Fluorescence Phenomena Research Articles

FRET-based Ratiometric Fluorescent Probes for Enzyme Detection: Current Insight

Over the decade many types of fluorescent sensors have been developed for detecting diverse types of analyte. The sensors developed using the phenomenon of fluorescence provide high sensitivity, selectivity, for the analyte that they are being developed for. This has led to a huge increase in development of sensors for biomarkers that are particularly of importance for early detection or diagnosis of life threatening diseases. In addition to the advantages of Fluorimetry there is continuous research going on to create sensors that are easy to construct, reproducible, cost and time efficient, along with maintaining sensitivity enough for accurate determination of the analyte of interest. As the research advanced, the dyes used as simple sensors were replaced with other molecules as a substrate for biomarker or other analyte sensing. Additionally, early scientists used single emission sensors for detection of analyte. Further, the single emission sensors were evolved to dual emission and then further advancement led to innovation of ratiometric sensors. These ratiometric sensors provide good internal standard referencing system which gives them good sensitivity as compared to other luminescent sensors. Through this review we aim to provide useful information on the subject of FRET, ratiometric fluorescence analysis, the types of materials used for developing the sensors and examples of biosensors used for enzyme detection.

MicroMetaSense: Coupling Plasmonic Metasurfaces with Fluorescence for Enhanced Detection of Microplastics in Real Samples.

Diverse analytical techniques are employed to scrutinize microplastics (MPs)─pervasive at hazardous concentrations across diverse sources ranging from water reservoirs to consumable substances. The limitations inherent in existing methods, such as their diminished detection capacities, render them inadequate for analyzing MPs of diminutive dimensions (microplastics: 1-5 μm; nanoplastics: < 1 μm). Consequently, there is an imperative need to devise methodologies that afford improved sensitivity and lower detection limits for analyzing these pollutants. In this study, we introduce a holistic strategy, i.e., MicroMetaSense, reliant on a metal-enhanced fluorescence (MEF) phenomenon in detecting a myriad size and types of MPs (i.e., poly(methyl methacrylate) (PMMA) and poly(ethylene terephthalate) (PET)) down to 183-205 fg, as well as validated the system with real samples (tap and lake) and artificial ocean samples as a real-world scenario. To obtain precise size distribution in nanometer scale, MPs are initially processed with an ultrafiltration on-a-chip method, and subsequently, the MPs stained with Nile Red dye are subjected to meticulous analysis under a fluorescence microscope, utilizing both a conventional method (glass substrate) and the MicroMetaSense platform. Our approach employs a metasurface to augment fluorescence signals, leveraging the MEF phenomenon, and it demonstrates an enhancement rate of 36.56-fold in detecting MPs compared to the standardized protocols. This low-cost ($2), time-saving (under 30 min), and highly sensitive (183-205 femtogram) strategy presents a promising method for precise size distribution and notable improvements in detection efficacy not only for laboratory samples but also in real environmental samples; hence, signifying a pivotal advancement in conventional methodologies in MP detection.

Brightening dark excitons in inorganic halide perovskites by local symmetry breaking

Thermally activated delayed fluorescence (TADF) phenomenon, defined as thermal activation of triplet dark-state excitons into singlet state excitons through reverse intersystem crossing (RISC), has become a prevalent principle for designing organic-based electro-/photo-luminescent molecules. However, activating dark excitons in inorganic metal halide perovskites (MHPs) still remains challenging. Herein, we report an efficient strategy of doping-induced local symmetry breaking to brighten dark excitons in inorganic halide perovskite Cs2ZrCl6, which result in a 500 % enhancement of low-temperature luminescence emission. Lattice symmetry breaking confirmed by the X-ray absorption spectroscopy measurements could disturb the local coordination environment at the Zr site. The singlet-triplet energy gap (ΔEST) fitted from temperature-dependent photoluminescence decay curves decreases from 0.087 to 0.071 eV, thus reducing up-conversion barrier of dark excitons. Theoretical calculation indicates large spatial separation of charge density favorable for enhanced TADF emission. Furthermore, a flexible X-ray scintillator screen with poly (dimethylsiloxane) as the matrix could obtain high-quality X-ray imaging with a 14 lp mm−1 resolution. This work gives insights into improving photoemission of inorganic MHPs via brightening dark excitons.

