FLUORESCENCE OF THE COENZYME ANALOG NICOTINAMIDE FORMYCIN DINUCLEOTIDE

Abstract— Emission spectra of unbound reduced nicotinamide formycin dinucleotide (NFDH) revealed the presence of two major conformations of the coenzyme in solution: when examined at excitation wavelengths at or below 307 nm, emission spectra contained peaks at 343 and 447 nm; when excited above 307 nm, an additional maximum appeared at 355 nm and the peak of the dihydronicotinamide emission band shifted from 447 to 440 nm. Both conformers are probably detected at the longer wavelengths since the emission peak at 343 nm was retained. Identical changes occurred in the emission spectra of NFD⁺, however, the dihydronicotinamide emission between 440 and 447 nm was absent. Several mechanisms which may account for the presence of these conformers have been considered. The choice has been narrowed to conformations with ring-ring interactions of the formycin and nicotinamide moieties resulting from (a) formycin tautomerization or (b) heterogeneity of glycosidic bond angles in the structures. The efficiency of intramolecular energy transfer from the formycin to the dihydronicotinamide moiety for free NFDH in aqueous solution was 84% and declined slightly (to 77%) when measured in 1,2-propanediol. NFD⁺ has coenzyme activity for NAD-specific isocitrate dehydrogenase (Plaut et al. , 1979). The emission spectrum of enzyme bound NFDH was altered markedly in the presence of manganese isocitrate; emission intensity at 343 and 355 nm decreased while the emission from the dihydronicotinamide ring at 433 nm increased, when NFDH was excited at 310 nm. This shift in emission intensity was indicative of an increase in energy transfer within the NFDH molecule, caused by a change in coenzyme conformation upon binding to the enzyme-substrate complex.     

A CHARACTERIZATION OF THE FLUORESCENT PROPERTIES OF CIRCULATING HUMAN EOSINOPHILS

Abstract— This paper presents a characterization of the fluorescence properties of human eosinophils isolated from peripheral blood of normal donors over a wide range of excitation and emission wavelengths. Circulating eosinophils possess three fluorescence excitation emission maxima: one at 280 nm excitation, 330 nm emission, attributable to tryptophan fluorescence, and currently unassigned peaks at 360 nm excitation, 440 nm emission and 380 nm excitation, 415 nm emission. Fluorescence microscopy studies show that the Huorescence of eosinophils may be site dependent; specifically, when observed at 365 nm excitation, circulating eosinophil Huorescence appears blue-violet, while the fluorescence of tissue-dwelling eosinophils appears amber-gold. These results should be considered in developing an optical biopsy technique to identify eosinophils in human tissue.     

A BLUE LIGHT-SENSITIVE CYTOCHROME-FLAVIN COMPLEX FROM CORN COLEOPTILES. FURTHER CHARACTERIZATION*

Abstract— The partially purified blue light-sensitive membrane-associated flavin-cytochrome complex from etiolated corn coleoptiles shows a unique sharp α-band at 555 nm in its light-minus-dark difference spectrum at liquid nitrogen temperature. This band is clearly distinguishable from the α-bands found in fractions enriched for mitochondria and endoplasmic reticulum respectively. The photoactive membrane fraction is shown to have ATPase activity that is not stimulated by K⁺ and that is not inhibited by oligomycin. Other than flavin fluorescence at 525 nm obtained upon excitation at 450 nm, there is a second fluorescent component with emission at 430 nm on excitation at 350 nm. The mid-point potential of the Triton X-100 solubilized b-cytochrome, measured by simultaneously monitoring the reduction of the pyocyanine 600-800 nm peak and the appearance of the 427 nm Soret peak of the b-cytochrome upon titration with dithionite in the presence of ferricyanide, is estimated to be ?65 mV. The kinetics of the blue light-induced reduction and dark rcoxidation of the 6-cytochrome suggest that the mid-point potential of the b-cytochrome is not affected by Triton X-100 solubilization.     

