Ratiometric fluorescence imaging of nuclear pH in living cells using Hoechst-tagged fluorescein

Small-molecule fluorescent sensors that allow specific measurement of nuclear pH in living cells will be valuable for biological research. Here we report that Hoechst-tagged fluorescein (hoeFL), which we previously developed as a green fluorescent DNA-staining probe, can be used for this purpose. Upon excitation at 405 nm, the hoeFL–DNA complex displayed two fluorescence bands around 460 nm and 520 nm corresponding to the Hoechst and fluorescein fluorescence, respectively. When pH was changed from 8.3 to 5.5, the fluorescence intensity ratio (F₅₂₀/F₄₆₀) significantly decreased, which allowed reliable pH measurement. Moreover, because hoeFL binds specifically to the genomic DNA in cells, it was applicable to visualize the intranuclear pH of nigericin-treated and intact living human cells by ratiometric fluorescence imaging.     

Plasmonically Tunable Blue-Shifted Emission from Coumarin 153 in Ag Nanostructure Random Media: A Demonstration of Fast Dynamic Surface-Enhanced Fluorescence

Enhancement of intensity and wavelength tunability of emission are desirable features for light-emitting device applications. We report on the large and tunable blue shift (60 nm) in emission from an environment-sensitive fluorophore (Coumarin153) embedded in Ag plasmonic random media. Coumarin 153 having emission at 555 nm, show a systematic blue shift (to 542, 503 and 495 nm) upon infiltration into random media fabricated by Ag nanowires of different aspect ratio (hence, surface plasmon resonances at 426, 445 and 464 nm). The blue shift is due to the fast dynamic surface-enhanced fluorescence mechanism and can be tuned by controlling the surface plasmon resonance and hotspot density in random media. Enhanced emission at desired wavelength is achieved by using nanostructures having higher extinction coefficient but same-surface plasmon resonance. Ag nanostructures of different aspect ratio used for fabricating the random media are synthesized by chemical route.     

Excitation energy transfer from phycobiliprotein to chlorophyll d in intact cells of Acaryochloris marina studied by time- and wavelength-resolved fluorescence spectroscopy.

The fluorescence decay spectra and the excitation energy transfer from the phycobiliproteins (PBP) to the chlorophyll-antennae of intact cells of the chlorophyll (Chl) d-dominated cyanobacterium Acaryochloris marina were investigated at 298 and 77 K by time- and wavelength-correlated single photon counting fluorescence spectroscopy. At 298 K it was found that (i) the fluorescence dynamics in A. marina is characterized by two emission peaks located at about 650 and 725 nm, (ii) the intensity of the 650 nm fluorescence depends strongly on the excitation wavelength, being high upon excitation of phycobiliprotein (PBP) at 632 nm but virtually absent upon excitation of chlorophyll at 430 nm, (iii) the 650 nm fluorescence band decayed predominantly with a lifetime of 70 +/- 20 ps, (iv) the 725 nm fluorescence, which was observed independent of the excitation wavelength, can be described by a three-exponential decay kinetics with lifetimes depending on the open or the closed state (F(0) or F(m)) of the reaction centre of Photosystem II (PS II). Based on the results of this study, it is inferred that the excitation energy transfer from phycobiliproteins to Chl d of PS II in A. marina occurs with a time constant of about 70 ps, which is about three times faster than the energy transfer from the phycobilisomes to PS II in the Chl a-containing cyanobacterium Synechococcus 6301. A similar fast PBP to Chl d excitation energy transfer was also observed at 77 K. At 77 K a small long-lived fluorescence decay component with a lifetime of 14 ns was observed in the 640-700 nm spectral range. However, it has a rather featureless spectrum, not typical for Chl a, and was only observed upon excitation at 400 nm but not upon excitation at 632 and 654 nm. Thus, this long-lived fluorescence component cannot be used as an indicator that the primary PS II donor of Acaryochloris marina contains Chl a.     

