeting nuclear proteins. R-CDs, simply prepared by electrochemical treatment of 1,2,4-benzenetriamine, exhibit strong emission at 635 nm when excited at 507 nm. The R-CDs can rapidly stain the nucleus of human SH-SY5Y, HepG2, and HUH-7 cells with a high signal-to-noise ratio owing to fluorescence enhancement after entering the nucleus. Compared to conventional cytosolic dyes such as Hoechst and DAPI, R-CDs are cheaper, more highly dispersed in water, and more stable (requiring no stringent storage conditions). The R-CDs show stable optical properties with insignificant photobleaching over 7 days and salt resistance up to 2 M of NaCl. More importantly, R-CDs, possessing a positive charge, allow rapid staining of live cells (3 min) and dead cells (10 s) in saline. According to kinetic variation, R-CDs can distinguish live cells from dead cells. Staining exhibits high efficiency in onion epidermal cells, Aspergillus niger, Caenorhabditis elegans, and human spermatozoa. The mechanism for efficient staining is based on their fast accumulation in the nucleus due to their small size and positive charge and strong interaction with nuclear proteins at amino acid residues of histidine and arginine, resulting in fluorescence enhancement by dozens of times. The developed R-CDs do not bind to DNA and would not cause genetic damage and will find various safe applications in biological and medical fields.

Evidence of Incomplete Feeding Behaviors among South Carolina Tick Populations.

Dynamic environmental conditions, such as climate change and host availability, have greatly influenced the expansion of medically relevant tick vectors into new regions throughout the southeastern United States of America. As tick populations migrate into new areas, it has been suggested they can exhibit a phenomenon known as incomplete feeding. With this phenomenon, tick vectors feed on more than one host at each life stage, thus increasing the likelihood of pathogen transmission. Although this behavior is not well understood, it presents an important threat to human health. Here we present evidence of incomplete feeding behaviors in multiple tick species in South Carolina. Engorged, blood-fed female ticks were collected from feral dogs at animal shelters across South Carolina in 2022. All ticks were tested for human blood meals using rapid stain identification blood tests. Approximately one third (33.78%) of all ticks tested positive for a human blood meal, with various patterns seen across species, geographic location, and collection month. The results of this pilot study follow the current national trend of increasing rates of tick-borne disease incidence in the southeastern United States of America and warrant further investigation into the relationship between seasonality, geographic distribution, species, and incomplete feeding among tick populations in South Carolina.

Microwave-assisted and methanol/acetic acid-free method for rapid staining of proteins in SDS-PAGE gels

We describe a microwave-assisted, methanol and acetic acid-free, inexpensive method for rapid staining of SDS-PAGE proteins. Only citric acid, benzoic acid, and Coomassie brilliant blue G-250 (CBG) were used. Microwave irradiation reduced the detection duration, and proteins in a clear background were visualized within 30 min of destaining, after 2 min of fixing and 12 min of staining. By using this protocol, comparable band intensities were obtained to the conventional methanol/acetic acid method.

Optimizing the ratio of coomassie and methylene blue dyes for a cost-effective and rapid staining of PET, PVC, PP, PS, LLDPE, LDPE, and HDPE

The ubiquitous presence of plastic brought on by the extensive use of plastic products calls for efficient and rapid plastic detection methods to detect and evaluate pollution. The commonly used Nile red dye takes many hours and is expensive while also not equally efficient across all the common plastic waste. To address this, we investigated the staining efficiency and optimized the ratio of a combined Coomassie brilliant blue and Methylene blue dye. In the optimisation process, Methanol-based Coomassie and Methylene blue dyes effectively stained the Polyethylene Terephthalate (PET), Polypropylene (PP), Polystyrene (PS), Linear Low-Density Polyethylene (LLDPE), Low-Density Polyethylene (LDPE), and Linear Low-Density Polyethylene (HDPE) plastics without compromising the plastic's integrity. Image analysis showed a generally better staining efficacy compared to Nile red. Through systematic experimentation, we identified specific optimal ratios of Coomassie (C) brilliant blue: Methylene (M) blue for various plastics: 5:5 (mass) for PVC, 7:3 (mass) for PET, and 8:2 (mass) for PP, LDPE, and HDPE. Additionally, the ratio of 10:0 (mass) was found suitable for PS and LLDPE. Given the cost-effectiveness, efficiency, and accessibility of the blue dyes in labs, the optimized ratio of the blue dyes makes it suitable for large-scale plastic staining across the six tested types of plastic, replacing Nile red.