An artificial intelligence handheld sensor for direct reading of nickel ion and ethylenediaminetetraacetic acid in food samples using ratiometric fluorescence cellulose paper microfluidic chip

User-friendly in-field sensing protocol is crucial for the effective tracing of intended analytes under less-developed countries or resources-limited environments. Nevertheless, existing sensing strategies require professional technicians and expensive laboratory-based instrumentations, which are not capable for point-of-care on-site analyses. To address this issue, artificial intelligence handheld sensor has been designed for direct reading of Ni2+ and EDTA in food samples. The sensing platform incorporates smartphone with machine learning-driven application, 3D-printed handheld device, and cellulose paper microfluidic chip stained with ratiometric red-green-emission carbon dots (CDs). Intriguingly, Ni2+ introduction makes green fluorescent (FL) of CDs glow but red FL fade because of the coordination of Ni2+ with CDs verified by density functional theory (DFT), concurrently manifesting continuous FL colour transition from red to green. Subsequent addition of EDTA renders FL of CDs-Ni2+ recover owing to the capture of Ni2+ from CDs by EDTA based on strong chelation effect of EDTA on Ni2+ confirmed via DFT, accompanying with a noticeable colour returning from green to red. Inspired by above FL phenomena, CDs-based cellulose paper microfluidic chips are first fabricated to facilitate point-of-care testing of Ni2+ and EDTA. Designed fully-automatic handheld sensor is utilized to directly output Ni2+ and EDTA concentration in water, milk, spinach, bread, and shampoo based on wide linear ranges of 0–48 μM and 0–96 μM, and low limits of detection of 0.274 μM and 0.624 μM, respectively. The proposed protocol allows for speedy straightforward on-site determination of target analytes, which will trigger the development of automated and intelligent sensors in near future.

Al2O Photochemistry

The chemistry within the interstellar medium (ISM) is notably influenced by the interplay between kinetics and photochemical processes, which play significant roles in both the formation and destruction of molecular species. This study focuses on theoretical investigations of Al2O photochemistry, aiming to elucidate the mechanisms underlying the production of AlO and Al in the VY-CMa star. Utilizing advanced theoretical methodologies, we explore the lowest electronic states with singlet and triplet spin multiplicities in linear Al2O. We investigated the photostability of Al2O in the near UV‒Vis region, revealing the low likelihood of photodissociation and photoconversion while suggesting the plausibility of fluorescence and phosphorescence phenomena. Calculations also identify three prominent peaks in the UV range at 261.5, 206.2, and 199 nm. Finally, Al2O is predicted to be photostable and cannot be the parent molecule of the diatomic AlO or even the astrochemical reservoir of atomic aluminum. These results contribute to improving the astronomical models in simulating aluminum chemistry in the ISM.

Iron oxide quantum dots‐based fluorescence probe for rapid and selective cytosine sensing

AbstractThe application of iron oxide quantum dots (IOQDs) in this study led to the development of a straightforward, easy, and selective approach for cytosine sensing. To examine the capability of IOQDs in detecting nucleobases, attempts have been made to gain insight into the characteristics of IOQDs, including UV/Vis spectroscopic analysis, infrared spectroscopy, fluorescence spectroscopy, transmission electron microscopy, and powder‐XRD. We introduced IOQDs with a mean particle size distribution of 5.71 ± 0.08 nm for detecting cytosine by harnessing a turn‐on fluorescence phenomenon. The fostering fluorescence response of IOQDs was triggered in the presence of cytosine. In comparison, the presence of other nucleobases did not further induce the fluorescence signal of IOQDs. It was concluded that the hydrogen bonding bridging the carboxylate and hydroxyl groups on the surface of QDs with the amine groups of cytosine results in the elevation of IOQDs fluorescence.

The dipole-radiation interaction revisited

The phenomena of light absorption, fluorescence and laser emission are accounted for within a unique picture. The atomic dipole is dealt with in a quantum framework, whereas the electromagnetic field is assumed to be a classical sine-wave. The on and off resonance cases are investigated. This is achieved owing to a novel application of Poynting’s theorem. Observable predictions are made, regarding absorption of the electromagnetic energy and the frequency bandwidth of light emitted either by fluorescence or laser effect.

