Confocal fluorescence lifetime imaging of free calcium in single cells

Ca²⁺ concentrations in biological cells are widely studied with fluorescent probes. The probes have a high selectivity for free calcium and exhibit marked changes in their photophysical properties upon binding. The differences in the fluorescent lifetime of the probes can now be used as a contrast mechanism for imaging purposes. This technique can be further exploited for the quantitative determination of ion concentrations within the cells. We describe the use of a fast fluorescence lifetime imaging method in combination with a standard confocal laser scanning microscope for the determination of Ca²⁺ concentrations in single rat cardiac myocytes using the intensity probe Calcium Green.     

Recent developments in monitoring calcium and protein interactions in cells using fluorescence lifetime microscopy

Time-resolved fluorescence lifetime microscopy (TRFLM) allows the combination of the sensitivity of fluorescence lifetime to environmental parameters to be monitored in a spatial manner in single living cells, as well as providing more accurate, sensitive, and specific diagnosis of certain clinical diseases and chemical analyses. Here we discuss two applications of TRFLM: (1) the use of nonratiometric probes such as Calcium Crimson, for measuring Ca²⁺; and (2) quantification of protein interaction in living cells using green and blue fluorescent protein (GFP and BFP, respectively) expressing constructs in combination with fluorescence resonance energy transfer microscopy (FRET). With respect to measuring Ca²⁺ in biological samples, we demonstrate thatintensity-based measurements of Ca²⁺ with single-wavelength Ca²⁺ probes such as Calcium Crimson may falsely report the actual Ca²⁺ concentration. This is due to effects of hydrophobicity of the local environment on the emission of Calcium Crimson as well as interaction of Calcium Crimson with proteins, both of which are overcome by the use of TRFLM. The recent availability of BFP (P4-3) and GFP (S65T) (which can serve as donor and acceptor, respectively) DNA sequences which can be attached to the carboxy-or amino-terminal DNA sequence of specific proteins allows the dual expression and interaction of proteins conjugated to BFP and GFP to be monitored in individual cells using FRET. Both of these applications of TRFLM are expected to enhance substantially the information available regarding both the normal and the abnormal physiology of cells and tissues.     

Fluorescent Determination of Succinylcholine Chloride by Naphthalimide/ Stilbazolium Dye ⊂ CP5A

Succinylcholine Chloride (SCC), a short-acting neuromuscular relaxant, is non-fluorescent in aqueous solutions. This property makes it impossible to be determined by direct fluorescent method. Naphthalimide dye (NA) exhibits very strong fluorescence emissions in aqueous solution, after complexing with carboxylatopillar[5]arene (CP5A) in aqueous solutions, the fluorescent quenched intensity of complex was observed. On the contraty, stilbazolium dye (SA) exhibits weak fluorescence emissions in aqueous solution, after being included by CP5A, a fluorescence enhancement was observed. However, adding SCC to the NA? CP5A or SA? CP5A complex solution led to the recovery of the fluorescence intensity, in the meantime, the color of SA? CP5A solution changed from dark yellow to light yellow. The competitive supramolecular interaction between SCC, NA and SA for CP5A was studied by spectrofluorometry, ¹H NMR. Herein, a FID (fluorescence indicator displacement) system to detect SCC based on NA? CP5A and SA? CP5A complex was developed.     

Calcium imaging using fluorescence lifetimes and long-wavelength probes

We describe imaging of calcium concentrations using the long-wavelength Ca²⁺ indicators, Calcium Green, Orange, and Crimson. The lifetimes of these probes were measured using the frequency-domain method and were found to increase from 50% to severalfold in response to calcium. The two-dimensional images of the calcium concentration were obtained using a new apparatus for fluorescence lifetime imaging (FLIM). We also describe procedures to correct for the position-dependent frequency response of the gain-modulated image intensifier used in the FLIM apparatus. Importantly, the FLIM method does not require the probe to display shifts in the excitation or emission spectra. Using the FLIM method, calcium imaging is possible using probes which display changes in lifetime in response to calcium. Consequently, calcium imaging is possible with excitation wavelengths ranging from 488 to as long as 620 nm, where autofluorescence and/or photochemical damage is minimal. These probes are also suitable for calcium measurements of single cells using lifetime-based flow cytometry.     

