Fluorescence Signal Research Articles

ProDiVis: a method to normalize fluorescence signal localization in 3D specimens.

A common problem in confocal microscopy is the decrease in intensity of excitation light and emission signal from fluorophores as they travel through 3D specimens, resulting in decreased signal detected as a function of depth. Here, we report a visualization program compatible with widely used fluorophores in cell biology to facilitate image interpretation of differential protein disposition in 3D specimens. Glioblastoma cell clusters were fluorescently labeled for mitochondrial complex I (COXI), P2X7 receptor (P2X7R), β-Actin, Ki-67, and DAPI. Each cell cluster was imaged using a laser scanning confocal microscope. We observed up to ∼70% loss in fluorescence signal across the depth in Z-stacks. This progressive underrepresentation of fluorescence intensity as the focal plane deepens hinders an accurate representation of signal location within a 3D structure. To address these challenges, we developed ProDiVis: a program that adjusts apparent fluorescent signals by normalizing one fluorescent signal to a reference signal at each focal plane. ProDiVis serves as a free and accessible, unbiased visualization tool to use in conjunction with fluorescence microscopy images and imaging software.

A label-free strategy for direct detection of Staphylococcus aureus in complex matrixes based on RCA and aptamer

Given the significant threat to human health posed by Staphylococcus aureus, rapid and accurate quantification of S. aureus is essential to prevent and diagnose foodborne diseases and to ensure the safety of food consumption. Here, we developed a novel, simple，and label-free biosensor for quantitatively detecting S. aureus by integrating an allosteric aptamer probe and a rolling circle amplification technique. Pathogen S. aureus-specific binding could induce the structural change of the allosteric aptamer beacon to expose the primer for the padlock probe. Then, the primer could trigger the RCA reaction to produce a long-chain singles-tranded DNA harboring repeating Thioflavin T (ThT) aptamer, leading to powerful fluorescence signals. The assay demonstrates sensitivity, specificity, and accuracy, showing its potential as an alternative tool for pathogen detection in different fields, such as food safety monitoring.

Neutrophil elastase-activated carbon dots for non-invasive early diagnosis of inflammatory bowel disease by near-infrared-II fluorescence imaging

Most near-infrared-II (NIR-II) fluorescent probes produce an “always-on” output with poor signal specificity for biomarkers. Neutrophil elastase (NE) is a critical factor in chronic inflammation or in the acute response to infection and injury, and NE activatable NIR-II fluorescent probes can aid in the early clinical diagnosis of inflammatory bowel disease (IBD). In this paper, we synthesized OPDG-PFA@PEI carbon dots (CDs) through a two-step reaction of solvothermal/surface covalent bond modification, which can “turn on” specific NIR-II fluorescence signals related to the level of NE produced by neutrophils and target sites of inflammation effectively, thus realizing the monitoring of in-situ NE in cells and in vivo. OPDG-PFA@PEI CDs fluorescent nanoprobe show high-performance real-time non-invasive NIR-II fluorescence imaging for early diagnostic evaluation of IBD in mouse models of DSS-induced IBD, providing a promising approach for the development of clinical non-invasive and more convenient early diagnosis of IBD.

TWINKLE: An open-source two-photon microscope for teaching and research.

Many laboratories use two-photon microscopy through commercial suppliers, or homemade designs of considerable complexity. The integrated nature of these systems complicates customization, troubleshooting as well as grasping the principles of two-photon microscopy. Here, we present "Twinkle": a microscope for Two-photon Imaging in Neuroscience, and Kit for Learning and Education. It is a fully open, high-performance and cost-effective research and teaching microscope without any custom parts beyond what can be fabricated in a university machine shop. The instrument features a large field of view, using a modern objective with a long working distance and large back aperture to maximize the fluorescence signal. We document our experiences using this system as a teaching tool in several two week long workshops, exemplify scientific use cases, and conclude with a broader note on the place of our work in the growing space of open-source scientific instrumentation.

3D Printed Microneedles for the Transdermal Delivery of NAD+ Precursor: Toward Personalization of Skin Delivery.