Iron Deficiency in the Blue-Green Alga Anacystis nidulans: Fluorescence and Absorption Spectra Recorded at 77°K

Low-temperature (77°K) fluorescence and absorption spectra have been determined for whole cells and photosystem I particles of Anacystis nidulans grown in iron-supplied or iron-deficient inorganic media. Iron deficiency induces a decrease of F720 relative to F685 and F695 in the fluorescence spectra of both whole cells and photosystem I particles. This change is correlated to a reduction of preferentially the long wavelength absorbing fraction of chlorophyll a. The relative fluorescence intensity at 755 nm is increased by iron deficiency. No significant effects of culture-age are found in the ratio between the three fluorescence bands (F685: F695: F720) of iron-supplied A. nidulans.     

Measuring cytosolic free calcium concentration in endothelial cells with indo-1: the pitfall of using the ratio of two fluorescence intensities recorded at different wavelengths

Indo-1 is a new fluorescent indicator of the intracellular free calcium concentration Cai++. Indo-1 may be used in a similar manner as its predecessor quin2 but offers the principal advantage that the Ca++ saturated form of the Ca++ chelator has a emission maximum different in wavelength from that of free indo-1 (400 nm versus 483 nm). Therefore, the ratio of the fluorescence intensity F emitted at 400 nm to that of the fluorescence intensity G emitted at 483 nm (or 500 nm) should be a measure of Cai++ independent of the total amount of intracellular dye. However, when indo-1 is loaded into endothelial cells (grown in culture on quartz coverslips) by incubation with the acetoxymethylester of indo-1 (indo-1/AM), the ester in not completely hydrolysed to indo-1 intracellularly. Fluorescence emitted by uncleaved indo-1/AM at wavelengths 483-500 nm interferes with the fluorescence of indo-1. Ester fluorescence is influenced not only by ester concentration but by the fluorescence emitted at 400 nm by Ca++ bound indo-1 as well. Therefore, the ratio F/G cannot reliably evaluate increases in Cai++ in endothelial cells although F/G would indicate a basal Cai++ constant with time. By contrast, the fluorescence F is a sensitive parameter of the intracellular concentration of Ca++ bound indo-1, in particular when the excitation wavelength is set to 332 nm. F was used to measure resting Cai++ in endothelial cells (132 +/- 22 nM; n = 22) and to demonstrate dose-dependent and reversible increases in Cai++ in response to stimulation with bradykinin.     

Distribution of UV-shielding of the epidermis of sun and shade leaves of the beech (Fagus sylvatica L.) as monitored by multi-colour fluorescence imaging

Plants can protect against damaging ultraviolet (UV) radiation by accumulating UV-absorbing substances in the epidermis of the leaves. Sun and shade leaves of a free standing beech tree (Fagus sylvatica L.) were studied for the differences in UV-shielding of the epidermis by means of multi-colour fluorescence images taken with UV and blue excitation. The distribution of the fluorescence intensity was detected over intact leaves in the emission maxima in the blue at 440 nm (F440), in the green at 520 nm (F520), in the red at 690 nm (F690) and in the far red at 740 nm (F740). Images of the logarithmic ratio between F690 excited in the blue and the UV (log (^BF690/^UVF690)) were calculated representing the relative absorption of UV in the epidermis and thus the degree of UV-shielding. It was found that UV-shielding is stronger for sun leaves than for shade leaves and better for the upper (adaxial) leaf side than for the lower (abaxial) leaf side of both leaf types. Within one leaf the highest value for the ratio log (^BF690/^UVF690) and thus the highest UV-shielding was found at the leaf rim which in broad leaves contains young tissue.     

Three-Step Photoconversion of Only Three Subunits of the Water-Soluble Chlorophyll-Binding Protein Tetramer from Chenopodium album