Characterization of the autofluorescence of polymorphonuclear leukocytes, mononuclear leukocytes and cervical epithelial cancer cells for improved spectroscopic discrimination of inflammation from dysplasia

Fluorescence spectroscopy has the potential to improve the in vivo detection of intraepithelial neoplasias; however, the presence of inflammation can sometimes result in misclassifications. Inflammation is a common and important pathologic condition of epithelial tissues that can exist alone or in combination with neoplasia. It has not only been associated with the presence of cancer but also with the initiation of cancer by damage induced due to the oxidative activity of inflammatory cells. Microscopic examination of cervical biopsies has shown increased numbers of polymorphonuclear and mononuclear leukocytes in inflamed tissues mostly confined to the stroma. The purpose of this study was to characterize the fluorescence properties of human polymorpho- and mononuclear leukocytes and compare their fluorescence to that of cervical cancer cells. Human neutrophils were purified from peripheral blood and their fluorescence characterized over an excitation range of 250-550 nm. There are four notable excitation emission maxima: the tryptophan peak at 290 nm excitation, 330 nm emission; the NAD(P)H peak at 350 nm excitation, 450 nm emission, the FAD peak at 450 nm excitation, 530 nm emission and an unidentified peak at 500 nm excitation, 530 nm emission. Treatment of these peripheral blood neutrophils with 40 nM phorbol myristate acetate or with the chemotactic peptide formyl-Met-Leu Phe (1 M) demonstrated a significant increase in NAD(P)H fluorescence. Isolated mononuclear cells have similar emission peaks for tryptophan and NAD(P)H and a small broad peak at 450 nm excitation, 530 nm emission suggestive of FAD. Comparison of the fluorescence from leukocytes to epithelial cancer cell fluorescence has demonstrated the presence of these fluorophores in different quantities per cell. The most notable difference is the high level of tryptophan in cervical epithelial cancer cells, thus offering the potential for discrimination of inflammation.     

Application of solid‐phase extraction for the concentration of chromophores,fluorophores, and photosensitizers from lens protein digests

Solid‐phase extraction was applied for the separation of protein digests obtained from aged human lenses, cataractous human lenses, calf lens proteins in vitro glycated with dehydroascorbic acid and native calf lens proteins. Four fractions were collected after stepwise elution with different solvents. The first fraction contained about 80% of the digested material possessing free amino groups. At the same time, the third and the fourth fractions were enriched in chromophores, fluorophores, and photosensitizing structures that originate mainly from advanced protein glycation. The comparison between the total digest and the fourth fraction based on their UV absorption at 330 nm, intensity of fluorescence (excitation/emission 350/450 nm), and production of singlet oxygen upon UVA irradiation argues that the solid‐phase extraction was capable of concentrating the advanced glycation end‐products about a hundredfold. Thus, this technique is a useful step for separation and concentration of fluorophores, chromophores, and photosensitizers from aged and glycated lens protein digests.     

BLUE AND ULTRAVIOLET-B LIGHT PHOTORECEPTORS IN PARSLEY CELLS

Abstract— Ultraviolet-B (UV-B) and blue light photoreceptors have been shown to regulate chalcone synthase and flavonoid synthesis in parsley cell cultures. These photoreceptors have not yet been identified. In the present work, we studied UV-B photoreception with physiological experiments involving temperature shifts and examined the possible role of flavin in blue and UV-B light photoreception. Cells irradiated with UV-B light (0.5–15 min) at 2°C have the same fluence requirement for chalcone synthase and flavonoid induction as controls irradiated at 25°C. This is indicative of a purely photochemical reaction. Cells fed with riboflavin and irradiated with 6 h of UV-containing white light synthesize higher levels of chalcone synthase and flavonoid than unfed controls. This effect did not occur with blue light. These results indicate that flavin-sensitization requires excitation of flavin and the UV-B light photoreceptor. The in vivo kinetics of flavin uptake and bleaching indicate that the added flavin may act at the surface of the plasma membrane. In view of the likely role of membrane-associated flavin in photoreception, we measured in vitro flavin binding to microsomal membranes. At least one microsomal flavin binding site was solubilized by resuspension of a microsomal pellet in buffer with high KPi and NaCl concentrations and centrifugation at 38000 g. The 38000 g insoluble fraction had much greater flavin binding and contained a receptor with an apparent K_D of about 3.6 μM and an estimated in vivo concentration of at least 6.7 × 10–⁸M. Flavin mononucleotide, roseoflavin, and flavin adenine dinucleotide can compete with riboflavin for this binding site(s), although each has lower affinity than riboflavin. Most microsomal protein was solubilized by resuspension of the microsomal pellet in non-denaturing detergents and centrifugation at 38 000 g ; however, this inhibited flavin binding, presumably because of disruption of the environment of the flavin receptor. The parsley microsomal flavin binding receptor(s) have a possible role in physiological photoreception.     