3D printing of microneedles (μNDs) for transdermal therapy has the potential to enable patient personalization based on the target disease, site of application, and dosage requirements. To convert this concept to reality, it is necessary that the 3D printing technology can deliver high resolution, an affordable cost, and large print volumes. With the introduction of benchtop 4K and 8K 3D printers, it is now possible to manufacture medical devices like μNDs at sufficient resolution and low cost. In this research, we systematically optimized the 3D printing design parameters such as resin viscosity, print angle, layer height, and curing time to generate customizable μNDs. We have also developed an innovative 3D coating microtank device to optimize the coating method. We have applied this to the development of novel μNDs to deliver an established NAD+ precursor molecule, nicotinamide mononucleotide (NMN). A methacrylate-based polymer photoresin (eSun resin) was diluted with methanol to adjust the resin viscosity. The 3D print layer height of 25 μm yielded a smooth surface, thus reducing edge-ridge mismatches. Printing μNDs at 90° to the print platform yielded 84.28 ± 2.158% (n = 5) of the input height thus increasing the tip sharpness (48.52 ± 10.43 μm, n = 5). The formulation containing fluorescein (model molecule), sucrose (viscosity modifier), and Tween-20 (surface tension modifier) was coated on the μNDs using the custom designed microtank setup, and the amount deposited was determined fluorescently. The dye-coated μND arrays inserted into human skin (in vitro) showed a fluorescence signal at a depth of 150 μm (n = 3) into the skin. After optimization of the 3D printing parameters and coating protocol using fluorescein, NMN was coated onto the μNDs, and its diffusion was assessed in full-thickness human skin in vitro using a Franz diffusion setup. Approximately 189 ± 34.5 μg (5× dipped coated μNDs) of NMN permeated through the skin and 41.2 ± 7.53 μg was left in the skin after 24 h. Multiphoton microscopy imaging of NMN-coated μND treated mouse ear skin ex vivo demonstrated significantly (p < 0.05) increased free-unbound NADPH and reduced fluorescence lifetime of NADPH, both of which are indicative of cellular metabolic rates. Our study demonstrates that low-cost benchtop 3D printers can be used to print high-fidelity μNDs with the ability to rapidly coat and release NMN which consequently caused changes in intracellular NAD+ levels.

Wavelength-Based luminescence sensing via Turn-On responses for acid detection in complex Environments

One possible way to address the shortcomings of current acid detection is to add fluorescence detection capabilities, however, there is little data regarding wavelength-based luminescence sensing, which makes possible for the naked eye to perceive various colors through wavelength variations. In this paper, we achieved turn-on response and wavelength-based luminescence sensing on acids detection by using donor-accepter complex Fe-L, which display significant fluorescence discoloration in the presence of a wide range of inorganic and organic acids, including benzoic acids, providing the fluorescence response for a more intuitive and convenient acid detection. The simultaneous achievement of fluorescence enhancement and color change as the acid concentration changes enables visual detection of the acid concentration, which provides a foundation for future research into convenient detection methods for various types of acids.

Bimetallic complexation for significant fluorescence enhancement of faecal pigment towards water quality testing

Systematic spectroscopic investigations revealed bimetallic complexation of faecal pigments (FP) in waterthrough their multiple binding sites. FP-Zn(II)/Gd(III) complexes showed narrow emission (FWHM ∼ 26 nm) with maximum intensity compared to different bimetallic complexes in water. The fluorescence intensity of bimetallic Zn(II)/Gd(III) complexes of FP increased ∼3–5 fold in aqueous media compared to the FP-Zn(II) complexes, i.e. well-known as Schlesinger’s test. The optimum Zn(II): Gd(III) stoichiometry was observed at 3:2 for maximum FP fluorescence enhancement. The bimetallic FP-Zn(II)/Gd(III) complexes also exhibited higher fluorescence lifetimes and quantum yield than FP-Zn(II) complexes. The photophysical investigations demonstrated that bimetallic Zn(II)/Gd(III) complexation increased the rigidity of chromophores present in FP. Consequently, a decrease in non-radiative decay rate constants (knr) by ∼ 1.5 times was observed for bimetallic complexes compared to FP-Zn(II) in water. Hence, a detection limit of the nanomolar concentration range of FP could be achieved in the aqueous media. The effect of diverse interfering factors (humic acid, water hardness, pH) on the fluorescence behaviour of FP-Zn(II)/Gd(III) complexes was also investigated with an approach mimicking real water samples. Finally, a simple non-extraction analytical approach for sensitive detection of FP in water was demonstrated while also considering the fluorescence matrix interference problem.

Development of a Modular miRNA-Responsive Biosensor for Organ-Specific Evaluation of Liver Injury.

