Weak Fluorescence Signal Research Articles

Multispectral multiplexed confocal FLIM for live cell imaging

Abstract Spectrally resolved fluorescence lifetime imaging with high spatial precision offers comprehensive information on species localization and behavior. It is challenging to resolve weak fluorescence signals in multiple dimensions (spatial, spectral, and temporal) at high frame rates, especially in dynamic live cell processes, as photobleaching and phototoxicity limit acceptable photon count rates. We developed a multiplexed confocal FLIM technique, which uses a prism-based imaging spectrometer to separate a 10x10 array of confocal foci into their spectral components. This allows the sampling of the spectra by a time-resolved image sensor to produce a multispectral time-resolved data set used for generating multispectral lifetime images. This system captures 300x300 pixel fluorescence lifetime images containing 12 unique spectral bands covering a 450-700 nm spectral range in 1.8 seconds of exposure. Its performance was demonstrated in fixed stained samples and in multispectral imaging of FLIM-FRET in live cells.

Orbital Adipose Tissue: The Optimal Control for Back-Table Fluorescence Imaging of Orbital Tumors.

Control tissue is essential for ensuring the precision of semiquantitative analysis in back-table fluorescence imaging. However, there remains a lack of agreement on the appropriate selection of control tissues. To evaluate the back-table fluorescence imaging performance of different normal tissues and identify the optimal normal tissue, a cohort of 39 patients with orbital tumors were enrolled in the study. Prior to surgery, these patients received indocyanine green (ICG) and following resection, 43 normal control tissues (34 adipose tissues, 3 skin tissues, 3 periosteal tissues, and 3 muscle tissues) were examined using back-table fluorescence imaging. The skin tissue demonstrated significantly elevated fluorescence intensity in comparison to the diseased tissue, whereas the muscle tissue exhibited a broad range and standard deviation of fluorescence signal intensity. Conversely, the adipose and periosteum displayed weak fluorescence signals with a relatively consistent distribution. Additionally, no significant correlations were found between the signal-to-background ratio (SBR) of adipose tissue and patients' ages, genders, weights, disease duration, tumor origins, dosing of administration of ICG infusion, and the time interval between ICG infusion and surgery. However, a positive correlation was observed between the SBR of adipose tissue and its size, with larger adipose tissues (>1 cm) showing an average SBR 27% higher than smaller adipose tissues (≤1 cm). In conclusion, the findings of this study demonstrated that adipose tissue consistently exhibited homogeneous hypofluorescence during back-table fluorescence imaging, regardless of patient clinical variables or imaging parameters. The size of the adipose tissue was identified as the primary factor influencing its fluorescence imaging characteristics, supporting its utility as an ideal control tissue for back-table fluorescence imaging.

Enhanced denoising for weak signal preservation in structured illumination microscopy

Structured illumination microscopy (SIM) is a powerful super-resolution technology in biological science because of its fast imaging speed, low phototoxicity, and full-field imaging. Despite this, SIM is hampered by out-of-focus background noise, which can obscure weak fluorescence signals and render them unrecognizable. Previous denoising algorithms tended to eliminate the noise along with the weak signals, causing a decrease in image quality. To address this issue, we propose a denoising algorithm based on out-of-focus plane information extraction (OPIE-SIM) that salvages the weak signal from the out-of-focus background noise. The OPIE-SIM algorithm enhances weak fluorescence signals by combining out-of-focus layer information with focal plane data and correcting the differences in point spread functions (PSF). This approach eliminates out-of-focus background noise and preserves the integrity of weak fluorescence structures while significantly reducing image acquisition time compared to traditional over-focusing imaging techniques. Through extensive simulations and experiments, we verified the feasibility of our approach. Compared with other denoising algorithms, our method generates images with a higher signal-to-noise ratio while maintaining the integrity of weak fluorescence structures.

Constructing Mechanochromic Fluorescent Interphase via Core-Shell Microcapsules: A Route for Interface Reinforcing and Damage Self-Reporting of CFRP Composites.