Enhanced aggregation induced emission by H-bond between the barbiturate molecules containing hydroxyl group

Barbiturates had been widely studied as a molecule with aggregation induced emission (AIE) phenomenon, but barbiturates as a group with multiple hydrogen bond donor and acceptor sites, the relationship between intermolecular hydrogen bond and AIE phenomenon has not been mainly studied. In this paper, a new barbiturate fluorescence molecule BNOH with hydroxyl group was designed and synthesized in order to analyze the relationship between hydrogen bond and molecular AIE phenomenon by increasing the functional group (–OH) that was easy to form hydrogen bond. By spectroscopic analysis, BNOH had structural torsion in aqueous mixed solution, which led to fluorescence decrease, while in aprotic mixed solvent, there was strong AIE phenomenon. DFT and TD-DFT calculations at the B3LYP/6-31G (d) were performed for a deeper understanding of optical transitions. In addition, in order to investigate the difference of its fluorescence phenomenon, two other barbiturate derivatives were synthesized and their spectra were compared with BNOH. The relationship between hydrogen bond and AIE phenomenon of barbiturates was proved, and the fluorescence mechanism of BNOH in organic solvent was proposed and determined. We believe that this study will not only increase the understanding of barbiturate AIE compounds, but also contribute to the study of the important role of hydrogen bonds in the fluorescence phenomenon of barbiturate AIE compounds.

Interpretation of the Seidel test for open eye injury: Experimental study

AIM: The study aimed to examine the phenomenon of fluorescence during the Seidel test on an experimental model of open eye injury (OEI). MATERIAL AND METHODS: The experimental study used cadaver pig eyes and was conducted in the Wetlab educational laboratory of the St. Petersburg branch of the S.N. Fedorov National Medical Research Center Interbranch Scientific and Technical Complex “Eye Microsurgery”. After creating a perforated hole, the cornea was stained with fluorescein and observed under a cobalt-blue slit-lamp light. After the test strips were removed, the experiment was timed. RESULTS: The fluorescence phenomenon was noted immediately after applying the stain to the ocular surface of cadaver eyes with OEI. Fluorescein was dissolved in the flowing intraocular fluid (IOF) and was restrained at the wound edges. Then, the color of the flowing IOF changed. In the experiment, two phases of the flowing IOF color were registered, that is, up to 2.95 s, a bright-green fluid flow was noted. After 2.95 s, the stain washed out, and the main stream of liquid was divided into several streams with varying degrees of staining intensity. At 4.12 s, a transparent stream of escaping liquid was noted in the center and green streams along the edges. CONCLUSION: The Seidel test consists of two successive phases, namely, the initial appearance of a “bright-green stream” of IOF flowing through the corneal wound, lasting no more than 3 s after the administration of fluorescein; and during phase 2, in the flow center, the fluid became transparent, and bright-green staining remained at the edges. The Seidel test should be performed in compliance with all conditions that ensure the manifestations of fluorescence, always using a cobalt slit-lamp filter, and the result should be evaluated considering the sequence of its phases.

Solvent effects on the ESIPT emission of salicylaldehyde Schiff base derivative: A theoretical reconsideration

Salicylaldehyde Schiff base derivatives have potential applications in bioimaging, chemical sensors and organic luminescence materials because of their high fluorescence quantum yield with the excited-state intramolecular proton transfer (ESIPT) process. Recently, Shu et al. studied the luminescent mechanism of the Et2N-substituted salicylaldehyde Schiff base compound (DDHAC) in solvents [Dyes and Pigments 195 (2021) 109708]. It showed double-peaked bands in an n-hexane solvent but a single-peaked band in an acetonitrile solvent. They suggested that the open-enol in the acetonitrile solvent was the dominant species. They considered that open-enol fluorescence and ESIPT fluorescence constituted a dual fluorescent phenomenon in the n-hexane solvent. However, because acetonitrile is a non-protonic solvent, DDHAC molecules in acetonitrile mainly existed as the lower-energy hydrogen-bonded enol form rather than the open-enol form alone. In addition, the open-enol did not undergo the ESIPT process in n-hexane. Therefore, the luminescence mechanism of the DDHAC molecules in n-hexane and acetonitrile solvents needs to be reconsidered. In this study, we investigated the DDHAC molecules in n-hexane and acetonitrile solvents using the density functional theory and time-dependent density functional theory. The potential energy surfaces and optimisation of structures elucidated that the DDHAC molecules in n-hexane and acetonitrile solvents underwent the ESIPT process. The calculated fluorescence peaks demonstrated that the single-peaked broad emission band in the acetonitrile solvent was formed by the combination of the hydrogen-bonded enol* and keto* forms rather than open-enol*. Moreover, the dual fluorescence peaks in the n-hexane solvent were reattributed to the open-enol* form and hydrogen-bonded enol* form. The lack of keto* fluorescence in the n-hexane solvent was attributed to diminished charge coupling in comparison to the enol* form. Our results revise the mechanism of the DDHAC molecules in n-hexane and acetonitrile solvents, providing guidance for designing efficient organic fluorescence probes.

