Fluorophore Research Articles

Nile Red Fluorescence: Where's the Twist?

Nile Red is a fluorescent dye used extensively in bioimaging due to its strong solvatochromism. The photophysics underpinning Nile Red's fluorescence has been disputed for decades, with some studies claiming that the dye fluoresces from two excited states and/or that the main emissive state is twisted and intramolecular charge-transfer (ICT) in character as opposed to planar ICT (PICT). To resolve these long-standing questions, a combined experimental and theoretical study was used to unravel the mechanism of Nile Red's fluorescence. Time-resolved fluorescence measurements indicated that Nile Red emission occurs from a single excited state. Theoretical calculations revealed no evidence for a low-lying TICT state, with the S1 minimum corresponding to a PICT state. Ultrafast pump-probe spectroscopic data contained no signatures associated with an additional excited state involved in the fluorescence decay of Nile Red. Collectively, these data in polar and nonpolar solvents refute dual fluorescence in Nile Red and definitively demonstrate that emission occurs from a PICT state.

Live-Cell Imaging of RNA G-Quadruplex with a Dual-Color Fluorescence Switch-on Probe.

In-cell imaging of the G-quadruplex (G4) formation of nucleic acids remains challenging for revealing G4's functions in cells. This study describes the cell imaging method of G4 nucleic acids using our unique tripodal quinone-cyanine fluorescent dye, QCy(MeBT)3, whose 600nm and 700nm fluorescence is enhanced independently upon the binding with double-stranded DNA and G4-DNA/RNA, respectively. The use of QCy(MeBT)3 enables us to visualize cytosolic G4 RNAs in fixed and living HeLa cells with near-infrared 700nm fluorescence.

Production, Characterization, and Application of Hydrophobin-Based IR780 Nanoparticles for Targeted Photothermal Cancer Therapy and Advanced Near-Infrared Imaging.

As a promising approach for breast cancer treatment, photothermal therapy (PTT) features high spatial selectivity, noninvasiveness, and minimal drug resistance. IR780 (a near-infrared fluorescent dye) serves as an effective photosensitizer in PTT cancer therapy. However, the clinical application of IR780 in PTT has been hindered by its poor water solubility and unstable photostability. In this study, a genetically engineered dual-functional fusion protein tLyP-1-MGF6 is successfully constructed and expressed, which presents a novel use of hydrophobin MGF6 for its amphiphilicity combined with the tumor-penetrating peptide tLyP-1 to create an innovative carrier for IR780. These results show this fusion protein serving as a biodegradable and biocompatible carrier, significantly improves the water solubility of IR780 when formulated into nanoparticles. These studies demonstrate that the IR780@tLyP-1-MGF6 nanoparticles significantly enhance tumor targeting and photothermal therapeutic efficacy in comparison with control in vitro and in vivo. These advancements highlight the potential of the unique combination hydrophobin-based IR780 delivery system as a multifunctional nanoplatform for integrated imaging and targeted photothermal treatment of breast cancer.

BN SO01 - Fluorescent Precision: Advancing Laparoscopic Cholecystectomy with Indocyanine Green ( ICG )

Abstract Background Laparoscopic cholecystectomy represents a significant advancement in surgical techniques for gallbladder disease. However, challenges persist in accurately identifying critical structures such as the bile ducts, leading to complications. Indocyanine Green (ICG), a fluorescent dye with unique properties, has emerged as a promising tool to enhance precision and safety in laparoscopic cholecystectomy. Originally developed for infrared photography, ICG's fluorescence capabilities have been harnessed in various medical applications, including ophthalmology and surgical procedures. Its ability to provide real-time visualization of the biliary anatomy facilitates better surgical navigation, reducing the risk of inadvertent injury and improving overall outcomes. Method Comprehensive review of existing literature and research on laparoscopic cholecystectomy, with a focus on the role of Indocyanine Green (ICG) in enhancing surgical precision and safety. Relevant articles, clinical trials, and meta-analyses were identified through databases such as PubMed, Google Scholar, and Scopus. Search terms included "laparoscopic cholecystectomy," "Indocyanine Green," "fluorescence imaging," and related terms. Results The results of this study highlight the significant role of Indocyanine Green (ICG) in laparoscopic cholecystectomy. Through a comprehensive review of existing literature and research, it was found that ICG integration enhances surgical precision and safety by providing real-time visualization of the biliary anatomy. The use of ICG reduces the risk of inadvertent injury and improves overall surgical outcomes. Despite challenges such as technical complexities and safety considerations, the evidence strongly supports the transformative potential of ICG in modern surgery. Conclusion In conclusion, the integration of Indocyanine Green (ICG) into laparoscopic cholecystectomy represents a pivotal advancement in surgical practice. The findings of this study underscore the importance of cautious navigation and collaboration in maximizing the benefits of ICG. By addressing challenges through innovation and evidence-based guidelines, ICG has the potential to revolutionize the landscape of minimally invasive surgery, offering patients safer procedures and better outcomes. As research continues to evolve, the journey of ICG in laparoscopic cholecystectomy holds promise for a future where precision and safety converge, ultimately shaping the standard of surgical excellence.