Perylenediimide-chitosan/γ-poly (glutamic acid) microcapsules sizing (PDI-CS/γ-PGA) core-shell microcapsule is designed and used to establish a novel interphase in carbon fiber/epoxy (CF/EP) composite, and the interfacial property, as well as the damage self-reporting of the composite, is compared with desized carbon fiber (CF-desized)/EP and commercial carbon fiber (CF-COM)/EP composite. The ruptured PDI-CS/γ-PGA microcapsule exhibits strong "turn-on" green fluorescence from the released PDI upon mechanical stimuli. The anchoring of PDI-CS/γ-PGA microcapsule on carbon fiber with PDI-CS/γ-PGA microcapsules sizing (CF@PDI-CS/γ-PGA) surface results in increased chemical activity and roughness, exhibiting a weak green fluorescence signal instead of non-fluorescence on CF-desized and CF-COM surface. The transverse fiber bundle tensile (TFBT) strength of CF@PDI-CS/γ-PGA composite is 80.97% and 31.09% higher than those of CF-desized/EP and CF-COM/EP composite, which is attributed to the mechanical interlocking and chemical bonding interaction between carbon fiber and epoxy matrix by introducing PDI-CS/γ-PGA microcapsule with spherular structure and active groups. After microdroplet testing, the strong "turn-on" green fluorescence signal of the released PDI from the microcapsules is detected in the interfacial debonding regions, realizing the microscopic damage self-reporting of CF@PDI-CS/γ-PGA composite.

Enhanced detection of UV fluorescence from food products and tissue using lanthanide-doped polymer composite membranes

We demonstrate polymer composite membranes that can be coupled to conventional spectroscopic tools for enhancing the detection of weak UV fluorescence signals. The membranes incorporate a down-conversion phosphor with lanthanide ions allowing for the detection of UV emission from biomolecules commonly found in food products. We also explore their capability to discern between healthy and wounded tissue through measurements of the UV autofluorescence emission from skin. Our results show that the composite membranes have great potential to improve UV fluorescence detection in applications related to the biomedical, pharmaceutical and food areas.

Derivatization-free CTAB-induced fluorescence sensing strategy for determination of daptomycin in environmental, pharmaceutical, and biological samples

A simple, economical, green, and sensitive spectrofluorimetric method was developed for the determination of the cyclic lipopeptide antibiotic daptomycin (DPT). The method is based on the sensitizing effect of the cationic surfactant cetyltrimethylammonium bromide (CTAB) on the weak fluorescent signal of DPT. After excitation at 373 nm, DPT-CTAB fluoresces at a maximum intensity of 458 nm. Under the optimized conditions, the method exhibits linearity over the concentration range of 0.50–10.0 μg/mL with a detection limit down to 0.11 μg/mL. The study revealed excellent selectivity towards DPT in the presence of the co-administered drugs. The proposed method was able to determine DPT in its commercial vials, and the results were comparable to those obtained by the reported method. The high sensitivity of the developed method allowed its estimation in water and biological fluids (spiked plasma and urine samples) with high % recoveries (95.89–105.99%). DPT was successfully determined in rat plasma after intra-peritoneal administration, and the method was applied for the calculation of its pharmacokinetic parameters (Cmax, t1/2, tmax, and AUC) in rat plasma. Comparison with the reported methods reveals many benefits of the proposed spectrofluorimetric method, such as greenness, simplicity, sensitivity, and reliability. Finally, different recent assessment tools confirmed the greenness and whiteness of the proposed method.