Imaging intracellular pH in live cells with a genetically encoded red fluorescent protein sensor

Intracellular pH affects protein structure and function, and proton gradients underlie the function of organelles such as lysosomes and mitochondria. We engineered a genetically encoded pH sensor by mutagenesis of the red fluorescent protein mKeima, providing a new tool to image intracellular pH in live cells. This sensor, named pHRed, is the first ratiometric, single-protein red fluorescent sensor of pH. Fluorescence emission of pHRed peaks at 610 nm while exhibiting dual excitation peaks at 440 and 585 nm that can be used for ratiometric imaging. The intensity ratio responds with an apparent pK(a) of 6.6 and a >10-fold dynamic range. Furthermore, pHRed has a pH-responsive fluorescence lifetime that changes by ~0.4 ns over physiological pH values and can be monitored with single-wavelength two-photon excitation. After characterizing the sensor, we tested pHRed's ability to monitor intracellular pH by imaging energy-dependent changes in cytosolic and mitochondrial pH.     

Resonance surface plasmon spectroscopy: low molecular weight substrate binding to cytochrome p450

A new detection mechanism has been developed for low molecular weight substrate binding to heme proteins based on resonance localized surface plasmon spectroscopy. Cytochrome P450 has strong electronic transitions in the visible wavelength region. Upon binding of a substrate molecule (e.g., camphor), the absorption band of cytochrome P450 shifts to shorter wavelength. The event of camphor binding to a nanoparticle surface modified with cytochrome P450 protein receptors is monitored using UV-vis spectroscopy. It is observed for the first time that the binding of the substrate molecules to the protein receptor induces a blue-shift in the localized surface plasmon resonance (LSPR) of the nanosensors. The coupling between the molecular resonance of the substrate-free and substrate-bound cytochrome P450 proteins and the nanoparticles' LSPR leads to a highly wavelength-dependent LSPR response. When the LSPR of the nanoparticles is located at a wavelength distant from the cytochrome P450 resonance, an average of approximately 19 nm red-shift is observed upon cytochrome P450 binding to the nanoparticles and a approximately 6 nm blue-shift is observed upon camphor binding However, this response is significantly amplified approximately 3 to 5 times when the LSPR of the nanoparticles is located at a slightly longer wavelength than the cytochrome P450 resonance, that is, a 66.2 nm red-shift upon cytochrome P450 binding and a 34.7 nm blue-shift upon camphor binding. This is the first example of the detection of small molecules binding to a protein modified nanoparticle surface on the basis of LSPR.     

UV-INDUCED DAMAGE OF PHOTORECEPTOR PROTEINS IN THE PARAFLAGELLAR BODY OF Euglena gracilis

Abstract— The paraflagellar body (PFB), the putative photoreceptor for phototaxis in the flagellate Euglena gracilis , was isolated still attached to the flagellum. After solubilization the proteins were separated and analyzed by two-dimensional gel electrophoresis. To discriminate the PFB-specific proteins from the flagella proteins, flagella without PFB were analyzed, isolated from the flagellate Astasia longa (a chlorophyll-free relative of E. gracilis ), which has no PFB and lacks phototaxis. The absorption spectra of solubilized PFB samples showed a maximum at 415 nm, two shoulders around 380 and 410 nm and two additional small peaks at 515 and 540 nm not present in the control sample without PFB. Two-dimensional gel electrophoresis showed eight PFB-specific proteins with molecular masses in the range of 25000–45000 and isoelectric points in the range of pH 3.5–7. Ultraviolet radiation strongly affects some of these PFB-specific proteins, but also flagella proteins are damaged by UV treatment. There is also a drastic decrease in the PFB-specific absorption maxima after UV irradiation.     