MicroRNAs (miRNAs) are increasingly being considered essential diagnostic biomarkers and therapeutic targets for multiple diseases. In recent years, researchers have emphasized the need to develop probes that can harness extracellular miRNAs as input signals for disease diagnostics. In this study, we introduce a novel miRNA-responsive biosensor (miR-RBS) designed to achieve highly sensitive and specific detection of miRNAs, with a particular focus on targeted organ-specific visualization. The miR-RBS employs a Y-structured triple-stranded DNA probe (Y-TSDP) that exhibits a fluorescence-quenched state under normal physiological conditions. The probe switches to an activated state with fluorescence signals in the presence of high miRNA concentrations, enabling rapid and accurate disease reporting. Moreover, the miR-RBS probe had a modular design, with a fluorescence-labeled strand equipped with a functional module that facilitates specific binding to organs that express high levels of the target receptors. This allowed the customization of miRNA detection and cell targeting using aptameric anchors. In a drug-induced liver injury model, the results demonstrate that the miR-RBS probe effectively visualized miR-122 levels, suggesting it has good potential for disease diagnosis and organ-specific imaging. Together, this innovative biosensor provides a versatile tool for the early detection and monitoring of diseases through miRNA-based biomarkers.

The Detection of Lymph Node Metastasis Metastases Using a Near-Infrared Fluorescent Probe Based on Tumor-Targeted Monoclonal Antibody Antibodies Specific: A Prospective Study in a Nude Mouse Model

Background: To identify the metastatic lymph nodes and remove them accurately, the fluorescent surgical navigation ability of the ovarian cancer-specific fluorescent probe COC183B2-800 was assessed to verify the metastatic lymph nodes in the nude mouse model. Methods: The nude mouse model related to lymph node metastases in human ovarian cancer was established using the SKOV3-ip1 cell line. Besides, the COC183B2-800 probe (IRDye800CW Ester conjugated COC183B2 antibody) was fabricated. Moreover, in vivo fluorescence imaging was performed to determine the ability of the COC183B2-800 fluorescent probe to identify metastatic lymph nodes in the nude mouse model. Results: The nude mouse model related to lymph node metastases in human ovarian cancer was successfully established. In vivo fluorescence imaging was performed 30 hours after the injection of the COC183B2-800 fluorescent probe (25 μg) into the animal model, which can achieve specific imaging of metastatic lymph nodes. All metastatic lymph nodes were detected in vivo and in vitro (8/8), and only 1 negative lymph node with reactive enlargement showed a false positive fluorescent signal. Conclusions: The targeted fluorescent probe COC183B2-800 can be employed to identify metastatic lymph nodes in the nude mouse model related to lymph node metastases in human ovarian cancer with high specificity and sensitivity. Targeted fluorescence imaging using COC183B2-800 is expected to become a method to achieve precise lymphadenectomy.

A fluorescence lifetime-based novel method for accurate lipid quantification of BODIPY vital-stained C. elegans

Lipid droplets (LDs) are organelles associated with lipid storage and energy metabolism; thus, their morphology and quantity are of significant research interest. While commercially available BODIPY dye effectively labels LDs in various cell types, it also labels lysosome-related organelles (LROs) in C. elegans, leading to non-specific LD quantification. Here, we report that the fluorescent signals of BODIPY exhibit distinct fluorescence lifetime patterns for LROs and LDs, which can be captured, visualized, and filtered by fluorescence lifetime imaging microscopy. Furthermore, we proposed and validated a method based on fluorescence lifetime that can improve the accuracy of fat storage quantification in BODIPY vital-staining worms, which holds broad applications, including rapid and accurate LD quantification in forward genetic screening. Additionally, our method enables observing dynamic LD-LRO interactions in living worms, a unique capability of BODIPY vital staining. Our findings highlight distinct BODIPY fluorescence lifetime characteristics of LDs and LROs, providing a valuable tool for future research on LDs, LROs, or their interactions.

An intelligent fluorescence sensing platform based on entropy-driven toehold-mediated strand displacement cycle reaction for point-of-care testing of miRNA

