Microscopic steady-state transient fluorescence spectroscopy

Fluorescence is a phenomenon of material photoluminescence. Substances that produce fluorescence require specific structural and electronic transition properties. Typically, molecules with conjugated double bond systems, rigid planar structures, and specific functional groups are more likely to produce fluorescence. From a microscopic perspective, when a substance molecule is irradiated with light, it will be excited upon absorbing photon energy, and the electrons inside the molecule will transition from the ground state to the excited state. The electrons in the excited state are unstable and will be excited in a very short time (usually within a short period of time)10⁻⁸to10⁻⁴secondReturning to the ground state and releasing excess energy in the form of light, this emitted light is called fluorescence.

image1 Fluorescence schematic diagram

fluorescence spectrumIn the time domainDivided into steady-state and transient fluorescence spectraSteady state fluorescence spectroscopy mainly studies the fluorescence signal of substances under continuous light source irradiation, and detects the fluorescence signal/intensity(y)It's wavelength(x)The function. Transient fluorescence spectroscopy mainly studies the fluorescence signal of substances after being irradiated by pulsed light sources/The variation of intensity over time is generally detectedThe fluorescence signal/intensity(y)It's time.(x)The function. Transient fluorescence spectroscopy, also known as fluorescence lifetime curve/Time-resolved fluorescence spectroscopy(TRPL)In the field of transient fluorescence, fluorescence lifetime is a specific interpretation of time-resolved fluorescence spectra, defined as the average time required for a fluorescent substance to return from the excited state to the ground state and release fluorescent photons after being excited to the excited state after absorbing light energy. It is the core temporal characteristic of fluorescence phenomena, reflecting the interaction between fluorescent molecules and their surrounding microenvironment, and has important scientific and practical value. Compared to the signal obtained by steady-state fluorescence measurement, which reflects the average behavior of the excited state particle ensemble, fluorescence lifetime measurement provides direct information on the decay dynamics of excited state molecules. The former mainly indicates the occurrence of specific photophysical processes or chemical reactions, while the latter can reveal in depth the specific physical mechanisms that lead to the occurrence of such processes by analyzing the rate constants of exciton state relaxation.

Application of steady-state transient fluorescence spectroscopy

1.materialscience

In the application of measuring the luminescence characteristics of semiconductor materials, laser excitation is commonly used to generate fluorescence, and the optical properties of the material are analyzed using fluorescence spectroscopy. Single layerMoS₂Due to the strong fluorescence Z caused by the direct bandgap characteristic, it transitions to an indirect bandgap with increasing number of layers, resulting in a sudden decrease in fluorescencePLSpectrum can be judgedMOS2Number of layers.(Reference)：Krishnaet al.,Nanoscale, 2014, 6, 13028–13035；）

image2 fluorescence spectrumRegarding imagingtoMOS2Qualitative analysis of thin films

Regarding vacuum depositionCH₃NH₃PbI₃（eMAPI）In the study of perovskite thin films, steady-state fluorescence and transient fluorescenceSpectral technologyPlayed a complementary and crucial role.Jointly revealed the vacuum deposition of perovskite thin films（eMAPI）The intrinsic relationship between the physical properties of intermediate light and device performance.image3. (A)The steady-state measurement results indicate that based oneMAPIThe solar cells have achieved up to20.6%Conversion efficiency and1.16 VThe open circuit voltage proves its excellent bulk quality, but this contradicts the conventional view that a long fluorescence lifetime is a prerequisite for high efficiency, becauseeMAPIThe fluorescence lifetime is extremely short. Picture3. (B)Transient fluorescence technology, as the core analytical method, solves this contradiction. The experiment first confirmedeMAPIThe ultra short fluorescence lifetime (only about)0.75 ns）Subsequently, through surface passivation experiments, it was found that the lifespan was significantly extended after passivation73.5 nsThis firmly proves that its short lifespan is not due to bulk non radiative recombination, but is dominated by rapid non radiative recombination on the surface of the film.

