Multiplexing Strategy Research Articles

Applications of CRISPR Technologies in Forestry and Molecular Wood Biotechnology.

Forests worldwide are under increasing pressure from climate change and emerging diseases, threatening their vital ecological and economic roles. Traditional breeding approaches, while valuable, are inherently slow and limited by the long generation times and existing genetic variation of trees. CRISPR technologies offer a transformative solution, enabling precise and efficient genome editing to accelerate the development of climate-resilient and productive forests. This review provides a comprehensive overview of CRISPR applications in forestry, exploring its potential for enhancing disease resistance, improving abiotic stress tolerance, modifying wood properties, and accelerating growth. We discuss the mechanisms and applications of various CRISPR systems, including base editing, prime editing, and multiplexing strategies. Additionally, we highlight recent advances in overcoming key challenges such as reagent delivery and plant regeneration, which are crucial for successful implementation of CRISPR in trees. We also delve into the potential and ethical considerations of using CRISPR gene drive for population-level genetic alterations, as well as the importance of genetic containment strategies for mitigating risks. This review emphasizes the need for continued research, technological advancements, extensive long-term field trials, public engagement, and responsible innovation to fully harness the power of CRISPR for shaping a sustainable future for forests.

A cost-effective protocol for single-cell RNA sequencing of human skin.

Single-cell RNA sequencing (scRNAseq) and flow cytometry studies in skin are methodologically complex and costly, limiting their accessibility to researchers worldwide. Ideally, RNA and protein-based analyses should be performed on the same lesion to obtain more comprehensive data. However, current protocols generally focus on either scRNAseq or flow cytometry of healthy skin. We present a novel label-free sample multiplexing strategy, building on the souporcell algorithm, which enables scRNAseq analysis of paired blood and skin samples. Additionally, we provide detailed instructions for simultaneous flow cytometry analysis from the same sample, with necessary adaptations for both healthy and inflamed skin specimens. This tissue multiplexing strategy mitigates technical batch effects and reduces costs by 2-4 times compared to existing protocols. We also demonstrate the effects of varying enzymatic incubation durations (1, 3, and 16 hours, with and without enzyme P) on flow cytometry outcomes. Comprehensive explanations of bioinformatic demultiplexing steps and a detailed step-by-step protocol of the entire experimental procedure are included. The protocol outlined in this article will make scRNAseq and flow cytometry analysis of skin samples more accessible to researchers, especially those new to these techniques.

Achieving a 35-Plex Tandem Mass Tag Reagent Set through Deuterium Incorporation.

Mass spectrometry-based sample multiplexing with isobaric tags permits the development of high-throughput and precise quantitative biological assays with proteome-wide coverage and minimal missing values. Here, we nearly doubled the multiplexing capability of the TMTpro reagent set to a 35-plex through the incorporation of one deuterium isotope into the reporter group. Substituting deuterium frequently results in suboptimal peak coelution, which can compromise the accuracy of reporter ion-based quantification. To counteract the deuterium effect on quantitation, we implemented a strategy that necessitated the segregation of nondeuterium and deuterium-containing channels into distinct subplexes during normalization procedures, with reassembly through a common bridge channel. This multiplexing strategy of "design independent sub-plexes but acquire together" (DISAT) was used to compare protein expression differences between human cell lines and in a cysteine-profiling (i.e., chemoproteomics) experiment to identify compounds binding to cysteine-113 of Pin1.

A 96-Well LED Array for Multiplexed Photoelectrochemical Detection of Nucleic Acids.