BackgroundMicroRNA (miRNA) has gradually become an emerging biomarker for early diagnosis and prognosis of various diseases due to its specific gene expression and high stability. With the development of molecular diagnosis and point-of-care testing (POCT) technology, developing simple, fast, sensitive, efficient, and low-cost miRNA sensors is of great significance for clinical applications and emergency rapid diagnosis. At present, entropy-driven toehold mediated chain displacement reaction, as a promising enzyme free isothermal amplification technique, is an important tool for ultra-sensitive biosensing applications. ResultsIn this study, we used gold nanoparticles (AuNPs) as carriers and quenchers, modified them using self-assembled triple chain composite substrates AuNPs@A@B1/B2, and used dual reporter molecules for cascade cyclic amplification to amplify fluorescence signals, which proposed a fluorescent biosensor based on this reaction and build an intelligent fluorescence sensing platform for rapid detection of miRNA. We designed a highly specific self-programmable sensor using the acute ischemic stroke (AIS) biomarker miRNA-125a-5p as a sample, and achieved sensitive detection of miRNA in the range of 0.01 μM∼10 μ M under optimal conditions. It broke through the traditional detection limitations of weak signals and liberated the fluorescence detection environment. SignificanceIn summary, this creative miRNA biosensor combined with POCT has demonstrated extraordinary detection potential, broad application prospects in the early diagnosis and prognosis monitoring of AIS, provides a novel miRNA universal detection strategy for the fields of biological and life sciences.

PUPAID: A R + ImageJ pipeline for thorough and semi-automated processing and analysis of multi-channel immunofluorescence data

PUPAID is a workflow written in R + ImageJ languages which is dedicated to the semi-automated processing and analysis of multi-channel immunofluorescence data. The workflow is designed to extract fluorescence signals within automatically-segmented cells, defined here as Areas of Interest (AOI), on whole multi-layer slides (or eventually cropped sections of them), defined here as Regions of Interest (ROI), in a simple and understandable yet thorough manner. The included (but facultative) R Shiny-based interactive application makes PUPAID also suitable for scientists who are not fluent with R programming. Furthermore, we show that PUPAID identifies significantly more cells, especially in high-density regions, as compared to already published state-of-the-art methods such as StarDist or Cellpose. For extended possibilities and downstream compatibility, single cell information is exported as FCS files (the standardized file format for single cell-based cytometry data) in order to be openable using any third-party cytometry analysis software or any analysis workflow which takes FCS files as input.

Development and Validation of the MAST ISOPLEX®VTEC Kit for Simultaneous Detection of Shiga Toxin/Verotoxin 1 and 2 (stx1/vt1 and stx2/vt2) with Inhibition Control (IC) in a Rapid Loop-Mediated Isothermal Amplification (LAMP) Multiplex Assay.

Loop-mediated isothermal amplification (LAMP) is a cost-effective, rapid, and highly specific method of replicating nucleic acids. Adding multiple targets into a single LAMP assay to create a multiplex format is highly desirable for clinical applications but has been challenging due to a need to develop specific detection techniques and strict primer design criteria. This study describes the evaluation of a rapid triplex LAMP assay, MAST ISOPLEX®VTEC, for the simultaneous detection of Shiga toxin/verotoxin 1 and 2 (stx1/vt1 and stx2/vt2) genes in verotoxigenic Escherichia coli (E. coli) (VTEC) isolates with inhibition control (IC) synthetic DNA using a single fluorophore-oligonucleotide probe, MAST ISOPLEX®Probes, integrated into the primer set of each target. MAST ISOPLEX®Probes used in the MAST ISOPLEX®VTEC kit produce fluorescent signals as they integrate with reaction products specific to each target, allowing tracking of multiple amplifications in real time using a real-time analyzer. Initial validation on DNA extracts from fecal cultures and synthetic DNA sequences (gBlocks) showed that the MAST ISOPLEX®VTEC kit provides a method for sensitive simultaneous triplex detection in a single assay with a limit of detection (LOD) of less than 100 target copies/assay and 96% and 100% sensitivity and specificity, respectively.

Multifunctional Ethyl Violet@NH2-MIL-88B(Fe) Hybrids: CRISPR-Cas12a-Assisted PEC-FL-CL Triple-Mode Sensitive Detection of HPV-16.