Quantitative Analysis of Intracellular Calcium and Mitochondrial Kinetic Fluorescence Changes in GSNO-induced Thymocyte Early Apoptosis

A fluorescence microscopy imaging technique was applied to observe the single-cell kinetic changes of intracellular Ca²⁺ concentration ([Ca²⁺]_i) and mitochondrial membrane potential (ΔΨ_m) during the early stage of S-nitrosoglutathione (GSNO)-induced thymocytes apoptosis. The kinetic features of [Ca²⁺]_i and ΔΨ_m were quantitatively analyzed and compared by fitting the fluorescence intensity data. The mathematical parameter, inflection point which indicated the time point when [Ca²⁺]_i or ΔΨ_m changed the most rapidly, was proposed to analyze the fitting curve. The results revealed that the inflection point of [Ca²⁺]_i always appeared prior to that of ΔΨ_m during apoptosis induced by a certain GSNO concentration. Both the [Ca²⁺]_i and ΔΨ_m changed in a GSNO concentration-dependent manner. Another parameter, half-max effect point was also employed and displayed the similar results. Such quantitative analyses of real-time observations at the single-cell level are useful for interpreting the sequence of the biological events operating in GSNO-induced thymocyte apoptosis.     

Multiple switching behaviors of poly(N-isopropylacrylamide) hydrogel with spironaphthoxazine and D-π-A type dye

A multi-switchable poly(NIPAM-co-SPO-co-D-π-A dye) hydrogel with a photochromic spironaphthoxazine and an electron donor-π-conjugated-electron acceptor (D-π-A) type dye was prepared by typical radical copolymerization. The low critical solution temperature (LCST) behavior was investigated by UV–vis spectroscopy, which allows the measurement of the phase transition from 20 to 40 °C in aqueous solution. The fluorescence intensity of the poly(NIPAM-co-SPO-co-D-π-A dye) was temperature-dependent. Reversible modulation of fluorescence intensity was achieved using alternating irradiation with UV and visible light. Prepared polymer hydrogel also exhibited spectra change when not only used Cu²⁺ cation but also an acid unit.     

Photophysical studies of xanthene dye in alkanols and in presence of inorganic ions

Eosin Y belongs to a xanthene group. It is an anionic fluorescent dye. The absorbance and fluorescence of Eosin Y have been investigated in a series of alkanols (methanol to propanol). When the solvents are added to the aqueous solution of Eosin Y (EY) the absorbance and fluorescence intensity are enhanced. The alkanols are found to affect the absorption and fluorescence spectra of the dye. On the basis of solvent adsorption model the binding constants of the dye with alkanols have been estimated. The interaction of solvent molecule with dye in aqueous solution is specific in nature. The fluorescence quenching of Eosin Y by the inorganic ions [Fe(CN)₆]⁻³, [Fe(CN)₆]⁻⁴ and Cl⁻ was also observed. The ions influenced the quenching process to different extents. The rate constants of quenching were calculated using the Stern-Volmer equation. The equilibrium constant of dye in presence of inorganic ions are determined by Scott equation.     

Enhancement of the fluorescence of triphenylmethane dyes caused by their interaction with nanoparticles from β-diketonate complexes

We have studied the absorption and fluorescence spectra of Malachite Green and Crystal Violet in aqueous and alcoholic-aqueous solutions in which nanoparticles from Ln(III) and Sc(III) diketonates are formed at concentrations of complexes in a solution of 5–30 μM. We have shown that, if the concentrations of the dyes in the solution are lower than 0.5 μM, dye molecules are incorporated completely into nanoparticles or are precipitated onto their surface. The fluorescence intensity of these incorporated and adsorbed Malachite Green and Crystal Violet molecules increases by several orders of magnitude compared to the solution, which takes place because of a sharp increase in the fluorescence quantum yields of these dyes and at the expense of the sensitization of their fluorescence upon energy transfer from β-diketonate complexes entering into the composition of nanoparticles. We have shown that, if there is no concentration quenching, the values of the fluorescence quantum yield of the Crystal Violet dye incorporated into nanoparticles and adsorbed on their surface vary from 0.06 to 0.13, i.e., are close to the fluorescence quantum yield of this dye in solid solutions of sucrose acetate at room temperature. The independence of the fluorescence quantum yield of Crystal Violet on the morphology of nanoparticles testifies to a high binding constant of complexes and the dye. The considerable fluorescence quantum yields of triphenylmethane dyes in nanoparticles and sensitization of their fluorescence by nanoparticle-forming complexes make it possible to determine the concentration of these dyes in aqueous solutions by the luminescent method in the range of up to 1 nM.     