Computational Model-Assisted Development of a Nonenzymatic Fluorescent Glucose-Sensing Assay.

Deep-red fluorescence was implemented in this fully injectable, nonenzymatic glucose biosensor design to allow for better light penetration through the skin, particularly for darker skin tones. In this work, a novel method was developed to synthesize Cy5.5 labeled mannose conjugates (Cy5.5-mannobiose, Cy5.5-mannotriose, and Cy5.5-mannotetraose) to act as the fluorescent competing ligand in a competitive binding assay with the protein Concanavalin A acting as the recognition molecule. Using fluorescence anisotropy (FA) data, a computational model was developed to determine optimal concentration ratios of the assay components to allow for sensitive glucose measurements within the physiological range. The model was experimentally validated by measuring the glucose response via FA of the three Cy5.5-labeled mannose conjugates synthesized with Cy5.5-mannotetraose demonstrating the most sensitive response to glucose across the physiological range. The developed method may be broadly applied to a vast range of commercially available fluorescent dyes and opens up opportunities for glucose measurements using nonenzymatic assays.

Development of a simple, low-cost, blue light-emitting diode illuminator for hands-on training of DNA detection experiments using agarose gel electrophoresis.

DNA detection by agarose gel electrophoresis (AGE) is commonly used in molecular biology. AGE is a separation method that provides opportunities for students to learn about the topology and size of DNA molecules. Recently, several fluorescent dyes have been used for DNA staining owing to their convenience, safety, reduced toxicity, and high sensitivity. A blue light-emitting diode (LED) transilluminator is required to detect DNA using fluorescent dyes; however, the associated high cost may limit its availability in classrooms or small laboratories. Therefore, we have designed a simple, low-cost blue LED illuminator to enable easy assembly for instructors and students. We evaluated the performance of the proposed illuminator by observing fluorescent dye-stained DNA markers using AGE, revealing clear DNA marker bands. Despite its limited functionality, the ease of construction and affordability of the proposed illuminator make it sufficient for hands-on molecular biology training in classrooms, thereby enhancing the learning environment and educational efficiency.

Abstract 4139187: Electrophysiological and Mechanical Characterization of Human Ventricular Myocardium from Patients with Primary or Secondary Cardiac Hypertrophy

Left-ventricular hypertrophy (LVH) is an adaptive condition to hemodynamic stress often involving the left ventricle (LV). This pathological condition is associated with clinical complications such as ventricular arrhythmias and diastolic dysfunction, the molecular and cellular mechanisms of which have been poorly investigated in the human heart. We collected myocardial samples from the upper interventricular septum of 132 patients with hypertrophic cardiomyopathy (HCM), 42 patients with aortic stenosis and severe LVH (AoS-LVH) and 12 non-failing non-hypertrophic patients with valve disease (NF-NH), who underwent myectomy operations at our cardiac surgery center. Samples were used to isolate single viable cardiomyocytes from the left ventricle to perform patch-clamp electrophysiological studies and intracellular calcium measurements with fluorescent dyes. Intact trabeculae were also dissected to perform isometric force measurements of electrically-stimulated twitches. Single-cell patch-clamp studies revealed a marked prolongation of action potential duration (APD) in HCM (N=82, mean APD at 90% repolarization=763±252ms at 0.5Hz) and AoS-LVH (N=22, APD90%=579±147ms) samples with respect to NF-NH (N=12, APD90%=447±88ms). In both HCM and AoS-LVH cardiomyocytes, APD prolongation was associated with increased late-Na + current with respect to NF-NH, while L-type Ca 2+ current was enlarged only in HCM samples. Ca 2+ -fluorescence studies revealed markedly slower Ca-transient (CaT) kinetics in myocardial samples from HCM (N=38, mean CaT 50% decay time at 0.5 Hz = 658±179ms) and AoS-LVH patients (N=9, CaT50%= 568±187ms), when compared with NF-NH samples (N=8, CaT50%=283±59ms), paralleled by elevated diastolic [Ca 2+ ]. Twitch force measurements in intact trabeculae revealed prolonged isometric contractions in HCM (N=78, overall twitch duration at 0.5Hz= 730±147ms) and in AoS-LVH patient-samples (N=24, TwD=669±116ms), as compared with NF-NH (N=7, TwD=511±73ms). Force-frequency relationship was flat in pathological samples, while NF-NH trabeculae showed a clear increase in twitch amplitude while increasing pacing rate to 2Hz. Our results suggest that the main features of functional cardiomyocyte remodeling (that is, changes in the expression and/or function of ion-channel and EC-coupling proteins) are qualitatively similar in primary vs. secondary LVH, albeit abnormalities are quantitatively more extensive in HCM samples.