Automated hematological cell count using sysmex XN-1000V in Testudo hermanni: Agreement with manual count

Mediterranean area represents the main habitat of Testudo hermanni. Clinical signs of disease of these tortoises are non-specific, making the hematology results crucial in revealing underlying pathological conditions. However, accurate automated identification of blood cell populations is hampered by the presence of nucleated erythrocytes (NRBC) and thrombocytes (Thr), necessitating manual methods such as counting chambers. The aim of the study was to assess the performance of the novel automated hematology analyzer Sysmex XN-1000 V, which includes a a specific channel (WNR) for counting NRBC, in accurately identify and quantify the different blood cell populations of Testudo hermanni. Additionally, its agreement with manual counts was evaluated. Fifty heparinized blood samples were initially counted using the Neubauer improved chamber and then analysed twice with Sysmex XN-1000 V. Thirteen out of 50 samples were instrumentally counted again after 48 h to assess the inter-assay precision. All WNR scattergrams were re-analysed using an ad hoc gate panel to differentiate two populations: NRBCs (weak fluorescence signal) and WBC + Thr (high fluorescence signal). Sysmex XN-1000 V demonstrated optimal intra- and inter-assay precision for NRBCs (CV 0.98% ± 1.96; 1.31% ± 2.98) and moderate precision for WBC + Thr (CV 9.24% ± 16.61; 12.69% ± 10.35). No proportional nor constant errors were observed between the methods for both the populations. The instrumental NRBC counts were consistently slightly lower, while WBC + Thr counts were slightly higher compared to manual counts. These findings suggest that Sysmex XN-1000 V can be used for analyzing cell populations in heparinized blood of Testudo hermanni. However, specific instrumental reference intervals are suggested.

Data-driven coordinated attention deep learning for high-fidelity brain imaging denoising and inpainting.

Deep learning offers promise in enhancing low-quality images by addressing weak fluorescence signals, especially in deep in vivo mouse brain imaging. However, current methods struggle with photon scarcity and noise within in vivo deep mouse brains, and often neglecting tissue preservation. In this study, we propose an innovative in vivo cortical fluorescence image restoration approach, combining signal enhancement, denoising, and inpainting. We curated a deep brain cortical image dataset and developed a novel deep brain coordinate attention restoration network (DeepCAR), integrating coordinate attention with optimized residual networks. Our method swiftly and accurately restores deep cortex images exceeding 800 μm, preserving small-scale tissue structures. It boosts the peak signal-to-noise ratio (PSNR) by 6.94 dB for weak signals and 11.22 dB for large noisy images. Crucially, we validate the effectiveness on external datasets with diverse noise distributions, structural features compared to those in our training data, showcasing real-time high-performance image restoration capabilities.

Preoperative PET imaging and fluorescence-guided surgery of human glioblastoma using dual-labeled antibody targeting ETA receptors in a preclinical mouse model: A theranostic approach.

Rationale: Glioblastoma (GBM) poses significant challenges regarding complete tumor removal due to its heterogeneity and invasiveness, emphasizing the need for effective therapeutic options. In the last two decades, fluorescence-guided surgery (FGS), employing fluorophores such as 5-aminolevulinic acid (5-ALA) to enhance tumor delineation, has gained attraction among neurosurgeons. However, some low-grade tumors do not show any accumulation of the tracers, and the lack of patient stratification represents an important limitation. Since 2000, endothelin axis has been extensively investigated for its role in cancer progression. More specifically, our team has identified endothelin A receptors (ETA), overexpressed in glioblastoma cancer stem cells, as a target of interest for GBM imaging. This study aims to evaluate the efficacy of a novel preclinical bimodal imaging agent, [89Zr]Zr-axiRA63-MOMIP, as a theranostic approach to: i) detect ETA + cells in an orthotopic model of human GBM, ii) achieve complete tumoral resection. Methods: Monomolecular multimodal imaging platform (MOMIP) - containing both a fluorophore (IRDye800CW) and a chelator for a positron-emitting radiometal (desferroxamine B, DFO) - was conjugated to the axiRA63 antibody targeting ETA receptors, overexpressed on the surface of GBM stem cells. Mice bearing orthotopic human GBM were imaged 48 h post injection of [89Zr]Zr-axiRA63-MOMIP via positron emission tomography (PET) and optical imaging. Subsequently, post-mortem proof-of-concept FGS was implemented as well as ex vivo analyses (H&E staining, autoradiography, serial block face imaging) on brains with resected or unresected tumor to assess the correlation between PET and fluorescence signals. Results: PET imaging of [89Zr]Zr-axiRA63-MOMIP enabled a clear detection of ETA + cells in an orthotopic model of human GBM. Intraoperative optical imaging allowed a near-complete tumor resection together with the visualization of a weak fluorescence signal, after a prolonged exposure time, that was attributed to residual tumor cells via H&E staining. Besides, a qualitative correlation between the signals of both modalities was observed. Conclusions: The use of [89Zr]Zr-axiRA63-MOMIP provides an effective theranostic approach to detect and treat GBM by surgery in a preclinical mouse model. Thanks to the high correlation between PET and fluorescence signal allowing patients stratification, this bimodal agent should have a great potential for clinical translation and should present a significant advantage over non-targeted fluorophores already used in the clinic.