Femtosecond laser-induced stimulation of a single neuron in a neuronal network

We demonstrated the stimulation of neurons at a single-cell level in cultured neuronal network by a focused femtosecond laser. When the femtosecond laser was focused on a neuron loaded with a fluorescent calcium indicator, the fluorescence intensity immediately increased at the laser spot, suggesting that intracellular Ca²⁺ increases in the neuronal cell due to the femtosecond laser irradiation. The probability of Ca²⁺ elevation at the laser spot depended on the average laser power, irradiation time, and position of the focal point along the optical axis, indicating that the femtosecond laser activates neurons because of multiphoton absorption. Moreover, after laser irradiation of a single neuron cultured on multielectrode arrays, the evoked electrical activity of the neurons was demonstrated by electrophysiological systems, which concluded that the focused femtosecond laser could achieve stimulating a single neuron in a neuronal network with high spatial and temporal resolution.     

Hydrostatic pressure effects on the fluorescence of tyrosine solution with different concentrations of copper ion

The effects of hydrostatic pressure on the fluorescence of tyrosine (Tyr) solution with different concentrations of copper ion (Cu²⁺) were investigated. The fluorescent intensity of pure Tyr aqueous solution enhanced with the increase of hydrostatic pressure, the fluorescent intensity increased by 8.8% when the pressure was up to 60 MPa. The fluorescence of Tyr aqueous solution was quenched obviously due to complex formation with Cu²⁺ and the quenching became stronger when the concentration of Cu²⁺ was higher. When the concentration ratio of Cu²⁺ and Tyr ([Cu²⁺]/[Tyr]) increased from 0 to 40, the fluorescent intensity decreased to 19.0% at 0.1 MPa and 24.2% at 60 MPa. It was also found that the effects of pressure on the fluorescent intensity of Tyr aqueous solution were different at various [Cu²⁺]/[Tyr]. The fluorescent intensity increased by 14.4% and 38.4% for 1 and 40 ratio respectively when the pressure was changed from 0.1 MPa to 60 MPa.     

A new calibration equation for ratiometric fluorescent ion indicators: Application to fura-2

The absolute values of intracellular ion concentrations as monitored by specific fluorescent indicators are determined by using calibration curves obtained underin vitro andin vivo conditions. In the derivation of the calibration curve by Grynkiewicz et al [(1985)J. Biol. Chem 260, 3440] it is implicitly assumed that the observed fluorescence signal is directly related to the concentrations of the free dye and the dye-ion complex in the ground state. We modified the calibration equation so that ion binding and dissociation in the excited state are taken into account. The extended calibration equation assumes the knowledge of the rate constants in the excited state. Expressions for the calibration curve assuming the absence or presence of an excited-state reaction are compared for the Ca²⁺ indicator Fura-2. The excited-state rate constants are determined by global compartmental analysis of time-resolved fluorescence decays of Fura-2 collected at various excitation and emission wavelengths using different Ca²⁺ concentrations. It is found that for Fura-2 there is negligible interference of the excited-state reaction so that the original calibration can used.     

Design and Synthesis of Highly Photostable Yellow–Green Emitting 1,8-Naphthalimides as Fluorescent Sensors for Metal Cations and Protons

Two highly photostable yellow–green emitting 1,8-naphthalimides 5 and 6, containing both N-linked hindered amine moiety and a secondary or tertiary cation receptor, were synthesized for the first time. Novel compounds were configured as “fluorophore–spacer–receptor” systems based on photoinduced electron transfer. Photophysical characteristics of the dyes were investigated in DMF and water/DMF (4:1, v/v) solution. The ability of the new compounds to detect cations was evaluated by the changes in their fluorescence intensity in the presence of metal ions (Cu²⁺, Pb²⁺, Zn²⁺, Ni²⁺, Co²⁺) and protons. The presence of metal ions and protons was found to disallow a photoinduced electron transfer leading to an enhancement in the dye fluorescence intensity. Compound 5, containing secondary amine receptor, displayed a good sensor activity towards metal ions and protons. However the sensor activity of dye 6, containing a tertiary amine receptor and a shorter hydrocarbon spacer, was substantially higher. The results obtained indicate the potential of the novel compounds as highly photostable and efficient “off–on” pH switchers and fluorescent detectors for metal ions with pronounced selectivity towards Cu²⁺ ions.     