Water-soluble chlorophyll (Chl)-binding proteins (WSCPs) have been found in various plants. WSCPs are categorized into two classes based on their photoconvertibility: Class I (photoconvertible) and Class II (non-photoconvertible). Based on their absorption peaks, which occur in the red wavelengths, the pre- and post-photoconverted forms of Chenopodium album WSCP (CaWSCP) are called CP668 and CP742, respectively. Although various biochemical and biophysical properties of CaWSCP have already been characterized, questions remain regarding the structural dynamics of the photoconversion from CP668 to CP742, and the relationship between the photoconversion activity and incident light wavelength. To address how the wavelength of incident light affects the photoconversion, we performed time-course analyses of CaWSCP photoconversion by using light-emitting diodes that emit either white light, or at the discrete wavelengths 670, 645, 525, 470, or 430 nm. The most efficient photoconversion was observed under irradiation at 430 nm. Less efficient photoconversion was observed under irradiation with 670, 645, 470, or 525 nm light, in that order. The relationship between photoconversion activity and wavelength corresponded with the absorption peak intensities of Chls in the CaWSCP complex. The observed time dependence of the A₇₄₂/A₆₆₈ ratio during photoconversion of the CaWSCP complex indicated that the photoconversion from CP668 to CP742 occurs in a three-step reaction, and that only three subunits in the complex could be photoconverted.     

Time-resolved spectral observations of cadmium-enriched cadmium sulfide nanoparticles and the effects of DNA oligomer binding

We measured the steady-state and time-resolved fluorescence spectral properties of cadmium-enriched nanoparticles (CdS-Cd2+). These particles displayed two emission maxima, at 460 and 580 nm. The emission spectra were independent of excitation wavelength. Surprisingly, the intensity decays were strongly dependent on the observation wavelength, with longer decay times being observed at longer wavelengths. The mean lifetime increased from 150 to 370 ns as the emission wavelength was increased from 460 to 650 nm. The wavelength-dependent lifetimes were used to construct the time-resolved emission spectra, which showed a growth of the long-wavelength emission at longer times, and decay-associated spectra, which showed the longer wavelength emission associated with the longer decay time. These nanoparticles displayed anisotropy values as high as 0.35, depending on the excitation and emission wavelengths. Such high anisotropies are unexpected for presumably spherical nanoparticles. The anisotropy decayed with two correlation times near 5 and 370 ns, with the larger value probably due to overall rotational diffusion of the nanoparticles. Addition of a 32-base pair oligomer selectively quenched the 460-nm emission, with less quenching being observed at longer wavelengths. The time-resolved intensity decays were minimally affected by the DNA, suggesting a static quenching mechanism. The wavelength-selected quenching shown by the nanoparticles may make them useful for DNA analysis.     

One-Step Preparation and Fluorescence Enhancement Effect in SiO2/Eu2+ Doped with Ag Nanowires

Eu²⁺ single-doped SiO₂ (SiO₂/Eu²⁺) and Eu²⁺, Ag nanowires co-doped SiO₂ (SiO₂/Eu²⁺–Ag) luminescent nanomaterials were prepared by an efficient one-step sol–gel method. Their microstructure and optical properties were characterized, and the fluorescence enhancement of Eu²⁺ by Ag nanowires was investigated. The experimental results indicate that the average diameter of Ag nanowires doped is 12.5 nm, and the length–diameter ratio is 30. The Ag nanowires cannot only enhance the light absorption of SiO₂/Eu²⁺ in the range of 230–350 nm, but also reduce the fluorescence lifetime of Eu²⁺. More importantly, the emission intensity is enhanced after doping Ag nanowires, and the red shift phenomenon of the emission spectrum is observed, red shift occurs between 10 and 56 nm. The highest fluorescence intensity is accessed under the Ag doping concentration of 0.10 %. Additionally, the emission of SiO₂/Eu²⁺ with 0.10 % of Ag doping at 456 nm is 16 times stronger than that of pure SiO₂/Eu²⁺. The present results indicate that the fluorescence enhancement is attributed to the local field enhancement and the increased radiative decay rates induced by Ag nanowires.     

Fluorescence lifetime and spectral study of the acid expansion of bovine serum albumin

The fluorescence lifetimes of the tryptophan residues of bovine serum albumin were measured in the native and acid-expanded conformation. A three-exponential process is required to fit the fluorescence decay data. The results are interpreted empirically in terms of two emitting species. The emission at longer wavelength (360 nm) has slower rates of decay than that at shorter wavelength (325 nm). For both emitting species the average lifetime decreases when the N-F transition occurs and shortens further when the protein expands. Rotational correlation times, derived from the decay of the fluorescence anisotropy of the tryptophan residues, suggest that longer emission wavelengths are associated with somewhat shorter correlation times. There is no certain indication of any independent motion of the tryptophans in any conformation, although some very fast process, perhaps Raman scattering, appears to occur. On acid expansion the long correlation times decrease to around 10 ns in the fully expanded form. Static quenching experiments using I- or acrylamide suggest a greater average exposure of the tryptophans when the protein is most greatly expanded. This is despite the fact that the fluorescence emission maximum shifts to shorter wavelength under these conditions. Also, there is no difference in accessibility to quenching between the longer and shorter wavelength emissions.     

