Calcium measurements in perfused mouse heart: quantitating fluorescence and absorbance of Rhod-2 by application of photon migration theory

Both theoretical and experimental results are presented for the quantitative detection of calcium transients in the perfused mouse heart loaded with the calcium-sensitive fluorescent dye Rhod-2. Analytical models are proposed to calculate both the reflected absorbance and fluorescence spectra detected from the mouse heart. These models allow correlation of the measured spectral intensities with the relative quantity of Rhod-2 in the heart and measurement of the changes in quantum yield of Rhod-2 upon binding calcium in the heart in which multiple scattering effects are predominant. Theoretical modeling and experimental results demonstrate that both reflected absorbance and fluorescence emission are attenuated linearly with Rhod-2 washout. According to this relation, a ratiometric method using fluorescence and absorbance is validated as a measure of the quantum yield of calcium-dependent fluorescence, enabling determination of the dynamics of cytosolic calcium in the perfused mouse heart. The feasibility of this approach is confirmed by experiments quantifying calcium transients in the perfused mouse heart stimulated at 8 Hz. The calculated cytosolic calcium concentrations are 368 +/- 68 nM and 654 +/- 164 nM in diastole and systole, respectively. Spectral distortions induced by tissue scattering and absorption and errors induced by the geometry of the detection optics in the calcium quantification are shown to be eliminated by using the ratio method. Methods to effectively minimize motion-induced artifacts and to monitor the oxygenation status of the whole perfused heart are also discussed.     

Correction for inner filter effects in turbid samples: fluorescence assays of mitochondrial NADH

Fluorescentdeterminations of NADH in porcine heart mitochondria were subject tosignificant errors caused by alterations in inner filter effects duringnumerous metabolic perturbations. These inner filter effects wereprimarily associated with changes in mitochondrial volume andaccompanying light scattering. The observed effects were detected in astandard commercial fluorometer with emission orthogonal to theexcitation light path and, to a lesser extent, in a light path geometrydetecting only the surface fluorescence. A method was developed todetect and correct for inner filter effects on mitochondrial NADHfluorescence measurements that were independent of the optical pathgeometry using an internal fluorescent standard and linearleast-squares spectral analysis. A simple linear correction with theinner fluorescence reference was found to adequately correct for innerfilter effects. This approach may be useful for other fluorescenceprobes in isolated mitochondria or other light-scattering media.

     

Protein scaffold-based molecular probes for cancer molecular imaging

Protein scaffold molecules are powerful reagents for targeting various cell signal receptors, enzymes, cytokines and other cancer-related molecules. They belong to the peptide and small protein platform with distinct properties. For the purpose of development of new generation molecular probes, various protein scaffold molecules have been labeled with imaging moieties and evaluated both in vitro and in vivo. Among the evaluated probes Affibody molecules and analogs, cystine knot peptides, and nanobodies have shown especially good characteristics as protein scaffold platforms for development of in vivo molecular probes. Quantitative data obtained from positron emission tomography, single photon emission computed tomography/CT, and optical imaging together with biodistribution studies have shown high tumor uptakes and high tumor-to-blood ratios for these probes. High tumor contrast imaging has been obtained within 1 h after injection. The success of those molecular probes demonstrates the adequacy of protein scaffold strategy as a general approach in molecular probe development.     

Comparison of chlorophyll fluorescence emission characteristics of wheat leaf tissue and isolated thylakoids as a function of excitation wavelength

Abstract. Chlorophyll fluorescence emission spectra and the kinetics of 685 mm fluorescence emission from wheat leaf tissue and thylakoids isolated from such tissue were examined as a function of excitation wavelength. A considerable enhancement of fluorescence emission above 700 nm relative to that at 685 nm was observed from leaf tissue when it was excited with 550 nm rather than 450 nm radiation. Such excitation wavelength dependent changes in the emission spectrum occurred over an excitation spectral range of 440–660 nm and appeared to be directly related to the total quantity of radiation absorbed at a given excitation wavelength. Experiments with isolated thylakoid preparations demonstrated that changes in the fluorescence emission spectrum of the leaf were attributable to the optical properties of the leaf and were not due to the intrinsic characteristies of the thylakoid photochemical apparatus. This was not the case for the observed excitation wavelength dependent changes in the 685 nm fluorescence induction curve obtained from leaf tissue infiltrated with DCMU. Excitation wavelength dependent changes in the ratio of the variable to maximal fluorescence emission and the shape of the variable fluorescence induction were observed for leaf tissue. Isolated thylakoid studies showed that such changes in the leaf fluorescence kinetics were representative of the way in which the photochemical apparatus in vivo was processing the absorbed radiation at the different excitation wavelengths. The results are considered in the context of the use of fluorescence emission characteristics of leaves as non-destructive probes of the photochemical apparatus in vivo.     

Biophotonic patterns of optical interactions between fish eggs and embryos

The optical (non-substantial) interactions between various biological samples have been evident in a number of cases mainly by the effects on their functional activity and developmental patterns. However, the mechanisms of these interactions have remained obscure. Effect of optical interaction has been observed on the intensity and Fourier patterns of biophoton emission of fish embryos. We demonstrate that: (1) the short-term optical interactions are accompanied by a gradual decrease of a total emission intensity of the interacting batches; (2) this effect is spread laterally to that part of a batch which does not have any direct optical contacts with its partner; and (3) the long-term optical contacts lead to a mutual exchange of spectral characteristics of interacting batches in which the total spectral density values are reversed (often with an overshoot). The reversal rate depends upon the developmental distance between the optical partners and the initial differences of their spectral characteristics. The results are discussed in terms of a sub-radiance and Le Chatelier principle.     