Nanoscatterer-Assisted Fluorescence Amplification Technique.

Weak fluorescence signals, which are important in research and applications, are often masked by the background. Different amplification techniques are actively investigated. Here, a broadband, geometry-independent and flexible feedback scheme based on the random scattering of dielectric nanoparticles allows the amplification of a fluorescence signal by partial trapping of the radiation within the sample volume. Amplification of up to a factor of 40 is experimentally demonstrated in ultrapure water with dispersed TiO2 nanoparticles (30 to 50 nm in diameter) and fluorescein dye at 200 μmol concentration (pumped with 5 ns long, 3 mJ laser pulses at 490 nm). The measurements show a measurable reduction in linewidth at the emission peak, indicating that feedback-induced stimulated emission contributes to the large gain observed.

Aberrant Expression of TET2 Accounts for DNA Hypomethylation in Varicocele.

Epigenetic modifications such as DNA methylation play a key role in male infertility etiology. This study aimed to explore the global DNA methylation status in testicular spermatogenic cells of varicocele-induced rats and consider their semen quality, with a focus on key epigenetic marks, namely 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC), as well as the mRNA and proteins of ten-eleven translocation (TET) methylcytosine dioxygenases 1-3. In this experimental study, 24 mature male Wistar rats (8 in each group) were assigned amongst the control, sham, and varicocele groups. Sperm quality was assessed, and DNA methylation patterns of testicular spermatogenic cells were investigated using reverse transcription-polymerase chain reaction (RT-PCR), western blot, and immunofluorescence techniques. Sperm parameters, chromatin and DNA integrity were significantly lower, and sperm lipid peroxidation significantly increased in varicocele-induced rats in comparison with control rats. During spermatogenesis in rat testis, 5-mC and 5-hmC epigenetic marks, and TET1-3 mRNA and proteins were expressed. In contrast to the 5-mC fluorescent signal which was presented in all testicular cells, the 5-hmC fluorescent signal was presented exclusively in spermatogonia and a few spermatids. In varicocele-induced rats, the 5-mC signal decreased in all cells within the tubules, whereas a strong signal of 5-hmC was detected in seminiferous tubules compared to the control group. As well, the levels of TET2 mRNA and protein expression were significantly upregulated in varicocele-induced rats in comparison with the control group. Also, our results showed that the varicocele-induced animals exhibited strong fluorescent signals of TET1-3 in testicular cells, whereas weak fluorescent signals were identified in the seminiferous tubules of the control animals. Consequently, we showed TET2 upregulation and the 5-hmC gain at testicular levels are associated with varicocele and sperm quality decline, and therefore they can be exploited as potential biomarkers of spermatogenesis.