Photoelectrochemical detection of nucleic acid-based cancer biomarkers offers opportunities for highly sensitive, selective, and fast quantitative detection using low-cost measurement instruments. In order to establish itself as a standard method for identifying and quantifying nucleic acids, we have developed a multiplexing strategy using LED technology for photoelectrochemical detection in 96 samples simultaneously. A dedicated setup based on the 96-well plate configuration with a custom-made 96-well LED array was developed. Subsequently, a proof-of-concept study was performed for three miRNAs that are associated with prostate cancer, i.e., miRNA-141, miRNA-145, and miRNA-375. First, measurements with photosensitizer chlorin e6 and redox reporter hydroquinone free in solution proved the proper functioning of the multiplexed detection. Second, the photoelectrochemical detection of the three miRNAs at 24 nM levels was successfully demonstrated. Thereafter, linear calibration curves (R2 > 0.9 for all analytes) were made with plasma spiked with 8-500 pM miRNA. This work presents the first system for multiplexed high-throughput photoelectrochemical detection, allowing it potentially to become a cost-effective and faster alternative to RT-qPCR and gene sequencing techniques in the future.

Biometry study of foveal isoplanatic patch variation for adaptive optics retinal imaging.

The change in ocular wavefront aberrations with visual angle determines the isoplanatic patch, defined as the largest field of view over which diffraction-limited retinal imaging can be achieved. Here, we study how the isoplanatic patch at the foveal center varies across 32 schematic eyes, each individualized with optical biometry estimates of corneal and crystalline lens surface topography, assuming a homogeneous refractive index for the crystalline lens. The foveal isoplanatic patches were calculated using real ray tracing through 2, 4, 6 and 8 mm pupil diameters for wavelengths of 400-1200 nm, simulating five adaptive optics (AO) strategies. Three of these strategies, used in flood illumination, point-scanning, and line-scanning ophthalmoscopes, apply the same wavefront correction across the entire field of view, resulting in almost identical isoplanatic patches. Two time-division multiplexing (TDM) strategies are proposed to increase the isoplanatic patch of AO scanning ophthalmoscopes through field-varying wavefront correction. Results revealed substantial variation in isoplanatic patch size across eyes (40-500%), indicating that the field of view in AO ophthalmoscopes should be adjusted for each eye. The median isoplanatic patch size decreases with increasing pupil diameter, coarsely following a power law. No statistically significant correlations were found between isoplanatic patch size and axial length. The foveal isoplanatic patch increases linearly with wavelength, primarily due to its wavelength-dependent definition (wavefront root-mean-squared, RMS <λ/14), rather than aberration chromatism. Additionally, ray tracing reveals that in strongly ametropic eyes, induced aberrations can result in wavefront RMS errors as large as λ/3 for an 8-mm pupil, with implications for wavefront sensing, open-loop ophthalmic AO, spectacle prescription and refractive surgery.

Aggregation Induced Emission-Based Covalent Organic Frameworks for High-Performance Optical Wireless Communication.

Here, we report the first utilization of covalent organic frameworks (COFs) in optical wireless communication (OWC) applications. In the solid form, aggregation-induced emission (AIE) luminogen often shows promising emissive characteristics that augment radiative decays and improve fluorescence. We have synthesized an AIE-COF through the Knoevenagel condensation reaction by taking advantage of the ability to carefully design and alter the COF structure by integrating an AIE luminogen with linear building blocks. The synthesized AIE-COF exhibited a high solid-state photoluminescence quantum yield (∼39%) and a short photoluminescence lifetime (∼1 ns), crucial for achieving modulation bandwidth for high-speed OWC applications. For comparison, we constructed an aggregation-caused quenching based COF, showing a similar lifetime but almost insignificant quantum yield. The orthogonal frequency-division multiplexing modulation strategy employed by the AIE-COF demonstrates remarkable high-rate data transmission, with a wide -3 dB modulation bandwidth of nearly 200 MHz and achieving high net data rates of 825 Mb/s, outperforming traditional materials. These results open new avenues for the ability to design and finetune new COF materials for their utilization as color converters in developing cutting-edge OWC components, enabling faster and more efficient data transfer.

Entropy production in communication channels.