A CCD-OMA device for the measurement of complete chlorophyll fluorescence emission spectra of leaves during the fluorescence induction kinetics

Summary A new device for the measurement of complete laser induced fluorescence emission spectra (maxima near 690 and 735 nm) of leaves during the induction of the chlorophyll fluorescence is described. In this the excitation light (cw He/Ne laser, 632.8 nm) is switched on by a fast electro-mechanical shutter which provides an opening time of 1 ms. The emitted fluorescence is imaged onto the entrance slit of a multichannel spectrograph through a red cut-off filter (> 645 nm). A charge coupled device (CCD) sensor with 2048 elements simultaneously detects the complete chlorophyll fluorescence emission spectrum in the 650–800 nm wavelength range. Scanning is accomplished electronically and the integration time for a complete fluorescence emission spectrum can be selected from 10 ms up to 260 ms. Shutter, detector system and data acquisition are controlled by an IBM-PC/AT compatible computer. A maximum of 32 spectra can be measured at selected times during the fluorescence induction kinetics with the shortest time resolution of 10 ms. The instrument permits the determination of various fluorescence parameters:a) the rise-time of the fluorescence to the maximum level fm,b) the changes in the shape of the fluorescence emission spectra during the induction kinetics,c) the induction kinetics in the fluorescence ratio F690/F735 as well asd) the fluorescence decrease ratio Rfd at any wavelength between 650 to 800 nm. These fluorescence parameters provide information about the functioning of photosynthesis. The ratio F690/F735 allows the non-destructive determination of the chlorophyll content of leaves. The application of this instrument in ecophysiological research and stress physiology of plants is outlined.     

Characterizing the photochemical degradation of aquatic humic substances from a dystrophic lake using excitation-emission matrix fluorescence spectroscopy and parallel factor analysis

Three-dimensional excitation-emission matrix (EEM) fluorescence and parallel factor analysis (PARAFAC) were used to monitor composition and reactivity changes caused by the photochemical degradation of aquatic humic substances (AHS) from a dystrophic lake in Kushiro Wetland, Japan. AHS-rich lake water was exposed to three treatments in summer and winter 2014: radiation with the full solar wavelength range, radiation with the >320-nm solar wavelength range, and no solar radiation. Irradiation caused AHS-like peaks to shift to shorter wavelengths in the EEM contour plots, implying that AHS photodegradation caused the formation of lower-molecular-weight fractions or more simply structured components. Three components were identified from PARAFAC analyses: AHS-1 (excitation/emission wavelengths of maxima: <252 and 315 nm/426 nm), AHS-2 (360 and 261 nm/489 nm), and AHS-3 (276 nm/403 nm). These components had different photosensitivities. AHS-1 was most sensitive to full solar radiation, while AHS-2 was most sensitive to >320-nm radiation. More photodegradation of these components occurred in the summer than in the winter, indicating that photodegradation depended on light intensity. AHS-3 was photoresistant. The different characteristics of the components reflected the in situ dynamics of the components. The AHS-3 fluorescence intensity was positively correlated with the dissolved organic carbon concentration but the AHS-1 and AHS-2 fluorescence intensities were not. The EEM–PARAFAC method was found to be a good tool for tracing AHS-like materials in situ and in the laboratory.     