Time-resolved fluorescence spectroscopy and imaging of proteinaceous binders used in paintings

The differentiation of proteins commonly found as binding media in paintings is presented based on spectrally resolved and time-resolved laser-induced fluorescence (LIF) and total emission spectroscopy. Proteins from eggs and animal glue were analysed with pulsed laser excitation at 248 nm (KrF excimer) and 355 nm (third harmonic of Nd:YAG) for spectrally resolved measurements, and at 337 nm (N₂) and 405 nm (N₂ pumped dye laser) for spectrally resolved lifetime measurements and fluorescence lifetime imaging (FLIM). Total emission spectra of binding media are used for the interpretation of LIF spectra. Time-resolved techniques become decisive with excitation at longer wavelengths as fluorescence lifetime permits the discrimination amongst binding media, despite minimal spectral differences; spectrally resolved measurements of fluorescence lifetime have maximum differences between the binding media examined using excitation at 337 nm, with maximum observed fluorescence at 410 nm. FLIM, which measures the average lifetime of the emissions detected, can also differentiate between media, is non-invasive and is potentially advantageous for the analysis of paintings. Figure The fluorescence of solid ox glue extracted from collagen can be visualised in this Total Fluorescence Spectrum; three different peaks from multiple fluorophores are present and allow the discrimination between collagen- and non-collagen proteinaceous binding media found in paintings     

Absorption and fluorescence characteristics of photo-activated adenylate cyclase nano-clusters from the amoeboflagellate Naegleria gruberi NEG-M strain

The spectroscopic characteristics of BLUF (BLUF = sensor of blue light using flavin) domain containing soluble adenylate cyclase (nPAC = Naegleria photo-activated cyclase) samples from the amoeboflagellate Naegleria gruberi NEG-M strain is studied at room temperature. The absorption and fluorescence spectroscopic development in the dark was investigated over two weeks. Attenuation coefficient spectra, fluorescence quantum distributions, fluorescence quantum yields, and fluorescence excitation distributions were measured. Thawing of frozen nPAC samples gave solutions with varying protein nano-cluster size and varying flavin, tyrosine, tryptophan, and protein color-center emission. Protein color-center emission was observed in the wavelength range of 360-900 nm with narrow emission bands of small Stokes shift and broad emission bands of large Stokes shift. The emission spectra evolved in time with protein nano-cluster aging.     

FLUOROMETRIC EVIDENCE FOR FLAVINS IN ISOLATED EYESPOTS OF EUGLENA GRACILIS VAR. BACILLARIS

Abstract— Fluorometric evidence suggesting the presence of flavins in isolated eyespots of Euglena gracilis var. bacillaris is reported for the first time. Fluorescence spectra of eyespots and flavin standards show maxima at 540nm and 530nm, respectively. Excitation spectra show matching major peaks at 360–370 nm and at 450nm. The addition of riboflavin standard to eyespot samples increases fluorescence intensity without major corresponding shifts in wavelength maxima. Photolysis of eyespot samples in the presence of EDTA effects a decrease in the fluorescence intensity; the fluorescence is quantitatively restored to its initial value by bubbling the photolyzed solution with air. Preliminary quantitative data, obtained by fluorescence measurements, indicate the presence of ca. 5 × 10^-4μg flavin/ml eyespot sample. While flavins have been hypothesized to be components of the photoreceptor system, they have been reported previously only in the paraflagellar bodies of intact cells. Emission and excitation data obtained by us for eyespots are similar to those previously reported by other investigators for paraflagellar bodies, but our studies now suggest the presence of flavins also in Euglena eyespots.     

A FLAVOPROTEIN MAY MEDIATE THE BLUE LIGHT-ACTIVATED BINDING OF GUANOSINE 5'-TRIPHOSPHATE TO ISOLATED PLASMA MEMBRANES OF Pisum sativum L.*

Abstract— A blue light photoreceptor has not been identified in higher plants. Most proposals for a blue light-absorbing chromophore lack evidence for a direct connection between the putative chromophdre and a biological effect. Fluorescence data for the plasma membrane from etiolated buds of Pisum sativum L. suggest that we are measuring fluorescence emission of flavin species, and probably not pterin species. Fluorescence data indicate that a putative flavin exists associated with a protein or protein complex in the plasma membrane. Excitation of plasma membranes that were boiled in the presence of 0.1% sodium dodecyl sulfate and treated with blue light yields a fluorescence band with a maximum of approximately 552 nm. This fluorescence emission can be rapidly quenched by the flavin antagonists phenylacetic acid (PAA) and KI. Blue light-enhanced binding of guanosine 5'-[Γ-thio]triphosphate (GTPγS) to a protein in the plasma membrane is strongly inhibited by PAA, KI, and NaN₃, all quenchers of flavin excited states, indicating that a chromophore for this photoreaction may be a flavin associated with a plasma membrane protein. The above evidence is consistent with the participation of a flavin as the chromophore for the light-induced GTP-binding reaction in pea plasma membrane.     