Temperature profiles of field-aged photovoltaic modules affected by optical degradation

Moisture ingress into PV module in the presence of ultraviolet radiation, high temperature, and other environmental stressors can affect the optical integrity of the PV module. Optical degradation can take the form of delamination, discolouration of encapsulant, metal grids corrosion, and trapped moisture or chemical species. This can influence the photon absorption and current transport properties in the PV module bulk, which can affect the module operating temperature. In the present work, the relationship between optical degradation and temperature sensitivity of 20-year-old multicrystalline silicon field-aged PV modules have been investigated. The selected PV modules were characterized using visual inspection, current-voltage (I–V) characterization, temperature coefficients profiling, current resistivity profiling, infrared (IR) thermal, ultraviolet fluorescence (UV–F), and electroluminescence (EL) imaging. PV modules affected by optical degradation show weak fluorescence and luminescence signal intensities. The average difference in cell temperature (ΔT) between the warmest and coldest cell for the PV modules investigated was found to be around 10 ± 2 °C and the average power degradation rate was approximately 0.8% per year. The underlying factor for the observed degradation is attributed to the degradation in the temperature coefficients of open circuit voltage (βVoc) and maximum power point voltage (βVmpp). The average temperature coefficient of efficiency (βηm) of the modules was found to be around −0.5%/°C. Finally, a temperature dependent resistivity method for extracting temperature coefficients from IR thermal data of PV modules has been proposed.

Highly Sensitive and Specific Detection of Influenza A Viruses Using Bimolecular Fluorescence Complementation (BiFC) Reporter System.

In this study, we developed a highly sensitive and specific bimolecular fluorescence complementation (BiFC)-based influenza A virus (IAV)-sensing system by combining a galactose/glucose-binding protein (GGBP) with an N-terminal large domain (YN1-172) and a C-terminal small domain (YC173-239) made up of enhanced yellow fluorescence protein (eYFP). The GGBP-based BiFC reporter exhibits the fluorescence reconstitution as a result of conformational changes in GGBP when lactose, which was derived from 6'-silalyllactose and used as a substrate for neuraminidase (NA), binds to GGBP in the presence of IAV. The system showed a linear dynamic range extending from 1 × 100 to 1 × 107 TCID50/mL, and it had a detection limit of 1.1 × 100 TCID50/mL for IAV (H1N1), demonstrating ultra-high sensitivity. Our system exhibited fluorescence intensity enhancements in the presence of IAV, while it displayed weak fluorescence signals when exposed to NA-deficient viruses, such as RSV A, RSV B, adenovirus and rhinovirus, thereby indicating selective responses for IAV detection. Overall, our system provides a simple, highly sensitive and specific IAV detection platform based on BiFC that is capable of detecting ligand-induced protein conformational changes, obviating the need for virus culture or RNA extraction processes.

Endoplasmic reticulum-targeted fluorescent probes for metal-free tracking of carbon monoxide in living cells

Endoplasmic Reticulum (ER) targetable fluorescent probes, ER-NapNO2 and ER-NapAllyl, have been designed and synthesized based on 1,8-naphthalimide fluorophore, for the selective and targeted detection of carbon monoxide (CO) in aqueous buffer medium of pH 7.4 at 37 °C. ER-targetable moiety, methyl sulfonamide unit, was attached with the one part with the fluorophore and another part was connected with CO reactive zone i.e. 4-nitrobenzyle and allyl group. Very weak fluorescence signals were observed with both the probes due to the possible PET process operating from naphthalimide moiety to electron withdrawing units i.e. -NO2 and allyl group. Interestingly, both of the probes exhibited their selective detection behavior towards CO in aqueous reaction buffer in absence of any metal ions e.g., palladium, rhodium, iron etc. The possible expulsion of highly fluorescent naphthalimide-derivative was identified from the reaction mixture during the reaction of both the probes and CO. The detection technique is found to highly selective towards CO over various reactive oxygen, nitrogen, sulfur, and other biological species with low detection limit, 0.080 µM and 0.161 µM of ER-NapNO2 and ER-NapAllyl respectively. Theoretical study using time dependent density functional theory (TDDFT) optimized the end-products formed after the reaction with CO and probes which excellently corroborate with the experimental observation. Also, the sensing reactions are exothermic with computed free energy changes for ER-NapNO2 and ER-NapAllyl are − 158.74 kcal/mol and − 28.51 kcal/mol respectively. Cellular confocal fluorescence microscopic experiments showed that these probes are also efficient to detect CO in ER specific regions in living cells.