Indocyanine Green Marking of Axillary Sentinel Lymph Nodes in Early Breast Cancer

AbstractAxillary sentinel lymph node excision (SLNE) in breast cancer patients with clinically node-negative disease may be carried out using different tracers. The standard tracer is technetium colloid (99mTc). Indocyanine green (ICG) can be used as an alternative. This study aimed to evaluate the clinical usefulness of this fluorescent dye in a standardized setting.A prospective, single-center cohort study carried out at the University Gynecological Hospital of Rostock from September 2023 to May 2024 carried out sentinel lymph node marking using only ICG in patients with breast malignancies. The ICG injection was administered immediately after the induction of anesthesia. Detection of the sentinel lymph node (SLN) was done using a laparoscopy system suitable for ICG. The aim was to determine the detection rate (DR) for SLNs marked exclusively using ICG and to record any complications. The costs of using ICG to mark SLNs were compared with those for 99mTc marking.During the study period, contraindications against marking with ICG were ascertained for five (3.8%) of 132 patients with planned SLNE. A total of 100 SLNEs were carried out after ICG marking in patients who met the inclusion criteria in the context of the study. A median of two SLNs were resected. The detection rate (DR) for SLNs was 98.0%. SLNs were identified in all obese patients. No serious systemic side effects occurred following ICG injection. Transient skin discoloration in the area around the injection site were observed in eight patients. The direct cost of ICG marking was 62.73 Euros, which was 170.36 Euros lower than the cost of 99mTc marking.The detection rate of axillary SLNs marked using ICG is high and the method is cost-effective, has few side effects and can also be used in obese patients. Contraindications against the administration of ICG are rare. Marking with ICG is a good alternative to the 99mTc method and offers advantages in terms of costs, logistics, no exposure to radiation, and patient comfort.

Dielectric microsphere sizing using fluorescence microscopy of whispering gallery mode resonances from adsorbed dyes

Whispering gallery mode (WGM) resonances in dielectric microspheres are very sensitive to their size and environment, which can be used for sensing but also as an indirect proxy to determine their size. By coating them with suitable fluorescent dyes and using fluorescence microscopy, we show that the WGM resonances of individual microspheres in solution can be easily studied with a high throughput. Brownian motion ensures that a representative sample is probed over time in the scattering volume. To analyze these WGM-imprinted fluorescent spectra, we propose a simple algorithm based on monitoring the spacing between resonances and comparing it to Mie theory predictions to infer their size. This allows us to measure the size distribution of typical polystyrene microsphere solutions. We also discuss the potential effects of dye concentration and choice of particle refractive index on the analysis. This method can be used, for example, for quality-testing microsphere solutions.

BRET Nano Q-Body: A Nanobody-Based Ratiometric Bioluminescent Immunosensor for Point-of-Care Testing.