In many complex systems, whether biological or artificial, the thermodynamic costs of communication among their components are large. These systems also tend to split information transmitted between any two components across multiple channels. A common hypothesis is that such inverse multiplexing strategies reduce total thermodynamic costs. So far, however, there have been no physics-based results supporting this hypothesis. This gap existed partially because we have lacked a theoretical framework that addresses the interplay of thermodynamics and information in off-equilibrium systems. Here we present the first study that rigorously combines such a framework, stochastic thermodynamics, with Shannon information theory. We develop a minimal model that captures the fundamental features common to a wide variety of communication systems, and study the relationship between the entropy production of the communication process and the channel capacity, the canonical measure of the communication capability of a channel. In contrast to what is assumed in previous works not based on first principles, we show that the entropy production is not always a convex and monotonically increasing function of the channel capacity. However, those two properties are recovered for sufficiently high channel capacity. These results clarify when and how to split a single communication stream across multiple channels.

ОЦІНКА ЧАСТОТНО-ФАЗОВИХ СПОТВОРЕНЬ В ОПТИЧНИХ ТЕЛЕКОМУНІКАЦІЯХ З OFDM

The article proposes a method for monitoring the peak-to-average power ratio (PAPR), and also discusses the results of assessing the sensitivity of optical-OFDM systems to phase-frequency distortions. The results of a study of the noise immunity of an optical-OFDM system for frequency shift relative to the spacing of optical subcarriers are presented. A description of the factors causing interference and frequency-phase distortions caused by chromatic dispersion (CD) and polarization mode dispersion (PMD) on the performance of optical multiplexing systems with orthogonal frequency division is carried out. Monitoring the PAPR in OFDM systems is relevant, in particular, in the context of fiber optic communication problems that are caused by the nonlinearity of the optical fiber. It is shown that when directly deploying optical networks, the presence of frequency distortions and sensitivity to phase noise are the two main disadvantages of OFDM. Both frequency distortion and phase noise lead to interchannel interference (ICI). Due to the relatively large symbol length compared to a single carrier, OFDM is susceptible to both frequency distortion and phase noise. An additional cumulative distribution function is used for a visual description of PARP. The effect of the oversampling coefficient on the PARP value is determined. It is proposed to use the oversampled signal to obtain a more accurate PARP value. The results of the study show that the effect of phase noise on a signal in optical OFDM channels is due to the existing long symbol period compared to signal transmission on a single carrier. In addition, with an increase in the modulation order, optical OFDM becomes more sensitive to phase noise, which encourages an increase in the signal-to-noise ratio to counteract this. The results of the research showed that for coherent OFDM optical systems, the line width of the optical quantum generator is a critical parameter, especially when switching to high-order modulation. The paper discusses a diagram describing the process of convergence of radio and optical technologies in the context of the use of OFDM modulation. This technique allows for the correct choice of channel multiplexing strategy in optical OFDM telecommunications with multi-position signals.

Optimizing Fi-Wi network performance through advanced multiplexing techniques: a comparative analysis for enhanced quality metrics

Abstract Fi-Wi networks, emblematic of the convergence between optical fibers and wireless access, stand resolutely at the vanguard of the transformative redefinition of communication paradigms. As advanced communication networks persistently redefine the contours of connectivity, characterized by their unparalleled speed, minimal latency, and augmented capacity, the exigency for innovative approaches undergoes heightened intensification. The crux of this study pivots upon the methodical application of multiplexing techniques, notably wavelength division multiplexing (WDM), optical code division multiplexing (OCDMA), and optical time division multiplexing (OTDM), each deployed with precision to elevate the nuanced performance of the Fi-Wi network. The multifaceted optimization of these techniques not only imparts an impetus to data transfer rates, mitigates latency, and augments spectral efficiency but concurrently instigates the realm of wireless connectivity. The research undertakes a technical exploration of the deployed multiplexing strategies, delineating their idiosyncratic advantages. A discerning comparative analysis vis-a-vis the hybrid (Fi-Wi)-single model, precisely serving as the baseline, unequivocally delineates the superior performance of the proposed methods across metrics of Q-factor, eye height, and logarithmic bit error rate-Q factor.