（References：Juboket al.,J. Phys. Chem. Ready. 2019.10.5167-5172）

image3 Non passivatingandIn the case of passivation,eMAPIGlass layerTransient Fluorescence

Substrate as twoDimension MaterialThe closely contacted supporting structure can significantly regulate the neighboring doping effectDimension MaterialOptical properties ofResearchers used fluorescence lifetime imaging microscopy technology to demonstrate the direct effect of neighboring dopingRegulating itMoS₂The generation and recombination dynamics of intermediate charge carriers. Specifically,ITOOn the substrateMoS₂Exhibiting significant reduction in carrier lifetime and fluorescence lifetime. This is because as the carrier density increases, the possibility of defects in the material capturing charges increases, and the captured carriers can remain for a longer period of time, reducing their contribution to charge transport and increasing the possibility of recombination, thereby shortening the carrier lifetime. The detection and characterization technology of steady-state transient fluorescence reveals the substrate's dependence on twoDimension MaterialThe regulation mechanism of optoelectronic properties for future design and optimization of high-performance IIDimension MaterialOptoelectronic devices have opened up new avenues and have important scientific significance and application prospects. (Reference: Locet al.,Laser & Photonics Reviews.2025.e00682）

image4 single layerMoS2On different substrates(a)Fluorescence characteristics(b)Fluorescence lifetime imaging experimental results

2.Biomedicine

Fluorescence spectroscopy has the advantage of non-destructive monitoring for early prevention and diagnosis of diseases. By using fluorescence spectroscopy to detect samples before and after cervical cancer lesions, the high sensitivity of the spectrum can reflect cellular metabolic abnormalities and detect small lesions that are difficult to detect by traditional pathology (reference:Jeonget al.,Journal ofBiophotonics2018; 11(5):e201700245）

image5. A precancerous lesionCINIIIThe patient showed differences in tissue morphology between two different sections,AFor ShangNo lesions,BFor lesions.CandDShow consistent spectral characteristics

In addition, transient fluorescence spectroscopy and its fluorescence lifetime imaging have an impact on tumor cellsofDiagnosis also provides a basis as a quickSensitive and minimally invasiveThe auxiliary diagnostic methods usually work together with medical devices such as endoscopes,convexShow the potential application of steady-state transient fluorescence spectroscopy.

image6. Fluorescence lifetime microscopy imaging of the tumor junction area in lung cancer patients

3.pharmacokinetics

Based on steady-state and transient fluorescence spectra, with high luminescenceCA/EDA-CDsAs a probe, for·OHSelective fluorescence response. The monitoring of aminophylline (a xanthine based bronchodilator) was achieved through fluorescence detection,CA/EDA-CDsDisplay high quantum yield（56.56%）And its emission peak is450 nmMaintain the positionStable, indicating its excitation independence and advantages as a probe, recorded using a transient fluorescence spectrometerCA/EDA-CDsDecline in lifespan, exposed to·OHLater, the introduction disrupted the conjugated system, resulting in an average fluorescence lifetime of15.11 nsdrop to12.08 ns, indicating·OHInduced non radiative energy dissipation.TCSPCThen the dynamic impact of this process on the excited state dynamics was quantified.(Reference:Yifet al.,Materials Today Bio 33:102019）

image7. （A）Probe andFe-XODEnzyme catalyzed system for detecting aminophylline（AMP）Feasibility analysis. （B）Electron paramagnetic resonance（ESR）. （C）Ultraviolet-Visible absorption spectrum. （D）CA/EDA-CDsSchematic diagram of fluorescence emission process and its explanationFe²⁺+XODcatalysisAMPgenerated·OHcaused byCA/EDA-CDsBand structure changes and fluorescence quenching mechanism.(E)CA/EDA-CDs, CA/EDA-CDs+AMP, CA/EDA-CDs+XOD, CA/EDA-CDs+Fe² ⁺+XODandCA/EDA-CDs+Fe² ⁺+ XOD+ AMPThe fluorescence lifetime decay curve.