We developed a nanobody-based homogeneous bioluminescent immunosensor to achieve a one-pot detection for point-of-care testing (POCT). This immunosensor was named BRET nano Q-body as its emission color changes via bioluminescence resonance energy transfer (BRET) upon antigen addition. NanoLuc luciferase and a cysteine-containing tag were fused to the N-terminus of the nanobody, which was labeled with a fluorescent dye via thiol-maleimide Michael addition. The nanobody employed in this proof-of-principle experiment recognizes methotrexate (MTX), a chemotherapeutic agent. After optimizing the fluorescent dye and linker, the BRET nano Q-body dose-dependently exhibited a greater than 7-fold increase in emission ratio (TAMRA/NanoLuc). Moreover, we found its superior thermostability endurance in organic solvents, reducing agents, and detergents due to the robust structure of nanobody, as well as accommodation in biological fluids, such as milk, serum, and whole blood without dilution, with limits of detection of 0.50, 1.6, and 3.7 nM, respectively. Furthermore, the BRET nano Q-body was subjected to lyophilization and fabricated into a paper device, which markedly improved its portability and enabled more than one month of storage at 25 °C. The paper device also performed appropriate functions in the biological fluids without any dilution and can be used for on-site therapeutic drug monitoring of MTX. Altogether, we developed a powerful tool, the BRET nano Q-body, for POCT, and demonstrated its applicability in several biological fluids. In addition, we confirmed the feasibility of paper devices, which are expected to be transformative for in situ detection in therapeutic, diagnostic, and environmental applications.

Rapid parallel reconstruction and specificity screening of hundreds of T cell receptors.

The ability to screen the reactivity of T cell receptors (TCRs) is essential to understanding how antigen-specific T cells drive productive or dysfunctional immune responses during infections, cancer and autoimmune diseases. Methods to profile large numbers of TCRs are critical for characterizing immune responses sustained by diverse T cell clones. Here we provide a medium-throughput approach to reconstruct dozens to hundreds of TCRs in parallel, which can be simultaneously screened against primary human tissues and broad curated panels of antigenic targets. Using Gibson assembly and miniaturized lentiviral transduction, individual TCRs are rapidly cloned and expressed in T cells; before screening, TCR cell lines undergo combinatorial labeling with dilutions of three fluorescent dyes, which allows retrieval of the identity of individual T cell effectors when they are organized and tested in pools using flow cytometry. Upon incubation with target cells, we measure the upregulation of CD137 on T cells as a readout of TCR activation. This approach is scalable and simultaneously captures the reactivity of pooled TCR cell lines, whose activation can be deconvoluted in real time, thus providing a path for screening the reactivity of dozens of TCRs against broad panels of synthetic antigens or against cellular targets, such as human tumor cells. We applied this pipeline to systematically deconvolute the antitumoral and antiviral reactivity and antigenic specificity of TCRs from human tumor-infiltrating lymphocytes. This protocol takes ~2 months, from experimental design to data analysis, and requires standard expertise in cloning, cell culture and flow cytometry.

Synthesis of Superhydrophobic Fluorescent Dyes Based on Coumarin-benzoxazole Derivatives and Their Dyeability Toward Unmodified Polypropylene Fibers

Synthesis of Superhydrophobic Fluorescent Dyes Based on Coumarin-benzoxazole Derivatives and Their Dyeability Toward Unmodified Polypropylene Fibers

Data-Driven Discovery of a New Fluorescent BASHY Dye for Bioimaging

Data-Driven Discovery of a New Fluorescent BASHY Dye for Bioimaging

Fluorescent primers amplification refractory mutation system qPCR (FP ARMS-qPCR) for MTHFR C677T SNP genotyping.

The currently used methods for the detection of methylene tetrahydrofolic acid reductase (MTHFR) C677T single nucleotide polymorphism (SNP) are either time-consuming or expensive. In this study, we devised an accurate, rapid and easy-to-use SNP detection system based on fluorescent primers amplification refractory mutation system qPCR, known as FP ARMS-qPCR. Fluorescent primers (FPs) modified by fluorescent dye or quencher near the 3' terminal thymine were designed. The reaction conditions were optimized and the performance was evaluated. Using commercial kits as controls, 242 samples were tested in parallel to verify the feasibility of the FP ARMS-qPCR assay. We demonstrated the good sensitivity and specificity of the FP ARMS-qPCR with optimized conditions. The assay was able to accurately distinguish between different SNP sites of MTHFR C677T in less than 2h using as low as 50 pg of template genomic DNA. Completely consistent genotyping results reveal that FP ARMS-qPCR is concordant with commercial kits. We established a specific, sensitive, and rapid FP ARMS-qPCR method for the detection of MTHFR C677T genotype. This could also serve as a potential diagnostic tool for a variety of diseases.