Modification of Rapid Papanicolaou Stain with Phloxine: A Study Assessing the Utility in Gynecological Smears.

Rapidity and reliability are the major advantages of cytopathology in tumor diagnosis. The need for minimal turnaround time for assessing cytological smears has encouraged innovations in staining techniques that require lesser staining time with unequivocal cell morphology. Rapid Pap staining was introduced as a hybrid to conventional Pap stain. It improves staining quality, gets over the staining time in restriction, and is a more efficient technique financially. In the present study, a modified staining technique was adopted where phloxine is added as one of the cytoplasmic stain components in rapid Pap stain kits. The aim of this study was to assess whether the modification of the existing procedure by adding phloxine as one of the components of the cytoplasmic stain intensifies the cytoplasmic differentiation and cytoplasmic staining in gynecological smears. This was a prospective study done on 50 cases of gynecological smears. Two smears were collected from each patient and fixed in 100% propanol and stained with the rapid Pap stain kit procedure and modified rapid Pap stain with phloxine. Slides were then analyzed by two pathologists blinded to the staining method used. Five parameters were considered and scored (background, cell morphology, nuclear staining, cytoplasmic differentiation, and cytoplasmic staining). The quality index for each method was calculated by finding out the ratio of the actual score obtained to the maximum score possible. Both the staining methods had comparable results. For both pathologists, the quality index calculated for modified Pap stain was found to be marginally higher than the quality index for rapid Pap. The mean quality index was comparable for modified Pap (0.91) and rapid Pap (0.89). The efficacy of modified Pap stain with phloxine to distinctly stain the cytoplasm is comparable with that of rapid Pap stain. In addition, the intensity of staining can be enhanced with little cost outlay, and this can be especially beneficial in low-resource settings.

Comparison of Intraoperative Imprint Cytology versus Frozen Section for Sentinel Lymph Node Evaluation in Breast Cancer. A study along with Systematic Review and Meta-analysis of literature.

Sentinel lymph node (SLN) is the first lymph node to drain the lymph from a particular region involved by cancer. The commonly performed intraoperative methods for SLN evaluation are touch imprint cytology (TIC) and frozen section (FS). The present study aimed to determine the sensitivity, specificity and accuracy of TIC and FS with histopathological diagnosis as gold standard. The nodes were bissected along their long axis and wet surface was imprinted on to clean glass slides followed by toluidine blue and rapid Papanicolaou staining. Subsequently the lymph node slices were cut at three levels using the cryostat machine and stained with Hematoxylin and eosin stain. The cytological and FS findings were compared and the specificity, sensitivity, accuracy, positive predictive value (PPV) and negative predictive value (NPV) of TIC and FS was evaluated taking histopathological diagnosis as gold standard. In addition, pooled sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy for touch imprint cytology and frozen section were assessed for the studies included in the meta-analysis. The specificity, sensitivity, diagnostic accuracy, positive predictive value and negative predictive value of touch imprint cytology were 100%, 88.2%, 90%, 100% and 60% respectively. The specificity, sensitivity, diagnostic accuracy, PPV and NPV of frozen section were 100%, 94.1%, 95%, 100% and 75% respectively. The sensitivity of TIC and FS for detection of micrometastasis was 60% and 80% respectively. The pooled sensitivity and specificity for touch imprint cytology were 85.24% (95% CI, 83.46%-86.90%), and 98.99% (95% CI, 98.69%-99.23%) respectively. The pooled sensitivity and specificity for frozen section examination were 90.45% (95% CI, 85.15%-94.34%), and 100% (95% CI, 99.24%-100%) respectively. Even though the sensitivity of FS was better than imprint cytology in detection of micrometastasis, TIC is a rapid inexpensive technique which can be utilized in remote areas in absence of cryostat machine. The sensitivity of the two techniques with respect to detection of macrometastasis was comparable. This meta-analysis highlights the accuracy of the touch imprint cytology and frozen section examination in the intra-operative detection of malignancy in breast cancer.