光系统Ⅱ核心复合物的稳态荧光光谱

在83K和160K两个温度下,通过激发波长对荧光发射谱的影响研究了光系统Ⅱ中核心复合物的荧光光谱特性。用不同波长的光激发,核心复合物的发射谱的最大发射峰值不变,用480、489、495和507nm的光分别激发核心复合物,其光谱最大峰值处的荧光强度随不同激发波长下β-胡萝卜素分子的吸收强度的增大而降低,在长波长区域光谱的变化依赖于首先被激发的色素分子。所以,激发波长的不同影响着核心复合物中能量传递的途径。通过高斯解析,分析出核心复合物中至少存在有7组叶绿素a组分,它们是Ch1 a660,Ch1 a670,Ch1 a680,Ch1 a682,Ch1 a684,Ch1 a687和Ch1 a690。     

Spectroscopic studies on origination of the peak at 730 nm in delayed fluorescence of chloroplasts.

The origination of the peak at 730 nm in the delayed fluorescence (DF) spectrum of chloroplasts was studied using various optical analysis methods. The DF spectrum showed that the main emission peak was at about 685 nm, with a small shoulder at 730 nm when the chloroplast concentration was < 7.8 microg/mL. The intensity of the peak at 685 nm decreased, while the intensity of the peak at 730 nm increased, when the chloroplast concentrations were increased from 7.8 to 31.2 microg/mL. With the concentration increasing, the peak at 730 nm became dominant while the peak at 685 nm finally disappeared. The DF decay kinetic curves showed that the intensity of the peak at 730 nm decayed as the same speed as the intensity of the peak at 685 nm during the entire relaxation process (0.5-30.5 s). With the excitation wavelength at 685 nm, the emission intensity was stronger in the excitation spectrum at 730 nm. The absorption spectrum demonstrated that the ratio A(685):A(730) remained almost constant when the chloroplast concentration increased. The results suggest that the peak at 730 nm appearing in DF is mainly contributed by the fluorescence of photosystem I (PSI), generated by the re-absorption of 685 nm band DF.     

Uptake of the Herbicide Diuron as Visualised by the Fluorescence Imaging Technique

A new fluorescence imaging system for monitoring the uptake of the PSII-herbicide diuron (OCMU) was tested in tobacco leaves. UV-laser-induced (Λ_exc = 355 nm) fluorescence images were collected for blue fluorescence F440 (Λ_em = 440 nm), green fluorescence F520 (Λ_em = 520 nm), red chlorophyll fluorescence F690 (Λ_em = 690 nm) and for far-red chlorophyll fluorescence F740 (Λ_em = 740 nm). Diuron-treated leaf parts exhibited a higher red and far-red chlorophyll fluorescence emission (F690 and F740) than untreated leaf halves, whereas the blue and green fluorescence, F440 and F520, remained unaffected. As a consequence, the fluorescence ratios blue/red (F440/F690) and blue/far-red (F440/F740) significantly decreased in diuron-treated leaf parts. The time course of diuron uptake into the leaf could be followed by fluorescence images taken 10 and 30 min after diuron application. The novel high resolution fluorescence imaging method supplies information on the herbicide uptake of each point of the leaf area. Its great advantage as compared to the point data fluorescence measurements applied so far is discussed.     

High spatial resolution identification of hematoma in inhomogeneous head phantom using broadband fNIR system

This paper presents a novel method for early detection of hematomas using highly sensitive optical fNIR imaging methods based on broadband photon migration. The NIR experimental measurements of inhomogeneous multi-layer phantoms representing human head are compared to 3D numerical modeling over broadband frequencies of 30–1000 MHz. A finite element method (FEM) simulation of the head phantom are compared to measurements of insertion loss and phase using custom-designed broadband free space optical transmitter (Tx) and receiver (Rx) modules that are developed for photon migration at wavelengths of 670 nm, 795 nm, 850 nm, though results of 670 nm are discussed here. Standard error is used to compute error between 3D FEM modeling and experimental measurements by fitting experimental data to the \( a\sqrt {frequency} + b \). Error results are shown at narrowband and broadband frequency modulation in order to have confidence in 3D numerical modeling. A novel method is established here to identify presence of hematoma based on first and second derivatives of changes in insertion loss and phase (?IL and ?IP), where frequency modulated photons sensitive to different sizes of hematoma is identified for wavelength of 670 nm. The high accuracy of this comparison provides confidence in optical bio-imaging and its eventual application to TBI detection.     