Development of a DualEmission Laser-Induced Fluorescence (DELIF) Method for Long-Term Temperature Measurements

The fluorescence intensity of fluorescent dyes typically employed in the dual-emission laser-induced fluorescence (DELIF) method gradually degrades as the excitation time increases, and the degradation rate depends on the type of fluorescent dye used. Therefore, the DELIF method is unsuitable for long-term temperature measurements. In this study, we focused on the fluorescence intensity ratio of a single fluorescent dye at two fluorescence wavelengths and developed a DELIF method for long-term temperature measurements based on this ratio. The fluorescence intensity characteristics of Fluorescein disodium and Rhodamine B, which are typically used in the DELIF method, in the temperature range of 10–60 °C were comprehensively investigated using two high-speed monochrome complementary metal-oxide semiconductor cameras and narrow bandpass filters. Interestingly, the ratio of the fluorescence intensity of each fluorescent dye at the peak emission wavelength of the fluorescence spectrum, λ, to the fluorescence intensity at wavelengths very close to the peak wavelength (λ ± 10 nm) was highly sensitive to temperature variations but not excitation time. Particularly, when Rhodamine B was used, the selection of the fluorescence intensity ratios at a wavelength combination of 589 and 600 nm enabled highly accurate temperature measurements with a temperature resolution of ≤0.042 °C. Moreover, the fluorescence intensity ratio varied negligibly throughout the excitation time of 180 min, with a measurement uncertainty (95% confidence interval) of 0.045 °C at 20 °C. The results demonstrate that the proposed DELIF method enables highly accurate long-term temperature measurements.

DLK1 Is Associated with Stemness Phenotype in Medullary Thyroid Carcinoma Cell Lines

Medullary thyroid carcinoma (MTC) is a rare and aggressive tumor, often requiring systemic treatment in advanced or metastatic stages, where drug resistance presents a significant challenge. Given the role of cancer stem cells (CSCs) in cancer recurrence and drug resistance, we aimed to identify CSC subpopulations within two MTC cell lines harboring pathogenic variants in the two most common MEN2-associated codons. We analyzed 15 stemness-associated markers, along with well-established thyroid stem cell markers (CD133, CD44, and ALDH1), a novel candidate (DLK1), and multidrug resistance proteins (MRP1 and MRP3). The ability to efflux the fluorescent dye Hoechst 3342 and form spheroids, representing CSC behavior, was also assessed. MZ-CRC-1 cells (p.M918T) displayed higher expressions of canonical markers, DLK1, and MRP proteins than TT cells (p.C634W). MZ-CRC-1 cells also formed more spheroids and showed less dye accumulation (p < 0.0001). Finally, we observed that DLK1+ cells (those expressing DLK1) in both cell lines exhibited significantly higher levels of stemness markers compared to DLK1− cells (those lacking DLK1 expression). These findings underscore DLK1’s role in enhancing the stemness phenotype, providing valuable insights into MTC progression and resistance and suggesting potential therapeutic implications.

Fluorescent Staining and Quantification of Starch Granules in Chloroplasts of Live Plant Cells Using Fluorescein.

Plants use CO2, water, and light energy to generate carbohydrates through photosynthesis. During daytime, these carbohydrates are polymerized, leading to the accumulation of starch granules in chloroplasts. The catabolites produced by the degradation of these chloroplast starch granules are used for physiological responses and plant growth. Various staining methods, such as iodine staining, have previously been used to visualize the accumulation of chloroplast starch granules; however, these staining methods cannot be used to image live cells and/or provide confocal images with non-specific signals. In this study, we developed a new imaging method for the fluorescent observation of chloroplast starch granules in living plant cells by staining with fluorescein, a widely available fluorescent dye. This simple staining method, which involves soaking a leaf disk in staining solution, shows high specificity in confocal images. Fluorescent images of the stained tissue allow the cellular starch content of living cells to be quantified with the same level of accuracy as a conventional biochemical method (amyloglucosidase/α-amylase method). Fluorescein staining thus not only enables the easy and clear observation of chloroplast starch granules but also allows for precise quantification in living cells. Key features • Visualizes chloroplast starch granules stained with fluorescein in living cells. • Requires only simple specimen preparation with no reagents needed other than the staining solution. • Fluorescein is readily available worldwide. • Highly specific method for identifying chloroplast starch granules in confocal images. • Enables estimation of cellular starch content using fluorescent images.