Quantitative Comparison of Ventilation Parameters of Different Approaches to Ventilator Splitting and Multiplexing.

Amid the COVID-19 pandemic, this study delves into ventilator shortages, exploring simple split ventilation (SSV), simple differential ventilation (SDV), and differential multiventilation (DMV). The knowledge gap centers on understanding their performance and safety implications. Our hypothesis posits that SSV, SDV, and DMV offer solutions to the ventilator crisis. Rigorous testing was anticipated to unveil advantages and limitations, aiding the development of effective ventilation approaches. Using a specialized test bed, SSV, SDV, and DMV were compared. Simulated lungs in a controlled setting facilitated measurements with sensors. Statistical analysis honed in on parameters like peak inspiratory pressure (PIP) and positive end-expiratory pressure. Setting target PIP at 15 cm H2O for lung 1 and 12.5 cm H2O for lung 2, SSV revealed a PIP of 15.67 ± 0.2 cm H2O for both lungs, with tidal volume (Vt) at 152.9 ± 9 mL. In SDV, lung 1 had a PIP of 25.69 ± 0.2 cm H2O, lung 2 at 24.73 ± 0.2 cm H2O, and Vts of 464.3 ± 0.9 mL and 453.1 ± 10 mL, respectively. DMV trials showed lung 1's PIP at 13.97 ± 0.06 cm H2O, lung 2 at 12.30 ± 0.04 cm H2O, with Vts of 125.8 ± 0.004 mL and 104.4 ± 0.003 mL, respectively. This study enriches understanding of ventilator sharing strategy, emphasizing the need for careful selection. DMV, offering individualization while maintaining circuit continuity, stands out. Findings lay the foundation for robust multiplexing strategies, enhancing ventilator management in crises.

Ultraviolet-visible spectroscopy with all-dielectric multi-foci dispersive metalens

Metasurfaces are a cutting-edge development in optical technology miniaturization, enabling the manipulation of light at a microscopic scale while providing unprecedented control over its intrinsic characteristics, such as amplitude, polarization, and phase. Researchers have been making significant progress in advancing and developing high-efficiency and functionally versatile metasurfaces, aiming to achieve miniaturization and overcoming the technological limitations of conventional components. This article is intended to develop a single-layered, highly transmissive all-dielectric multi-foci metalens that serves as a compact spectrometer for portable investigative measurements within the ultraviolet to the visible spectrum, especially for 290–460nm band. The proposed metalens can achieve top-notch performance by precisely mapping the wavelength information into subwavelength diffraction-limited focusing spots. This approach helps in accomplishing simultaneous spectral splitting and focusing on the same plane. The proposed metalens consists of an array of intelligently optimized nanoantennas made of a CMOS-compatible silicon nitride material. It exploits a unique phase and wavelength multiplexing strategy to create highly dispersive multi-foci metalens, making it an ideal choice for an ultra-compact, high-resolution, and dual-band meta-spectrometer. We expect our proposed framework to have numerous applications in the industrial and medical fields, enabling diagnosis, detection, and assessment within a compact platform.

Enhancing grid-connected inverter performance under non-ideal grid conditions: a multi-functional multiplexing control strategy

Compared to the traditional thermal power generation, new energy sources, such as wind and photovoltaic systems, are more vulnerable to the effects of non-ideal power grids due to their limited capacity. This susceptibility can jeopardize the safe operation of power equipment, degrade power output quality, and lead to non-compliance with grid-connected specifications. The LCL-type grid-connected inverter is a typical nonlinear system that weakens the controllability of the grid-connected energy. To address these challenges, this study employs feedback linearization theory to transform the inverter into a standard linear system. Subsequently, it utilizes linear system methodologies to develop robust control laws, ultimately introducing a multi-functional multiplexing control strategy for grid-connected inverters based on feedback linearization and Hamilton-Jacobi-Issacs inequality. Simulation results demonstrate that this multi-functional strategy outperforms traditional grid-connected inverter control schemes, effectively mitigating issues related to low short-circuit ratios, voltage fluctuations, imbalances, harmonics, and other non-ideal grid conditions. Furthermore, it significantly expands the system’s adaptability to varying weak grid impedances.