Fluorescence measurement by a streak camera in a single-photon-counting mode

We describe here a recently developed fluorescence measurement system that uses a streak camera to detect fluorescence decay in a single photon-counting mode. This system allows for easy measurements of various samples and provides 2D images of fluorescence in the wavelength and time domains. The great advantage of the system is that the data can be handled with ease; furthermore, the data are amenable to detailed analysis. We describe the picosecond kinetics of fluorescence in spinach Photosystem (PS) II particles at 4–77 K as a typical experimental example. Through the global analysis of the data, we have identified a new fluorescence band (F689) in addition to the already established F680, F685, and F695 emission bands. The blue shift of the steady-state fluorescence spectrum upon cooling below 77 K can be interpreted as an increase of the shorter-wavelength fluorescence, especially F689, due to the slowdown of the excitation energy transfer process. The F685 and F695 bands seem to be thermally equilibrated at 77 K but not at 4 K. The simple and efficient photon accumulation feature of the system allows us to measure fluorescence from leaves, solutions, single colonies, and even single cells. The 2D fluorescence images obtained by this system are presented for isolated spinach PS II particles, intact leaves of Arabidopsis thaliana, the PS I super-complex of a marine centric diatom, Chaetoceros gracilis, isolated membranes of a purple photosynthetic bacterium, Acidiphilium rubrum, which contains Zn-BChl a, and a coral that contains a green fluorescent protein and an algal endosymbiont, Zooxanthella.     

激光照射血液荧光光谱的初步研究

采用ＯＭＡ—ＩＩ微弱信号检测系统研究了人血液荧光光谱在激光照射下的变化情况。结果表明：在６３２．８ｎｍＨｅＮｅ激光诱导下,不同血液在６７０ｎｍ,７３０ｎｍ,９８１ｎｍ附近出现三个荧光峰;荧光强度在一定范围内与照射激光功率呈线性变化关系;随着激光照射时间的增加,三个峰位上的荧光强度下降,８分钟后趋于稳定值;在激光照射过程中,三个峰位出现不同数值的移动,同时在６７０ｎｍ和７３０ｎｍ两个荧光峰之间出现了竞争。     

Benzoxazinone — kanamycin derivative: a new fluorescent probe for flow cytometry analysis of bacteria (Agrobacterium tumefaciens)

Abstract A new polycation fluorescent dye (BVC-kinamycin-conjugate) has been synthesized and used to detect alive bacteria by flow cytometry. This fluorescent chromophore has the noteworthy property of being excited at the same wavelength as fluoresceinylated conjugates (488 nm) and to show a much longer emission wavelength (616 nm) than fluoresceinylated derivates (520 nm); furthermore its fluorescence intensity is not quenched at low pH in contrast with fluorescein. In such conditions, bacteria can easily be detected with cytofluorimeter equipped with a single excitation wavelength beam. The binding of fluoresceinylated lectins and antibodies onto a strain of Agrobacterium tumefaciens has been studied by this method.     

Significance of Over-Mature and Decaying Trees for Carbon Stocks in a Central European Natural Spruce Forest

Old-growth forests are important stores for carbon as they may accumulate C for centuries. The alteration of biomass and soil carbon pools across the development stages of a forest dynamics cycle has rarely been quantified. We studied the above- and belowground C stocks in the five forest development stages (regeneration to decay stage) of a montane spruce (Picea abies) forest of the northern German Harz Mountains, one of Central Europe’s few forests where the natural forest dynamics have not been disturbed by man for several centuries. The over-mature and decay stages had the largest total (up to 480 Mg C ha^?1) and aboveground biomass carbon pools (200 Mg C ha^?1) with biomass C stored in dead wood in the decay stage. The soil C pool (220–275 Mg C ha^?1, 0–60 cm) was two to three times larger than in temperate lowland spruce forests and remained invariant across the forest dynamics cycle. On the landscape level, taking into account the frequency of the five forest development stages, the total carbon pool was approximately 420 Mg C ha^?1. The results evidence the high significance of over-mature and decaying stages of temperate mountain forests not only for conserving specialized forest organisms but also for their large carbon storage potential.