Simultaneous 2-photon and 3-photon excitation with a red fluorescent protein-cyanine dye probe pair in the 1700-nm excitation window for deep in vivo neurovascular imaging

In vivo imaging of the neurovascular network is considered to be one of the most powerful approaches for understanding brain functionality. Nevertheless, simultaneously imaging the biological neural network and blood vessels in deep brain layers in a non-invasive manner remains to a major challenge due to the lack of appropriate labeling fluorescence probe pairs. Herein, we proposed a 2-photon and 3-photon fluorescence probe pair for neurovascular imaging. Specifically, the red fluorescence protein (RFP) with an absorption maximum of around 550 nm is used as a 3-photon excited probe to label neurons, and a cyanine derivative dye Q820@BSA has a NIR absorption maximum of 825 nm as a 2-photon excited probe to label the vasculature, enabling single wavelength excitation at 1650 nm for neurovascular imaging with high emission spectral separation (>250 nm). In particular, the 2-photon action cross-section of Q820@BSA was found to be about 2-fold larger than that of indocyanine green (ICG), a commonly used red 2-photon fluorescence labeling agent, at the same excitation wavelength. Benefiting from the long wavelength advantage in reducing scattering in both 2 and 3-photon excitation of the fluorescence pairs, we demonstrated in vivo neurovascular imaging in intact adult mouse brains through white matter and deep into the hippocampus in the somatosensory cortex.

A novel NIR-activatable and photodegradable mitochondria-targeted nano-photosensitizer for superior photodynamic therapy

Photodynamic therapy (PDT) has garnered significant interest as a promising approach for cancer treatment due to its effectiveness, versatility, and non-invasiveness. However, its clinical application is hindered by several factors, such as suboptimal efficacy, poor targeting capabilities, shallow penetration depth, and safety concerns. Herein, we introduce B-SQ, a novel nano-photosensitizer that addresses these challenges. B-SQ is a self-assembling, cancer mitochondria-targeting agent that is both activatable and photodegradable, designed by strategically modifying a near-infrared fluorescent squaraine dye. We extensively assessed the PDT potential of B-SQ in various cancer cell models and explored the molecular mechanisms of PDT-induced cell death. Importantly, B-SQ demonstrated high specificity for cancer cells, and its PDT efficiency was activatable, with near-infrared fluorescence to distinguish cancer cells from normal cells. This work provides the first evidence of B-SQ inducing the intrinsic mitochondrial apoptosis pathway and cell cycle arresting in cancer cells via PDT. Additionally, its photodegradable nature is likely to minimize side effects after treatment. This research introduces an innovative class of nano-photosensitizers that are activatable, targeting cancer mitochondria, and are photodegradable with near-infrared fluorescence. These qualities present new possibilities for safer and more effective clinical cancer treatments in the future.

Artificial Light-Harvesting Systems with a Three-Step Sequential Energy Transfer Mechanism for Efficient Photocatalytic Minisci-Type Late-Stage Functionalization.

The natural process of photosynthesis involves a series of consecutive energy transfers, but achieving more steps of efficient energy transfer and photocatalytic organic conversion in artificial light-harvesting systems (ALHSs) continues to pose a significant challenge. In the present investigation, a range of ALHSs showcasing a sophisticated three-step energy transfer mechanism is designed, which are meticulously crafted using pillar[5]arene (WP[5]) and p-phenylenevinylene derivative (PPTPy), utilizing host-guest interactions as energy donors. Three distinct types of fluorescent dyes, namely Rhodamine B (RhB), Sulforhodamine 101 (SR101), and Cyanine 5 (Cy5), are employed as acceptors of energy. Starting from PPTPy-2WP[5], energy is sequentially transferred to RhB, SR101, and Cy5, successfully constructing a multi-step continuous energy transfer system with high energy transfer efficiency. More interestingly, as energy is progressively transferred, the efficiency of superoxide anion radical (O2 •-) generation gradually increased, while the efficiency of singlet oxygen (1O2) generation decreased, achieving the transformation from type II photosensitizer to type I photosensitizer. Furthermore, in order to fully utilize the energy harvested and reactive oxygen species (ROS) obtained, the ALHSs employ a multi-step sequential energy transfer process to enhance Minisci-type alkylation reactions with aldehydes through photocatalysis for late-stage functionalization in an aqueous environment, achieving a 91% yield.