Development of UV-Excitable Red and Near-Infrared Fluorescent Labels and Their Application for Simultaneous Multicolor Bioimaging by Single-Wavelength Excitation

We report a new type of UV-excitable red/NIR-emissive fluorescent dyads (PKF series). Conjugation of a pyrene and a novel bright red/near-infrared (NIR) fluorophore resulted in large quasi-Stokes shift while retaining intense fluorescence emission and sharp spectral bands. Labeling of PKF dyads to biomolecules was performed by means of introduction of a succinimidyl ester. Simultaneous Ca²⁺/albumin dual-color intracellular imaging by PKF in combination with fura-2 (UV-excitable/VIS-emissive Ca²⁺ indicator) reveals its usefulness as a new bioimaging tool.     

Fluorescence of Crowned Butadienyl Dye and Its Metal Complexes

The absorption and fluorescence spectra of complexes of butadienyl dye (1) with lithium, sodium, magnesium, and calcium cations in MeCN were investigated. The addition of Li, Na, Mg, and Ca perchlorates to the solution of dye 1 in acetonitrile results in a significant (up to 5900-cm^–1) short-wavelength shift of the absorption spectrum and a small (about 200-cm^–1) short-wavelength shift of the fluorescence spectrum. The recoordination reaction in metal complexes of 1 takes place by intramolecular mechanism. The fluorescence quantum yield of 1Li⁺, 1Na⁺, and 1Ca²⁺ is approximately two times higher than that for 1. It was supposed that Li⁺ cation coordinates predominantly with oxygen atoms of macrocycle and, hence, influences weakly macrocycle nitrogen atoms conjugated with a molecule -system.     

The effect of alkaline earth metallic ions on the photoluminescence properties of sol-gel silica glass

Mg²⁺-, Ca²⁺-, Sr²⁺- and Ba²⁺-doped silica glasses have been prepared using sol-gel processing by employing Si(OC₂H₅)₄, MgCl₂6H₂O, CaCl₂2H₂O, SrCl₂6H₂O and BaCl₂2H₂O as precursors, with HCl as a catalyst. The UV–visibleabsorption spectra of the doped samples are almost the same as those of the undoped sample. The absorption bands of alkaline earth metallic ions have not been observed in the doped samples. Strong visible light has been observed from sol- gel silica glasses doped with alkaline earth metallic ions. The relative fluorescence intensity of the Sr²⁺-doped (the impurity mole ratio of Sr²⁺ was 0.268%) and the Ba²⁺-doped (the impurity mole ratio of Ba²⁺ was 0.448%) samples was about 4 times that of the undoped sample. The relative fluorescence intensity of the Mg²⁺-doped (the impurity mole ratio of Mg²⁺ was 0.069%) sample was about 2.5 times that of the pure glass sample. The relative fluorescence intensity of the Ca²⁺- doped (the impurity mole ratio of Ca²⁺ was 0.179%) sample was about 3 times that of the pure glass sample. Alkaline earth metallic ions affect the formation and conversion of luminescent defects in sol-gel silica glass. Thus, the relative fluorescence intensity of the doped samples increases more than that of the undoped sample. Received: 17 April 2001 / Accepted: 6 June 2001 / Published online: 30 August 2001     

Fluorescence lifetime imaging of intracellular calcium

Fluorescence lifetime imaging microscopy (FLIM) is a new methodology for studying the spatial and temporal dynamics of macromolecule, molecules, and ions in living cells. In FLIM image contrast is derived from the mean fluorescence lifetime at each point in a two-dimensional image. In our case the lifetime was measured by the phase-modulation method. We describe our FLIM apparatus, which consists of a fluorescence microscope, high-speed gated proximity focused MCP image intensifier, and slow-scan CCD camera. To accomplish subnanosecond time-resolved imaging, the gain of the image intensifier is modulated with a high-frequency signal, resulting in stationary phase-sensitive intensity images on the image intensifier. These images are recorded using a cooled slow-scan CCD camera and stored in an image processor. The lifetime images are created from a series of phase-sensitive images at various phase shift of the gain-modulation signal. We demonstrate calcium concentration imaging in living COS cells based on Ca²⁺-induced lifetime changes of Quin-2. The phase-angle image is mapped to the Ca²⁺ concentration image using anin vitro-determined calibration curve. The Ca²⁺ concentration was found to be uniform throughout the cell. In contrast, the intensity image shows significant spatial differences, which likely reflect variations in the thickness and distribution of probe within the cell.     