Abstract 6879: Development of a rapid multiplex immunohistochemistry for characterizing tumor-immune microenvironment

Abstract Intratumoral immune profiles have been related to prognosis and therapeutic efficacy; this could result in personalized treatment based on biomarkers. Although techniques for multifactorial evaluation of different tumor-immune microenvironments have been established, their practical implementation in clinical settings still requires further improvement, where the time required for testing needs to be reduced and the technology needs to be clinically validated. To develop a multiplex, quantitative, and rapid tissue evaluation method based on clinically established standard immunohistochemistry (IHC), we attempted to develop 6-marker rapid multiplex IHC based on our previously reported 14-marker multiplex IHC via the reduction of the number of labels and speeding up the staining procedure (Tsujikawa et al. Cell Reports, 2017). First, we confirmed whether immunological features that were found to be associated with prognosis in 14-marker multiplex IHC analyses could be identified with fewer labels. Quantitative results by selected six markers exhibited significant correlation with those by 14 markers in terms of immune classifications based on the balance of lymphoid and myeloid cells. Next, we developed a rapid staining protocol by optimizing the reaction temperature, chromogen, and washing time, allowing the completion of 6-marker analysis in 5 hours and 49 minutes as opposed to several days required for the conventional multiplex IHC. By using six markers, validation in benign tonsil tissue and head and neck cancer tissue exhibited significant correlation between rapid and standard multiplex IHC in terms of staining intensities, densities of T cells, macrophages, lymphoid/myeloid immune cells ratios, and spatial profiles of intratumoral immune infiltrates. This method may enable a quantitative assessment of tumor-immune microenvironment on a clinically feasible time scale, which promote the development of tissue biomarker-guided therapeutic strategies. Citation Format: Alisa Kimura, Takahiro Tsujikawa, Hiroki Morimoto, Junichi Mitsuda, Sumiyo Saburi, Shigeyuki Mukudai, Hikaru Nagao, Nana Sakurai, Nanako Murakami, Aya Miyagawa-Hayashino, Hiroshi Ogi, Saya Shibata, Eiichi Konishi, Kyoko Itoh, Shigeru Hirano. Development of a rapid multiplex immunohistochemistry for characterizing tumor-immune microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6879.

Development of 1,3a,6a-triazapentalene derivatives as practical erasable fluorescent cell staining reagents

1,3a,6a-Triazapentalene (TAP) derivatives were developed as erasable fluorescent cell staining reagents that can be used with a confocal laser microscope. To enhance the utility of previously developed erasable fluorescent cell staining reagents such as 2-biphenyl-TAP (1a) for practical applications in confocal laser microscopy, a carbonyl group with strong electron-withdrawing properties was introduced between the TAP core skeleton and the biphenyl group, leading to a longer wavelength shift in absorption and emission maxima. While the analog 1b, featuring only the carbonyl group insertion, declined significantly in fluorescence intensity, the two-carbon extended ketone analog 1d showed a desirable shift in absorption maximum towards longer wavelengths, green fluorescence, and high fluorescence intensity. Cell staining experiments using 1d demonstrated its usefulness, including rapid staining, erasable fluorescent staining, and compatibility with confocal laser microscopy employing a standard filter.

Target-activated multicolor fluorescent dyes for 3D imaging of plasma membranes and tracking of apoptosis.

Real-time tracking of dynamic changes in the three-dimensional morphology of the cell plasma membrane is of great importance for a deeper understanding of physiological processes related to the cell plasma membrane. However, there is a lack of imaging dyes that can specifically be used for a long term labelling of plasma membranes, especially for plant cells. Here, we have used molecular engineering strategies to develop a series of target-activated multicolour fluorescent dyes that can be used for long-term and three-dimensional imaging of plant cell plasma membranes. By combining different electron acceptors and donors, four molecular backbones with different emission colours from green to NIR have been obtained. In the designed styrene-based dyes, referred to as the SD dyes, several functional groups were introduced into the backbones to achieve the properties of target-activated fluorescence, rapid and wash-free staining, high plasma membrane targeting ability and long-term imaging function. Using onion epidermal cells as a platform, these dye molecules can provide high-quality imaging of the plasma membrane for up to 6 hours, providing a powerful tool for long-term monitoring of plasma membrane-related biological events. Calcium-mediated apoptosis of plant cells has been tracked for the first time by monitoring the morphological changes of the plasma membrane in real time using SD dyes. These dyes also exhibit excellent 3D imaging performance of the plasma membrane and were further used to track in real time the 3D morphological changes of the plasma membrane during plasmolysis of plant cells, providing a powerful imaging tool for three-dimensional (3D) biology. This work provides a set of multi-colour dye tools for long-term and three-dimensional imaging of plant cell plasma membranes, and also provides molecular design principles for guiding the transmembrane transport of small molecules.