Multiparameter flow cytometric detection and analysis of rare cells in in vivo models of cancer metastasis.

Rapid and reliable circulating tumor cell (CTC) and disseminated tumor cell (DTC) detection are critical for rigorous evaluation of in vivo metastasis models. Clinical data show that each step of the metastatic cascade presents increasing barriers to success, limiting the number of successful metastatic cells to fewer than 1 in 1,500,000,000. As such, it is critical for scientists to employ approaches that allow for the evaluation of metastatic competency at each step of the cascade. Here, we present a flow cytometry-based method that enables swift and simultaneous comparison of both CTCs and DTCs from single animals, enabling evaluation of multiple metastatic steps within a single model system. We present the necessary gating strategy and optimized sample preparation conditions necessary to capture CTCs and DTCs using this approach. We also provide proof-of-concept experiments emphasizing the appropriate limits of detection of these conditions. Most importantly, we successfully recover CTCs and DTCs from murine blood and bone marrow. In Supplemental materials, we expand the applicability of our method to lung tissue and exemplify a potential multi-plexing strategy to further characterize recovered CTCs and DTCs. This approach to multiparameter flow cytometric detection and analysis of rare cells in in vivo models of metastasis is reproducible, high throughput, broadly applicable, and highly adaptable to a wide range of scientific inquiries. Most notably, it simplifies the recovery and analysis of CTCs and DTCs from the same animal, allowing for a rapid first look at the comparative metastatic competency of various model systems throughout multiple steps of the metastatic cascade.

Expedient Synthesis and Characterization of π-Extended Luciferins.

Bioluminescence imaging enables the sensitive tracking of cell populations and the visualization of biological processes in living systems. Bioluminescent luciferase/luciferin pairs with far-red and near-infrared emission benefit from the reduced competitive absorption by blood and tissue while also facilitating multiplexing strategies. Luciferins with extended π-systems, such as AkaLumine and recently reported CouLuc-1 and -3, can be used for bioluminescence imaging in this long wavelength regime. Existing synthetic routes to AkaLumine and similar π-extended compounds require a multistep sequence to install the thiazoline heterocycle. Here we detail the development of a two-step strategy for accessing these molecules via a Horner-Wadsworth-Emmons reaction and cysteine condensation sequence from readily available aldehyde starting materials. We detail an improved synthesis of AkaLumine, as well as the corresponding two-carbon homologues, Tri- and Tetra-AkaLumine. We then extended this approach to prepare coumarin- and naphthalene-derived luciferins. These putative luciferins were tested against a panel of luciferases to identify capable emitters. Of these, an easily prepared naphthalene derivative exhibits photon emission on par with that of the broadly used Akaluc/AkaLumine pair with similar emission maxima. Overall, this chemistry provides efficient access to several bioluminescent probes for a variety of imaging applications.

Two-Time Multiplexed Targeted Next-Generation Sequencing Might Help the Implementation of Germline Screening Tools for Myelodysplastic Syndromes/Hematologic Neoplasms.