The multimode assay based on multiple response mechanisms has received great attention to effectively improve the accuracy of a sensing platform. However, multifunctional sensing materials for simultaneously satisfying the multiple-mode detections are still in shortage due to the incompatibility of the signal transduction mechanisms in different modes. Here, taking human papillomavirus 16 (HPV-16) DNA (TDNA) as the model due to its important role in cervical cancer, a novel multifunctional material, ethyl violet (EV)@NH2-MIL-88B(Fe) (ENM) hybrids, have been successfully prepared, which could simultaneously satisfy CRISPR-Cas12a-assisted photoelectrochemical (PEC)-fluorescent (FL)-colorimetric (CL) triple-mode detection of TDNA. Based on the TDNA-induced trans-cleavage ability of CRISPR-Cas12a and efficient separation of magnetic beads, ENM was obtained from the single-stranded DNA-surrounded streptavidin-modified magnetic beads-ENM (SMB-ssDNA-ENM) and decomposed by pyrophosphate to get free EV, 2-aminoterephthalic acid (NH2-BDC), and Fe3+. Thus, TDNA was sensitively detected based on the EV-enhanced PEC signal of SnS2 nanosheets (PEC mode), fluorescent signal of NH2-BDC (FL mode), and characteristic absorption peak at about 720 nm of Fe3+-induced Prussian blue (PB) (CL mode). The designed PEC-FL-CL triple-mode biosensing platform had good performance for the detection of TDNA with a wide linear range (0.1 fM-100 nM) and ultralow detection limits (0.07 fM for PEC, 0.03 fM for FL and 0.09 fM for CL). Additionally, the developed PEC-FL-CL triple-mode biosensing platform has great potential for applications in early disease diagnosis and bioanalysis, as it can be easily extended to other DNA assays through modification of the crRNA sequence within the CRISPR-Cas12a system.

An aggregation-induced fluorescence probe for H2S and its application in living cells

Hydrogen sulphide (H2S) plays an important role in the regulation of a number of important physiological and pathological processes. Therefore, the development of new fluorescent probes for H2S is of great importance and has recently attracted much attention. In this paper, a novel coumarin-based fluorescent probe named (1E)-1-(1-(6-chloro-2-oxo-2H-chromen-3-yl)ethylidene)thiosemicarbazide (1), which contained a CN moiety as a specific reaction site for nucleophilic addition of H2S, was prepared. Upon addition of H2S, probe 1 exhibited significant fluorescence enhancement with a detection limit as low as 0.54 μM, accompanied by a colour change from cyan to colourless. The mechanism was confirmed by ESI-MS, Job’s plot, DFT and the probe 1 was successfully applied to the imaging of H2S in HeLa cells.

A novel triple-signal biosensor based on ZrFe-MOF@PtNPs for ultrasensitive aflatoxins detection

The development of more sensitive, stable, and portable biosensors is crucial for meeting the growing demands of diverse and complex detection environments. MOF-based nanozymes have emerged as excellent optical reporters, making them ideal signal donors for constructing multi-signal lateral flow immunoassays (LFIA). In this study, a ZrFe-MOF@PtNPs nanocomposite was synthesized by uniformly depositing platinum nanoparticles (PtNPs) onto the surface of ZrFe-MOFs using an impregnation-reduction method. The ZrFe-MOF@PtNPs exhibited broad absorption spectra, excellent peroxidase-like activity (SA = 21.77 U/mg), high solvent stability, and efficient antibody binding capability. A portable LFIA platform was developed based on ZrFe-MOF@PtNPs and a smartphone for the targeted detection of carcinogenic aflatoxins. This method enabled the readout of colorimetric, fluorescent, and catalytic signals, significantly enhancing detection sensitivity, ensuring result accuracy, and expanding the dynamic detection range. For aflatoxin M1, the calculation of the detection limit of the three signal modes reached as low as 0.0062 ng/mL, which is two orders of magnitude more sensitive than AuNPs-LFIA (0.1839 ng/mL). This study provides effective guidance for multifunctional modification of MOFs and serves as a reference for the application of MOF-based nanozymes in point-of-care biosensors.

Impact of the Graphene Production Methods Sonication and Microfluidization on In Vitro and In Vivo Toxicity, Macrophage Response, and Complement Activation.

Graphene, a material composed of a two-dimensional lattice of carbon atoms, has due to its many unique properties a wide array of potential applications in the biomedical field. One of the most common production methods is exfoliation through sonication, which is simple but has low yields. Another approach, using microfluidization, has shown promise through its scalability for commercial production. Regardless of their production method, materials made for biomedical applications need to be tested for biocompatibility. Here, we investigated the differences in toxicity, macrophage response, and complement activation of similar-sized graphene flakes produced through sonication and microfluidization, using in vitro cell assays and in vivo assays on zebrafish larvae. In vitro toxicity testing showed that sonicated graphene had a high toxicity, with an EC50 of 100 μg mL-1 for endothelial cells and 60 μg mL-1 for carcinoma cells. In contrast, microfluidized graphene did not reach EC50 at any of the tested concentrations. The potency to activate the complement system in whole blood was 10-fold higher for sonicated than for microfluidized graphene. In zebrafish larvae, graphene of either production method was found to mainly agglomerate in the caudal hematopoietic tissue; however, no acute toxic effects were found. Sonicated graphene led to an increase in macrophage count and a macrophage migration to the ventral tail area, while microfluidized graphene led to a transient reduction in macrophage count and fewer cells in the ventral trail area. The observed reduction in macrophages and change in macrophage distribution following exposure to microfluidized graphene was less pronounced compared to sonicated graphene and contributed to masking of the fluorescent signal rather than cytotoxic effects. Summarized, we observed higher toxicity, macrophage response, and complement activation with graphene produced through sonication, which could be due to oxygen-containing functional groups introduced to the edge of the carbon lattice by this production method. These findings indicate that microfluidization produces graphene more suitable for biomedical applications.