In Vitro Tracking of Human Umbilical Vein Endothelial Cells Using Ultra-Sensitive Quantum Dot-Embedded Silica Nanoparticles

The nanoscale spatiotemporal resolution of single-particle tracking (SPT) renders it a powerful method for exploring single-molecule dynamics in living cells or tissues, despite the disadvantages of using traditional organic fluorescence probes, such as the weak fluorescent signal against the strong cellular autofluorescence background coupled with a fast-photobleaching rate. Quantum dots (QDs), which enable tracking targets in multiple colors, have been proposed as an alternative to traditional organic fluorescence dyes; however, they are not ideally suitable for applying SPT due to their hydrophobicity, cytotoxicity, and blinking problems. This study reports an improved SPT method using silica-coated QD-embedded silica nanoparticles (QD2), which represent brighter fluorescence and are less toxic than single QDs. After treatment of QD2 in 10 μg/mL, the label was retained for 96 h with 83.76% of labeling efficiency, without impaired cell function such as angiogenesis. The improved stability of QD2 facilitates the visualization of in situ endothelial vessel formation without real-time staining. Cells retain QD2 fluorescence signal for 15 days at 4 °C without significant photobleaching, indicating that QD2 has overcome the limitations of SPT enabling long-term intracellular tracking. These results proved that QD2 could be used for SPT as a substitute for traditional organic fluorophores or single quantum dots, with its photostability, biocompatibility, and superior brightness.

Ratiometric fluorescence detection of artemisinin based on photoluminescent Zn-MOF combined with hemin as catalyst

Artemisinin (ART) is a type of frontline drug to treat drug-resistant falciparum malaria. Simple, accurate and selective determination of ART is significant to monitor its clinical pharmaceutical efficacy. Herein, a new ratiometric fluorescence method has been designed for the determination of ART with Zn-MOF as fluorescence reference and hemin as catalyst, respectively. Zn-MOF possesses intrinsic fluorescence at 443 nm owing to 2-aminoterephthalic acid ligand. When o-phenylenediamine (OPD) is mixed with hemin, a weak fluorescent signal at 570 nm ascribed to oxidized product of OPD (oxOPD) is observed. In the presence of ART, hemin can catalyze ART to break its peroxide bridge and release a large number of reactive oxygen species, which effectively oxidize OPD into luminescent oxOPD. Therefore, the fluorescence at 570 nm is enhanced significantly while the fluorescence of Zn-MOF remains basically unchanged. Thus, a ratiometric fluorescence sensing platform has been constructed for the detection of ART. This method exhibits wider linear range (0.15 μM-150 μM) with detection limit of 50 nM. This novel and selective method has been used to detect ART in compound naphthoquinone phosphate tablets.

Modulation of G4 DNA sensing behavior by triphenylamine-imidazole based D-D-π-A-type organic fluorophores with subtle structural change

Two unsymmetrical D-D-π-A-type cyanine dyes TG and TGS using triphenylamine-imidazole as the electron donor moiety (D-D), ethylene unit as π conjugated spacer (π), and quinolinium as the electron acceptor (A) were developed. Their ground-state geometries, electronic properties as well as fluorescence responses to with various polynucleotide structures, including G-quadruplexes (G4s), double-stranded and single-stranded oligonucleotides were investigated by a combined experimental and theoretical study. The high structural similarity between the two dyes makes them undergo a similar internal conversion process to form an ICT state, giving significantly large Stokes shifts. However, they behave quite different polynucleotide structures sensing behaviors. That is, TG with a non-planar D-D moiety can selectively bind with G4s, and emit turn-on fluorescence possibly because of the restriction of its molecular rotations, while TGS with a planar D-D moiety failed to interact with G4s and remained the weak fluorescence signal. Moreover, TG has the potential to be used in visualizing the cellular nucleus in cells. Our findings suggest that a subtle structural change of dyes may lead to distinctly different fluorescence behaviors in G4s sensing event.