Investigation on Fluorescence Origin and Spectral Heterogeneity in Carbon Dots: A Dynamic Perspective

AbstractHeterogeneity in the fluorescence of carbon dots (CDs) has been hard to figure out so far. This could potentially be related to structural factors, size or intermediate fluorophores, especially when employing the bottom‐up synthesis. Herein, we unveil the origin of fluorescence and spectral heterogeneity of CDs using a simple dynamic method. This work reports the room light‐excited green fluorescence and dual‐emissive N‐doped CDs synthesized using a hydrothermal method. Studies were carried out considering the factors of fluorescence phenomena, such as excitation‐independent emission, fluorescent impurities, aggregation and solvation dynamics. The new steady‐state, Excitation‐resolved area‐normalized emission spectroscopy (ERANES) and ensemble measurements, including time‐resolved studies reveal that a heterogeneous environment exists in the ground state. Eventually, the results show the existence of core, edge and surface states in the CDs. Additionally, the fluorescence characteristics depend on the structural functionalization that occurs in both the intrinsic and extrinsic states of the CDs and it is proven that this does not violate the Kasha‐Vavilov rule. Although a highly purified material could still exhibit heterogeneity due to the ensemble emissive states and structural variations. Our results provide insights into the enduring debates about fluorescence and a deeper understanding of the structure‐ property relationship of CDs.

A novel fluorescence sensor based on Al3+-mediated aggregation of gold nanoclusters for determination of citric acid in beverages.

This paper revealed a new strategy for citric acid (CA) detection using aggregation-induced emission (AIE)-based fluorescent gold nanoclusters (AuNCs). AuNCs was synthesized using glutathione (GSH) as the template and reducing agent and used as the fluorescent probe to detect CA under aluminum ion (Al3+) mediation. The fluorescence intensity of AuNCs increased about 4 times with the addition of Al3+, but the enhanced fluorescence was quenched after the addition of CA. Based on this fluorescence phenomenon, an "on-off" fluorescence strategy was designed for the sensitive determination of CA and a linear detection range for CA was achieved within 0-80.0 μM. In addition, the developed probe exhibited high selectivity and accuracy for determination of CA. The mechanism of fluorescence enhancement and quenching of AuNCs was explored in detail. The established probe was used successfully for CA detection in beverages. The spiked recoveries from 97.50% to 103.67% were gratifying, which indicated the probe had potential prospects for detecting CA in food.

Detection of green fluorescence in serum albumin upon excitation with 375 nm light: revealing new fluorescent properties

The novel data on the fluorescence behavior of bovine serum albumin and human serum albumin under 375 nm excitation are presented, introducing a distinctive fluorescence phenomenon that holds promising implications for protein research. Extensive studies of these proteins are conducted to understand their intrinsic fluorescence properties, typically observed upon excitation with light in the range of 280–300 nm, resulting in fluorescence emission around 345 nm.

Thienopyrimidine-derived multifunctional fluorescence sensor for the detection of Cu2+, Fe3+, and PPi in different solvents.

Hydrazine substituted thienopyrimidine, a new fluorophore, was used to synthesize a novel Schiff base R1 as a chemosensor via the condensation with p-formyltriphenylamine, and the structure was confirmed using nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS) analysis. When treated with Cu2+ in dimethylsulfoxide (DMSO)/H2O buffer, R1 showed a phenomenon of fluorescence quenching, which was reversible with the action of ethylenediaminetetraacetic acid (EDTA). When treated with Fe3+ in dimethylformamide (DMF)/H2O buffer, R1 exhibited the same phenomenon, but fluorescence was recovered with inorganic pyrophosphate (PPi) quantitatively. The complexation ratios for R1-Cu2+ and R1-Fe3+ were both 1:2, which were manifested by MS titrations and corresponding Job's plots. The limits of detection of R1 for Cu2+ and Fe3+ were 3.11 × 10-8 and 1.24 × 10-7M, respectively. The sensing mechanism of R1 toward Cu2+ and Fe3+ was confirmed using density functional theory calculations and electrostatic potential analysis. Test strips of R1 were fabricated successfully for on-site detection of Cu2+ and Fe3+. In addition, R1 was applied to recognize Cu2+ and Fe3+ in actual water samples with satisfactory recovery.

Revisiting fulgide photochromism: Mechanistic decoding and electron transport from computational exploration.