MICROSPECTROFLUOROMETRIC APPROACH TO THE STUDY OF FREE/BOUND NAD(P)H RATIO AS METABOLIC INDICATOR IN VARIOUS CELL TYPES

Under excitation at 365 nm, the cell fluorescence is mainly due to bound and free NAD(P)H, plus a small contribution from flavins. Resolution is first attempted in the simplest case. i.e. the increase spectrum (δI_f) due to microinjection of glucose-6-phosphate (G6P) into EL2 ascites cells. Above 510 nm, δI_F is identical to the spectrum of free NADH. Below 510 nm. the presence of a second component is suggested, i.e. the intensity of the free NADH spectrum is lower than the measured δI_F level. The difference between δI_f and the free NADH spectrum (maximum at 475 nm) yields a spectrum suggestive of bound NADH with maximum at 450 nm. Thus, with free and bound NADH, the entire δI_F can be reconstructed, with some assumptions as to the relative quantum yields of the two components. This seems to leave no place for a flavin component. The questions raised by the lack of such a component are answered using a new microspectrofluorometer, which aiiows correlated monitoring of NAD(P)H and flavins with excitations at 365 and 436 nm, respectively. As detected by excitation at 436 nm, injections of G6P, malate, ADP, and treatments with azide, cyanide or partial anaerobiosis, all indeed show a redox change of flavins, in the sense of decreased emission. It is understandable, however, that such a change which is not very large even using 436 nm excitation should remain undetected when flavins are excited at 365 nm, i.e. using the tail of their excitation spectrum. In contrast to the increased δI_F spectrum recorded in response to injected substrate, the initial spectrum (I_f) of the cell prior to a metabolic perturbation reveals a third component, even with 365 nm excitation. The position and reactivity of this component shows flavin-like properties. The structural resolution attainable makes it possible to obtain the evaluation of free vs. bound NAD(P)H and flavin fluorochromes in the mitochondrial and cytosolic compartments of the intact cell.     

ENERGY TRANSFER FROM CHLOROPHYLL b TO CHLOROPHYLL a IN A HYDROPHOBIC MODEL SYSTEM

Abstract— Chlorophyll a and chlorophyll b purified by high-performance liquid chromatography (HPLC) were subsequently adsorbed on the surface of a pellicular reverse phase packing normally used in HPLC. The granule surface is reacted with octadecyl groups and furnishes an hydrophobic substrate for pigment adsorption. Reflectance spectra of chlorophyll a and chlorophyll b , each adsorbed at average spacings of about 11 nm² per molecule, had red region maxima at 664 and 643nm respectively. Fluorescence excitation spectra for 740nm emission from these surfaces peaked at about 420nm for chlorophyll a and 460nm for chlorophyll b. Adsorbed pigments excited at either of the two wave lengths had a single fluorescence emission peak at 683nm for chlorophyll a and at 664nm for chlorophyll b. A surface having both pigments adsorbed in approximately equal amounts with an overall average spacing of about 5.6nm² per molecule also had peaks at 420 and 460nm in the excitation spectrum. However, excitation of adsorbed molecules on this (latter) surface, at either 420 or 460nm, produced emission with the single chlorophyll a peak at 683nm. It is concluded that, under the conditions of our experiment, exciting adsorbed chlorophyll b contributes strongly to emission from adsorbed chlorophyll a.     

Excitation wavelength dependence of the proton-transfer reaction of the green fluorescent protein

Picosecond time-correlated single-photon counting was used to measure the proton-transfer rate of green fluorescent protein (GFP) excited by several wavelengths between 266 and 405 nm. When samples of GFP in water and D2O are excited at short wavelengths, lambda(ex) < 295 nm, the fluorescence properties are largely modified with respect to excitation at a wavelength around 400 nm, the peak of the absorption band of the S0 --> S1 transition of the ROH form of the chromophore. The shorter the excitation wavelength, the longer the decay time of the ROH emission band at 450 nm and the longer the rise time of the RO- emission band at 512 nm. The proton transfer is slower by an order of magnitude and about a factor of 3 when GFP in water and D2O are excited by 266 nm, respectively.     