Background inhibited and speed-loss-free volumetric imaging in vivo based on structured-illumination Fourier light field microscopy.

Benefiting from its advantages in fast volumetric imaging for recording biodynamics, Fourier light field microscopy (FLFM) has a wide range of applications in biomedical research, especially in neuroscience. However, the imaging quality of the FLFM is always deteriorated by both the out-of-focus background and the strong scattering in biological samples. Here we propose a structured-illumination and interleaved-reconstruction based Fourier light field microscopy (SI-FLFM), in which we can filter out the background fluorescence in FLFM without sacrificing imaging speed. We demonstrate the superiority of our SI-FLFM in high-speed, background-inhibited volumetric imaging of various biodynamics in larval zebrafish and mice in vivo. The signal-to-background ratio (SBR) is improved by tens of times. And the volumetric imaging speed can be up to 40 Hz, avoiding artifacts caused by temporal under-sampling in conventional structured illumination microscopy. These suggest that our SI-FLFM is suitable for applications of weak fluorescence signals but high imaging speed requirements.

Fluorescent transgenic mouse models for whole-brain imaging in health and disease.

A paradigm shift is occurring in neuroscience and in general in life sciences converting biomedical research from a descriptive discipline into a quantitative, predictive, actionable science. Living systems are becoming amenable to quantitative description, with profound consequences for our ability to predict biological phenomena. New experimental tools such as tissue clearing, whole-brain imaging, and genetic engineering technologies have opened the opportunity to embrace this new paradigm, allowing to extract anatomical features such as cell number, their full morphology, and even their structural connectivity. These tools will also allow the exploration of new features such as their geometrical arrangement, within and across brain regions. This would be especially important to better characterize brain function and pathological alterations in neurological, neurodevelopmental, and neurodegenerative disorders. New animal models for mapping fluorescent protein-expressing neurons and axon pathways in adult mice are key to this aim. As a result of both developments, relevant cell populations with endogenous fluorescence signals can be comprehensively and quantitatively mapped to whole-brain images acquired at submicron resolution. However, they present intrinsic limitations: weak fluorescent signals, unequal signal strength across the same cell type, lack of specificity of fluorescent labels, overlapping signals in cell types with dense labeling, or undetectable signal at distal parts of the neurons, among others. In this review, we discuss the recent advances in the development of fluorescent transgenic mouse models that overcome to some extent the technical and conceptual limitations and tradeoffs between different strategies. We also discuss the potential use of these strains for understanding disease.

A Highly Integrated and Diminutive Fluorescence Detector for Point-of-Care Testing: Dual Negative Feedback Light-Emitting Diode (LED) Drive and Photoelectric Processing Circuits Design and Implementation.

As an important detection tool in biochemistry, fluorescence detection has wide applications. Quantitative detection can be achieved by detecting fluorescence signals excited by excitation light at a specific wavelength range. Therefore, the key to fluorescence detection is the stable control of the excitation light and the accurate acquisition of weak photoelectric signals. Moreover, to improve portability and instantaneity, devices are developing in miniaturization and integration. As the core of such devices, fluorescence detectors should also have these features. Under this circumstance, we designed a highly integrated and diminutive fluorescence detector and focused on its excitation light driving and photoelectric signal processing. A current–light dual negative feedback light-emitting diode (LED) driving circuit was proposed to obtain constant current and luminance. In addition, a silicon photodiode (PD) was used to receive and convert the fluorescence signal to an electric signal. Then, amplifying, filtering, and analog-to-digital (A/D) converting were applied to make the detection of weak fluorescence signals possible. The test results showed that the designed circuit has wonderful performance, and the detector shows good linearity (R2 = 0.9967) and sensitivity (LOD = 0.077 nM) in the detection of fluorescein sodium solution. Finally, a real-time fluorescence polymerase chain reaction (real-time PCR) of Legionella pneumophila was carried out on a homemade platform equipped with this detector, indicating that the detector met the requirements of real-time PCR detection.