The photochromic behavior of the fulgide molecule relies on ring-closure and ring-opening processes involving conical intersections during excited state transformation between isomers. The precise location and topography of these conical intersections significantly shape the decay process and fluorescence phenomena inherent to the molecule. This work combines electronic structure theory calculations using the density functional theory and wavefunction methods, as well as surface hopping simulation to analyze the photochemical behavior of an experimentally synthesized fulgide molecule, (E)-p-methylacetophenylisopropylidenesuccinic anhydride (1E). Our study reveals the conical intersection between the first excited state (S1) and the ground electronic state (S0), which emerges beyond the S1 minimum of 1E to the ring-closing side. The distinctive topography of this conical intersection appears to be sloped. These findings suggest a reduced quantum yield for the formation of the closed isomer, indicating a higher likelihood of reformation of the open isomer(s). The surface hopping simulation further supports this observation, revealing a mere ∼8% quantum yield for the formation of the closed isomer. In addition, the photoisomerization of the fulgide molecule initiates a cascade of conduction switching and holds great potential for applications in molecular electronics. Delving into the realm of molecular electronics, we have further examined the electron transport properties, disclosing the higher conductivity of the closed isomer.

Intriguing Role of Weak Electron Donor in Dictating the Luminescent Characteristics of DS‐A‐DW Materials

AbstractThe short wavelength (SW) emission region of the materials employing strong donor (DS) phenothiazine (PTZ) is generally correlated to its quasi‐axial (QAPTZ) conformer arrangement. This propels to investigate the specific role of weak donors (DW) such as carbazole (CZ) in determining the emission characteristics of molecular heredity (MH) type structural design encompassing dual donors of varying strength. For this purpose, a common DS(PTZ)–Acceptor unit is linked to the DW(CZ) unit via different linkage positions to form M1, M2, and M3, which not only alters the absorption and dual emission characteristics but also influences the delayed fluorescence phenomenon. The SW emission originates by minimizing the aggregation factor rather than by literature envisaged QAPTZ conformer. Incorporating M1 as a single molecular white light emitter (SMWLE) in near UV light emitting diode (LED) devices results in cold white light emission with CRI of 68 and CIE of (0.24, 0.33), while its corresponding solution‐processed organic light‐emitting diode devices delivers warm white electroluminescence with an EQE of 5.8%. Overall, this work demonstrates the significant contribution of the DW in attaining the white light emission characteristics of MH molecular design not only at the molecular level but also in practical light‐emitting devices.

From Cs[C2N3] to Cs3[C6N9] – a thermal and structural investigation

Abstract Cesium dicyanamide Cs[C2N3] (≡ Cs[N(CN)2] or Cs[dca]) was obtained by a metathesis reaction in form of transparent colorless platelets. The results of single-crystal X-ray structure measurements and refinements (C2/c, Z = 8) with the monoclinic cell parameters a = 932.31(8), b = 1274.67(9), c = 824.94(7) pm, β = 110.803(3)° at −70 °C and a = 939.59(7), b = 1281.58(8), c = 827.57(6) pm, β = 110.610(3)° at 20 °C corroborate earlier results for this compound. The Raman and IR spectra of Cs[C2N3] are presented for the first time and the result compares well with those of NaCs2[C2N3]3. The heat-driven cyclotrimerization process of Cs[C2N3] was studied by thermal analyses (DSC) and temperature-dependent X-ray powder diffraction methods. At 370 °C, its trimerization product Cs3[C6N9] is formed, crystallizing in the orthorhombic space group Pbam with the cell parameters a = 3043.0(3), b = 1052.4(1) and c = 415.21(4) pm for Z = 4. The IR spectrum of this cesium tricyanomelaminate (Cs3[C6N9] or Cs3[TCM]) is presented, but a well-resolved Raman spectrum could not be acquired owing to fluorescence phenomena. An overview about the cyclotrimerization reactions of all pseudo-binary alkali-metal dicyanamides (A[C2N3]) to their corresponding tricyanomelaminates (A 3[C6N9]) with A = Li–Cs gives a basis for a discussion of the different thermal and structural characteristics.