PTERIN- AND FLAVIN-LIKE FLUORESCENCE ASSOCIATED WITH ISOLATED FLAGELLA OF Euglena gracilis

Fluorescence excitation- and emission spectra indicate the presence of pterin(s) and flavin(s) in isolated flagella of the phytoflagellate Euglena gracilis. These compounds appear to bind at least in part non-covalently to the molecular framework of the paraflagellar body, which is the presumed photoreceptor organelle and which is attached to the isolated flagella. A compound with pterin-like fluorescence excitation and emission spectrum could he extracted with methanol from isolated flagella and could he recovered on thin-layer silica gels. Besides the previously assumed photoreceptor function of flavins, our results suggest also a role for pterins in the photosensory transduction chain of Euglena gracilis.     

Dimers of polyene antibiotic amphotericin B detected by means of fluorescence spectroscopy: molecular organization in solution and in lipid membranes

Fluorescence emission from amphotericin B dissolved in 2-propanol-water was recorded in the spectral region 500-650 nm. The fluorescence excitation spectrum corresponds to the absorption spectrum of the monomeric drug. The large energy shift between the excitation and emission bands indicates that emission takes place from an energy level different than that responsible for absorption. These levels were attributed to the 2(1)A(g) and 1(1)B(u) states, respectively. Excitation of the same sample with short wavelength radiation (below 350 nm) yields light emission between 400 and 550 nm. The fluorescence excitation spectrum corresponding to this emission band displays distinct maxima at 350, 334 and 318 nm. This band was analyzed in terms of the exciton splitting theory and assigned to amphotericin B in a dimeric form, in which chromophores are spaced by 4.9 A. The binding energy of the dimers, determined to be 4.9 kJ/mol, indicates that the structures are stabilized by van der Waals interactions. The same type of molecular structures was also detected in the lipid membranes formed with dipalmitoylphosphatidylcholine. Linear dichroism of amphotericin B embedded in lipid multibilayers indicates that molecules are distributed between two fractions: parallel (38%) and perpendicular (62%) with respect to the membrane. The biological importance of such membrane organization is discussed.     

Visible radiation effects on flavocytochrome b2 in dilute aqueous solution: a steady-state and laser flash photolysis study

Irradiation of flavocytochrome b2 by visible radiation at 450 nm in dilute aqueous solution is found to have a devastating effect not only on its activity but also on the important flavin mononucleotide (FMN) constituents. The active site and the substrate binding site are also found to be largely modified on exposure to visible radiation. This has a telling effect on the constituent aromatic amino acids, tryptophan and tyrosine, and therefore justifies the role of FMN as a very potent photosensitizer. Partial unfolding of the irradiated enzyme molecule is also observed. Damage is much greater in deaerated conditions, which indicates that molecular oxygen plays a protecting role in this particular system. The inactivation is mediated through rapid electron transfer from tryptophan and tyrosine to excited flavin, forming flavin semiquinone and tryptophanyl and tyrosinyl radicals, which in turn cause permanent damage at the molecular level.     

Highly fluorescent cotton fiber based on luminescent carbon nanoparticles via a two-step hydrothermal synthesis method

A kind of highly fluorescent cotton fibers, in which the luminescent carbon nanoparticles (CNPs) are generated in the lumen and the mesopores directly, have been prepared by the method of hydrothermal synthesis in situ using citric acid and urea as raw materials, and hexadecyl trimethyl ammonium bromide and tributyl phosphate as active agents. The CNPs/cotton fibers were characterized by thermogravimetry-differential thermal analysis (TG-DTA), X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM) and X-ray photoelectron spectroscopy (XPS), respectively. The optical properties are investigated by fluorescence spectrofluorometry and PR-305 long afterglow phosphors tester. The results showed that the CNPs were self-assembled successfully in the lumen as well as in the mesopores of cotton fibers. The CNPs/cotton fibers could emit bright and colorful photoluminescence under excitation lights of different wavelengths. The afterglow decay process could be divided into fast decay and slow decay stages and the emission of CNPs/cotton fiber had two peaks at 450 nm and 570 nm respectively when the wavelength of excitation changed from 310 nm to 500 nm. The preparation of highly fluorescent cotton fibers by self-assembly method has great significance to the functionalization of cotton fibers.