4.fluorescent probe

The combined application of transient and steady-state fluorescence spectroscopy technology provides multidimensional and in-depth analysis methods for the development and optimization of fluorescence probesIn this study,Through steady-state and transient fluorescence spectroscopy techniques研究了8-methoxy-3-CarboxyethylCoumarin（8EMOHCC）Fluorescence behavior quenched by aniline in different solvents.Steady state fluorescence measurementAs shown in the figure8.（B）As shownAs the concentration of aniline increases,8EMOHCCThe fluorescence intensity significantly decreased, but the spectral shape and peak did not shift, indicating the absence of excited group complex formation.steady stateS-VThe curve is linear in all solvents, consistent with dynamic quenching characteristics, and has a quenching constantKSVAs the dielectric constant of the solvent increases, it shows a nonlinear increase, indicating that the polarity of the solvent affects the quenching efficiency by regulating the charge transfer process.

Transient Fluorescence ExperimentAs shown in the figure8.（C）As shown,The fluorescence lifetime decay curve was recorded using time-dependent single photon counting technology, and it was found that the decayPresenting a single indexForm, prove that the quenching process is uniform. transientS-VThe curve is the sameLinear, further confirming that quenching is dynamically dominant. By combining steady-state and transient fluorescence in a multi solvent system, it was clarified that8EMOHCCThe quenching mechanism is a dynamic process of synergistic diffusion and activation, and reveals the key role of solvent dielectric constant. This method and conclusion have important reference value for the design of fluorescent probes, the study of biomolecule interactions, and the exploration of photophysical mechanisms.(Reference:Keerthanaet al.,JFluoresc(2021) 31:393–400）

image8. （A）8EMOHCCmolecular structure（B）8EMOHCCTypical fluorescence spectra of aniline at different concentrations in propanol solvent（C）8EMOHCCFluorescence attenuation of aniline with different concentrations in propanol solventlightscore.

5.environmental monitoring

The coexistence of multiple pollutants in complex environmental matrices poses a significant threat to ecosystems and public healthThrough fluorescence characteristicsPeak canQuickly and sensitively screen target pollutants by using three unique ligand binding affinity compounds: lysine copper nanoclusters, cysteine copper nanoclusters, and amino acid copper nanoclustersCopper nanoclusters of Heli（Cu NCs）Combined with machine learning algorithms, it can distinguish between various heavy metal ions and pesticides. (Reference:Vargheseet al.,Biosensors and Bioelectronics,2025,287(117706)）

image9. Three types of copper nanoparticlesclusterAnd its fluorescence excitation spectrum and emission spectrum

6.food safety

In food science,LumineOptical spectroscopy technology, as a fast and non-destructive analysis tool, is systematically applied to monitoringIn foodChemical composition changesAs shown in the picture10As shown,Spectral data of milk at different time points during the acidification process were obtained through synchronous scanning and fixed-point excitation, throughSpectral analysis of chemical composition changes during milk acidification processAnd thenEstablish a linear model between fluorescence indicators and acidity to accurately predict the degree of acidification，To provide theoretical basis for the development of real-time monitoring equipment and assist in quality control and optimization of the dairy industry.(Reference:Sudhakaret al.,Agriculture 2023, 13, 1054）

image10. Milk under photoluminescence excitationfluorescenceSpectral characteristics

Fluorescence spectroscopy is used as a core analytical tool to explore the properties of special grade samplesextractOlive oil（EVOO）Fluorescence characteristics and adulteration identification under laser induction. research adoption405 nmContinuous wave（CW）The diode laser was used as the excitation light source, and the system measured itEVOOAnd its adulterated samples (such as those mixed with sunflower seed oil)SO）Fluorescence spectra at different detection angles. By analyzing the intensity distribution of fluorescence spectra andSpectral deformationResearchers have identified the transformationEVOOMain fluorescent groups in the middle (such as chlorophyll derivatives, carotenoids, and vitamins)E）The Contribution.(Reference:Duonget al.,Microchemical Journal 214 (2025) 114023）

image11. （A）Adulterated olive oil（B）Special Grade InitialextractOlive oil sampleIn405nmUnder laser excitationfluorescencespectrumandSpectrum detected at different detection anglesAnd its analysis