Next-generation sequencing (NGS) tools have importantly helped the classification of myelodysplastic syndromes (MDS), guiding the management of patients. However, new concerns are under debate regarding their implementation in routine clinical practice for the identification of germline predisposition. Cost-effective targeted NGS tools would improve the current standardized studies and genetic counseling. Here, we present our experience in a preliminary study detecting variants using a two-time multiplexed library strategy. Samples from different MDS patients were first mixed before library preparation and later multiplexed for a sequencing run. Two different mixes including a pool of three (3×) and four (4×) samples were evaluated. The filtered variants found in the individually sequenced samples were compared with the variants found in the two-time multiplexed studies to determine the detection efficiency scores. The same candidate variants were found in the two-time multiplexed studies in comparison with the individual tNGS. The variant allele frequency (VAF) values of the candidate variants were also compared. No significant differences were found between the expected and observed VAF percentages in both the 3× (p-value 0.74) and 4× (p-value 0.34) multiplexed studies. Our preliminary results suggest that the two-time multiplexing strategy might have the potential to help reduce the cost of evaluating germline predisposition.

A multi-laser hybrid absorption sensor for simultaneous measurement of NH3, NO, and temperature in denitrification flue gas

The real-time monitoring of NH3, NO, and flue gas temperatures is critical to controlling NOx emissions from coal-fired power plants, reducing ammonia slip, and realizing accurate ammonia injection. However, few previous studies have utilized laser absorption sensors to measure these three indicators simultaneously. In this paper, a multi-laser hybrid sensor is designed based on tunable diode laser absorption spectroscopy (TDLAS) for simultaneous measurement of NH3, NO, and flue gas temperature. The sensor uses a distributed feedback (DFB) laser to target the NH3 and the H2O lines, and a quantum cascade laser (QCL) is used to target the NO line. In the measurements of NH3 and NO using calibration-free wavelength-modulated spectroscopy (CF-WMS), we employ a method to obtain the laser wavelength response by fitting the demodulated signal of a standard gas. Additionally, temperature measurements are obtained using the time-division multiplexing (TDM) strategy and direct absorption spectroscopy (DAS) techniques with the help of the absorption spectrum of H2O. Under static conditions, the relative accuracy of NH3 and NO detected by the sensor is 4.2% and 2%, respectively, and the maximum relative standard deviation of temperature is 1.3%. When tested under flowing conditions, the sensor can accurately capture NH3, NO, and temperature changes. The relative accuracy of NH3 and NO is 4.7% and 2.2%, and the relative standard deviation of temperature is 0.9%. The sensor has great potential in optimizing denitrification processes in thermal power plants.

Deep learning-empowered moving cascaded acoustic holography for high-fidelity and high-capacity acoustic holographic reconstruction

Acoustic holograms offer a promising platform for reconstructing complex acoustic fields with high resolution in three dimensions. However, traditional design algorithms based on single acoustic holograms have limitations in terms of their information capacity. To address this challenge, our study proposes a novel strategy called deep learning-empowered moving cascaded acoustic holography. This strategy enables high-capacity target patterns to be encoded onto two moving cascaded acoustic phase plates at a set of movable distances along the forward diffractive path of the incident acoustic wave. To achieve the high-fidelity and high-capacity acoustic holographic reconstruction, we develop a physics-enhanced moving cascaded acoustic hologram deep neural network (PhysNet_MCAH) method, which employs a physics-driven neural network to inversely design the phase offset distributions of two phase plates. Our results demonstrate that the PhysNet_MCAH method effectively generates high-fidelity and high-capacity moving cascaded acoustic holograms that are capable of rendering different complex target patterns at different design movable distances. Additionally, we show that moving cascaded acoustic holograms significantly improve the holographic storage capacity and enhance acoustic field reconstruction accuracy, surpassing the capabilities of moving single acoustic holograms. Remarkably, we further demonstrate that the proposed PhysNet_MCAH method achieves a superior performance in terms of the trade-off between reconstruction quality and holographic storage capacity when compared to existing unsupervised deep learning methods and multiplexing strategies for designing the multi-plane or cascaded holograms. Our method offers a new paradigm for high-capacity acoustic volumetric display, dynamic particle manipulation, and other applications.

Optical Nonlinearity Enabled Super-Resolved Multiplexing Microscopy.