Split Reporters Facilitate Monitoring of Gene Expression and Peptide Production in Linear Cell-Free Transcription-Translation Systems.

Cell-free transcription-translation (TXTL) systems expressing genes from linear dsDNA enable the rapid prototyping of genetic devices while avoiding cloning steps. However, repetitive inclusion of a reporter gene is an incompressible cost and sometimes accounts for most of the synthesized DNA length. Here we present reporter systems based on split-GFP systems that reassemble into functional fluorescent proteins and can be used to monitor gene expression in E. coli TXTL. The 135 bp GFP10-11 fragment produces a fluorescent signal comparable to its full-length GFP counterpart when reassembling with its complementary protein synthesized from the 535 bp fragment expressed in TXTL. We show that split reporters can be used to characterize promoter libraries, with data qualitatively comparable to full-length GFP and matching in vivo expression measurements. We also use split reporters as small fusion tags to measure the TXTL protein and peptide production yield. Finally, we generalize our concept by providing a luminescent split reporter based on split-nanoluciferase. The ∼80% gene sequence length reduction afforded by split reporters lowers synthesis costs and liberates space for testing larger devices while producing a reliable output. In the peptide production context, the small size of split reporters compared with full-length GFP is less likely to bias peptide solubility assays. We anticipate that split reporters will facilitate rapid and cost-efficient genetic device prototyping, protein production, and interaction assays.

Construction of novel π-bridged fluorescent probes for Fe2+ monitoring in living cells and foods.

Fe2+ plays a crucial role in biological systems as an essential trace element in the body. Iron is absorbed by the body through food and Fe2+ is also an important component of living cells; therefore, quantitative testing of Fe2+ in food and in living cells is of great importance. This paper presents the development of a novel π-bridge EDOT-based N-oxide turn-on fluorescent probe, designated as FeE, for the detection of Fe2+. The probe has been shown to exhibit excellent sensitivity, a favorable linear relationship between fluorescence signal intensity and Fe2+ concentration, and an effective immunity to interference. The probe has low cytotoxicity and has been successfully used to detect Fe2+ in cells using laser confocal fluorescence microscopy. It is also possible to determine the presence of Fe2+ in animal blood, spinach, apple juice, red wine, mineral water and metal cans.

Fluorescent In Situ Hybridization Chain Reaction for RNA in the Drosophila Embryonic and Larval Central Nervous System.

In the Drosophila nerve cord, much is known about the generation of neurons from neuronal stem cells. Over the lifetime of a neuron, the cumulative expression of genes within that neuron determines its fate. Furthermore, gene expression in mature neurons determines their functional characteristics. It is therefore useful to visualize neural gene expression, which is often done via staining with antibodies to a protein of interest. In cases where there is no antibody to a desired gene product, or when it is useful to detect RNA rather than protein products, fluorescent in situ hybridization chain reaction for RNA (HCR RNA-FISH, or HCR for this protocol) can be used to detect and quantify RNA expression. RNA molecules reside predominantly in the cell soma, so HCR can facilitate determining neuron identity because somata position within the nerve cord is stereotyped across animals. HCR provides high-amplitude, high-fidelity signals. In principle, HCR can be broken down into a detection/hybridization stage and an amplification stage. During detection/hybridization, a probe set hybridizes to multiple sequences within a target gene. In the amplification step, concatemerized fluorescent hairpins bind to the hybridized probes. This two-step process increases the specificity of the fluorescent signal and helps reduce the likelihood of background fluorescence compared to traditional in situ hybridization techniques where the hybridizing probe itself contains the fluorescent signal. Here, we describe a protocol for using HCR to study gene expression in the Drosophila embryonic and larval nerve cord. We also describe how to combine HCR with immunofluorescence staining.