Exploring the mechanisms of excited-state intramolecular proton transfer in hydroxyphenyl-benzimidazole derivatives: A theoretical perspective

We conducted a comprehensive theoretical exploration for the ESIPT mechanism in a set of HBI derivatives in THF solution (Cpd-A and Cpd-B, and three isomers: Cpd-1, Cpd-2 and Cpd-3), employing time-dependent density functional theory (TD-DFT) methods for the excited-state calculations. An energy scan for the excited-state proton transfer pathway indicates that both Cpd-A and Cpd-B exhibit a low transition barrier from Enol* to Keto*, with the energy of Keto* much lower than Enol* state, which implies a rapid ESIPT process. The high instability of Enol* state contradicts the experimental observed dual emission bands for Cpd-A, where the emission band predominantly contributed from Keto* state. We propose a new mechanism to account for this dual fluorescence phenomenon of Cpd-A, where the overall fluorescence band can be ascribed to the emissions from both Keto* and a stable rotamer of Cpd-A. Calculations suggest that Cpd-A can exist in both syn and anti rotamers, with a relatively low isomerization barrier of 14.0 kcal/mol. The coexistence of two stable isomers in solution gives rise to the phenomenon of dual emission for Cpd-A. Further investigation for the substituent effects on the ESIPT dynamics of three isomers (Cpd-1, Cpd-2 and Cpd-3) indicates that varying substituent positions can have a substantial impact on both ESIPT process and excited-state charge transfer. The methoxy group substitution proximate to the hydroxy functional group decreases the energy barrier for Enol*-Keto* transition, and also promotes a more substantial charge transfer. Our study demonstrates a new mechanism of the dual fluorescence observed for the target HBI derivatives, offering a novel understanding for the ESIPT mechanism in solvent.

On-Off Ratiometric Fluorescence Europium(III) Metal-Organic Framework for Quantitative Detection of the Inflammatory Marker Neopterin.

Benefiting from the unique photoluminescence behavior of the lanthanide(III) ions and organic ligands, a lanthanide(III) metal-organic framework (Ln-MOF) material can simultaneously demonstrate photoluminescence of lanthanide(III) cations and organic molecules and endow its superior applications of fluorescence sensing behaviors. Herein, we present a europium(III) MOF material {[Eu2(BPTA)·(CH3COO)2·3DMA]·0.5DMA·3H2O}n (1) (where H4BPTA is 3,3',5,5'-biphenyltetracarboxylic acid) for photoluminescence performance of quantitatively sensing the inflammatory marker neopterin (Neo). The obtained 1 comprises Eu2(COO)4 paddlewheel secondary building units, which could be bridged by BPTA4- ligands to extend a 2D framework. The fluorescence titration indicates 1 can achieve simultaneous fluorescence behavior of Eu3+ ions and Neo via on-off ratiometric effects and thus could be exploited as the ratiometric fluorescence sensor matrix. Such a fluorescence phenomenon of 1 as a ratiometric sensor for quantitative detection of Neo via an on-off ratiometric effect is never observed in MOF chemistry. Moreover, naked-eye visible color variations of the fluorescence for 1 could be observed from red to blue with increasing concentrations of Neo, confirmed by fluorescent test strips as well as portable fluorescent hydrogels. And 1 also shows a low detection limit of 15.11 nM. A synergetic contribution of the competitive absorption, fluorescence resonance energy-transfer, and photoinduced electron-transfer mechanisms between Neo and the framework of 1 realizes the on-off ratiometric fluorescence behavior for Neo detection, supported by the UV-vis spectral overlap experiment and DFT calculations.

Chlorophyll Fluorescence Imaging for Environmental Stress Diagnosis in Crops.

The field of plant phenotype is used to analyze the shape and physiological characteristics of crops in multiple dimensions. Imaging, using non-destructive optical characteristics of plants, analyzes growth characteristics through spectral data. Among these, fluorescence imaging technology is a method of evaluating the physiological characteristics of crops by inducing plant excitation using a specific light source. Through this, we investigate how fluorescence imaging responds sensitively to environmental stress in garlic and can provide important information on future stress management. In this study, near UV LED (405 nm) was used to induce the fluorescence phenomenon of garlic, and fluorescence images were obtained to classify and evaluate crops exposed to abiotic environmental stress. Physiological characteristics related to environmental stress were developed from fluorescence sample images using the Chlorophyll ratio method, and classification performance was evaluated by developing a classification model based on partial least squares discrimination analysis from the image spectrum for stress identification. The environmental stress classification performance identified from the Chlorophyll ratio was 14.9% in F673/F717, 25.6% in F685/F730, and 0.209% in F690/F735. The spectrum-developed PLS-DA showed classification accuracy of 39.6%, 56.2% and 70.7% in Smoothing, MSV, and SNV, respectively. Spectrum pretreatment-based PLS-DA showed higher discrimination performance than the existing image-based Chlorophyll ratio.