Optical multiplexing for nanoscale object recognition is of great significance within the intricate domains of biology, medicine, anti-counterfeiting, and microscopic imaging. Traditionally, the multiplexing dimensions of nanoscopy are limited to emission intensity, color, lifetime, and polarization. Here, a novel dimension, optical nonlinearity, is proposed for super-resolved multiplexing microscopy. This optical nonlinearity is attributable to the energy transitions between multiple energy levels of the doped lanthanide ions in upconversion nanoparticles (UCNPs), resulting in unique optical fingerprints for UCNPs with different compositions. A vortex beam is applied to transport the optical nonlinearity onto the imaging point-spread function (PSF), creating a robust super-resolved multiplexing imaging strategy for differentiating UCNPs with distinctive optical nonlinearities. The composition information of the nanoparticles can be retrieved with variations of the corresponding PSF in the obtained image. Four channels multiplexing super-resolved imaging with a single scanning, applying emission color and nonlinearity of two orthogonal imaging dimensions with a spatial resolution higher than 150nm (1/6.5λ), are demonstrated. This work provides a new and orthogonal dimension - optical nonlinearity - to existing multiplexing dimensions, which shows great potential in bioimaging, anti-counterfeiting, microarray assays, deep tissue multiplexing detection, and high-density data storage.

Demonstration of 75 km-fiber quantum clock synchronization in quantum entanglement distribution network

The quantum entanglement distribution network, serviced as the communication infrastructure which distributes quantum information among remote users, enables many applications beyond the reach of classical networks. Recently, the applications such as quantum key distribution and quantum secure direct communication, have been successfully demonstrated in the quantum entanglement distribution network. In this article, we propose a multi-user round-trip quantum clock synchronization (QCS) scheme in the quantum network, which can be implemented with one single entangled photon source located at the server. The server distributes the entangled photons to remote multiple users with the wavelength division multiplexing strategy, and each user feeds partial received photons back to the server. The clock difference between the server and each user is calculated from the one-way and round-trip propagation times, which are determined according to the time correlation of entangled photons. Afterwards, the demonstration has been conducted between the server and a user over a 75-km-long fiber link, where the measured clock difference uncertainty is 4.45 ps, and the time deviation is 426 fs with an average time of 4000 s. Furthermore, the proposed QCS scheme is linearly scalable to many users, with respect to user hardware and number of deployed fibers.

Evaluation of Three-Level Code Division Multiplexing for High-Quality Radio over Fiber Communication

Radio over Fiber system (RoF) technology facilitates the transmission of wireless signals over optical fibers, which provides many benefits, including low signal loss, and immunity to electromagnetic interference. Despite these benefits, conventional RoF systems grapple with signal impairments and limited user capacity, as each user must be allocated a specific frequency. To address these limitations, this paper explores the implementation of the Three-Level Division Multiplexing (3LCDM) technique with RoF, a superior multiplexing strategy that allows two users to share a single channel, thereby doubling capacity. This innovative combination was evaluated for various cases using 2.4 GHz, 5 GHz, and the combination of 2.4 GHz and 5 GHz carrier frequencies, and its performance was compared with traditional RoF systems. Simulations were carried out using OptiSystem in cooperation with the MATLAB simulator. The findings of this study reveal that the 3LCDM-RoF system can accommodate twice the number of users as the conventional RoF system without the need for amplifiers. Furthermore, it supports higher bit rates and longer transmission distances. Specifically, the 3LCDM-RoF system achieved maximum data rates at maximum fiber length of 2 x 1.1 Gbps at 60 km, (2.4 GHz), 2 x 2 Gbps at 40 km (5 GHz), and 2 x 0.5 Gbps per channel at 43 km (combined 2.4 GHz and 5 GHz) respectively. Meanwhile, the conventional RoF with 2.4 GHz, 5GHz and the combination of 2.4 GHz and 5 GHz can only achieve maximum data rate at a maximum fiber length of 1.1 Gbps at 45 km, 1.6 Gbps at 35 km, and 0.4 Gbps at 35 km respectively