A Chemosensing Ensemble for the Detection of Cysteine Based on the Inner Filter Effect Using a Rhodamine B Spirolactam

A fluorescent chemosensing ensemble for the detection of cysteine is designed based on the fluorescence inner filter effect. The method employs the coordination of Cu²⁺ ion with salicylaldehyde rhodamine B hydrazone (I), a colorless and non-fluorescent rhodamine B spirolactam derivative to form I-Cu(II), a pink color but weakly fluorescent complex. When rhodamine B was introduced to the I-Cu(II) complex solution, the fluorescence signal of rhodamine B is dramatically decreased because of the fluorescence inner filter effect (IFE). Upon adding cysteine to the above solution, it can complex preferentially to Cu²⁺ compared to I, and the I-Cu(II) complex dissociates, which thus decreases the fluorescence IFE of the solution, and in turn leading to the fluorescence increase of the chemosensing system. Based on the above mechanism, a fluorescent chemosensing ensemble for cysteine is developed. The fluorescence increase is linearly with cysteine concentration up to 10.0 μ mol L⁻¹, with a detection limit of 1.4 × 10⁻⁷ mol L⁻¹ (3σ). The optimal conditions of the proposed method were studied and the selectivity of the proposed method was investigated in this paper.     

Single-molecule Detection of Reactive Oxygen Species: Application to Photocatalytic Reactions

In this review, we have focused on the oxidation reactions of single dye molecules by reactive oxygen species (ROS). The methodologies for the single-molecule detection of ROS, such as hydroxyl radical (HO^•), singlet oxygen (O₂(a¹Δ_g)), and hydrogen peroxide (H₂O₂), have been introduced together with examples. In particular, a successful application using the single-molecule fluorescence technique for the investigation of the TiO₂ photocatalytic oxidation reactions is demonstrated in detail.     

Photophysical studies of PET based acridinedione dyes with globular protein: Bovine serum albumin

Interaction of acridinedione dyes with model transport proteins, bovine serum albumin (BSA) in aqueous solution were investigated by fluorescence spectral studies. A fluorescence enhancement was observed on the addition of BSA to photoinduced electron transfer (PET) based acridinedione dyes, which posses C₆H₄(p-OCH₃) in the 9th position of the basic acridinedione ring. On the contrary, the addition of BSA to non-PET based acridinedione dyes with methyl or phenyl substitution in the 9th position does not result in any fluorescence enhancement. The enhancement in the fluorescence intensity is attributed to the suppression of PET process through space between -OCH₃ group and the acridinedione moiety is elucidated by steady state fluorescence measurements. The fluorescence anisotropy value (r) of 0.40 reveals that the motion of the dye molecule is highly constrained and is largely confined to the rigid microenvironment of the protein molecule. The binding constant (K) was found to be in the order of 6.0×10³ [M]⁻¹, which implies the existence of hydrophobic interaction between the PET based dye and BSA. Time resolved fluorescence lifetime measurements reveal that the PET based acridinedione dye preferably binds in the hydrophobic interior of BSA.     

Effect of Pendant Group Length Upon Metal Ion Complexation in Acetonitrile by Di-Ionized Calix[4]Arenes Bearing Two Dansyl Fluorophores

A series of three di-ionizable calix[4]arenes with two pendant dansyl (1-dimethylaminonaphthalene-5-sulfonyl) groups linked to the lower rims was synthesized. Structures of the three ligands were identical except for the length of the spacers which connected the two dansyl groups to the calix[4]arene scaffold. Following conversion of the ligands into their di-ionized di(tetramethylammonium) salts, absorption and emission spectrophotometry were utilized to probe the influence of metal cation (Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺, Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺, Ag⁺, Cd²⁺, Co²⁺, Fe²⁺, Hg²⁺, Mn²⁺, Pb²⁺, Zn²⁺ and Fe³⁺) complexation in acetonitrile. Upon complexation with these metal cations, emission spectra underwent marked red shifts and quenching of the dansyl group fluorescence for the di-ionized ligand with the shortest spacer. A similar effect was noted for the di-ionized ligand with an intermediate spacer for all of the metal ions, except Ba²⁺. For the di-ionized ligand with the longest spacer, the metal cations showed different effects on the emission spectrum. Li⁺, Mg²⁺, Ca²⁺ and Ba²⁺ caused enhancement of emission intensity with a red shift. Other metal cations produce quenching with red shifts in the emission spectra. Transition metal cations interacted strongly with all three di-ionized ligands. In particular, Fe³⁺ and Hg²⁺ caused greater than 99% quenching of the dansyl fluorescence in the di-ionized ligands.