Selection of quantum dot wavelengths for biomedical assays and imaging

Fluorescent semiconductor nanocrystals (quantum dots [QDs]) are hypothesized to be excellent contrast agents for biomedical assays and imaging. A unique property of QDs is that their absorbance increases with increasing separation between excitation and emission wavelengths. Much of the enthusiasm for using QDs in vivo stems from this property, since photon yield should be proportional to the integral of the broadband absorption. In this study, we demonstrate that tissue scatter and absorbance can sometimes offset increasing QD absorption at bluer wavelengths, and counteract this potential advantage. By using a previously validated mathematical model, we explored the effects of tissue absorbance, tissue scatter, wavelength dependence of the scatter, water-to-hemoglobin ratio, and tissue thickness on QD performance. We conclude that when embedded in biological fluids and tissues, QD excitation wavelengths will often be quite constrained, and that excitation and emission wavelengths should be selected carefully based on the particular application. Based on our results, we produced near-infrared QDs optimized for imaging surface vasculature with white light excitation and a silicon CCD camera, and used them to image the coronary vasculature in vivo. Taken together, our data should prove useful in designing fluorescent QD contrast agents optimized for specific biomedical applications.     

Comparison of kinetic and optical changes during guanidine denaturation of intestinal alkaline phosphatase

The modifications of the activity of calf intestinal Alkaline Phosphatase treated with moderate amounts of guanidinium chloride are compared with the conformational changes observed by ultraviolet absorbance and intrinsic fluorescence. The time course of catalytic and optical properties of the treated enzyme develops through two distinct steps: an instantaneous and a time-dependent one. The immediate effect of guanidine is to lower emission yield, to shift the emission maximum of the enzyme to longer wavelengths and to enhance the absorbance of the protein. The rapid conformational transition determines a paradoxical activation at low effector concentration (below 0.88 M) and an inhibition at higher amounts. The following marked decay of enzyme activity with time is related to spectroscopically detectable changes. Temperature influences both kinetic and structural aspects of the process and facilitates guanidine action.     

Optical signals from surface and T system membranes in skeletal muscle fibers. Experiments with the potentiometric dye NK2367

Absorbance signals were recorded from cut single skeletal muscle fibers stained with the nonpenetrating potentiometric dye NK2367 and mounted in a three-vaseline-gap voltage clamp. The characteristics of the optical signals recorded under current and voltage-clamp conditions were studied at various wavelengths between 500 and 800 nm using unpolarized light. Our results indicate that the absorbance signals recorded with this dye reflect potential changes across both the surface and T system membranes and that the relative contribution of each of these membrane compartments to the total optical change is strongly wavelength dependent. A peak intensity change was detected at 720 nm for the surface membrane signal and at 670 nm for the T system. Evidence for this wavelength-dependent separation derives from an analysis of the kinetics and voltage dependence of the optical signals at different wavelengths, and results obtained in detubulated fibers. The 670-nm optical signal was used to demonstrate the lack of potential control in the T system by the voltage clamp and the effect of a tetrodotoxin (TTX)-sensitive sodium conductance on tubular depolarization.     

Intramural multisite recording of transmembrane potential in the heart

Heart surface optical mapping of transmembrane potentials has been widely used in studies of normal and pathological heart rhythms and defibrillation. In these studies, three-dimensional spatio-temporal events can only be inferred from two-dimensional surface potential maps. We present a novel optical system that enables high fidelity transmural recording of transmembrane potentials. A probe constructed from optical fibers is used to deliver excitation light and collect fluorescence from seven positions, each 1 mm apart, through the left ventricle wall of the rabbit heart. Excitation is provided by the 488-nm line of a water-cooled argon-ion laser. The fluorescence of the voltage-sensitive dye di-4-ANEPPS from each tissue site is split at 600 nm and imaged onto separate photodiodes for later signal ratioing. The optics and electronics are easily expandable to accommodate multiple optical probes. The system is used to record the first simultaneous measurements of transmembrane potential at a number of sites through the intact heart wall.     

New near-infrared optical probes of cardiac electrical activity

Styryl voltage-sensitive dyes (e.g., di-4-ANEPPS) have been widely and successfully used as probes for mapping membrane potential changes in cardiac cells and tissues. However, their utility has been somewhat limited because their excitation wavelengths have been restricted to the 450- to 550-nm range. Longer excitation/emission wavelength probes can minimize interference from endogenous chromophores and, because of decreased light scattering and lower absorption by endogenous chromophores, improve recording from deeper tissue layers. In this article, we report efforts to develop new potentiometric styryl dyes that have excitation wavelengths ranging above 700 nm and emission spectra extending to 900 nm. Three dyes for cardiac optical mapping were investigated in depth from several hundred dyes containing 47 variants of the styryl chromophores. Absorbance and emission spectra in ethanol and multilamellar vesicles, as well as voltage-dependent spectral changes in a model lipid bilayer, have been recorded for these dyes. Optical action potentials were recorded in typical cardiac tissues (rat, guinea pig, pig) and compared with those of di-4-ANEPPS. The voltage sensitivities of the fluorescence of these new potentiometric indicators are as good as those of the widely used ANEP series of probes. In addition, because of molecular engineering of the chromophore, the new dyes provide a wide range of dye loading and washout time constants. These dyes will enable a series of new experiments requiring the optical probing of thick and/or blood-perfused cardiac tissues.     

Subzero temperature study of the inner mitochondrial membrane and related phospholipid membrane systems with the fluorescent probe, trans-parinaric acid.

The fluorescence intensity of trans-parinaric acid as a function of the temperature indicates a phase transition in bovine heart mitochondrial inner membranes below 0 degrees C. The comparison of the dye fluorescence intensity in intact inner mitochondrial membranes and in vesicles from extracted phospho lipids of mitochondria revealed a similar intensity increase with decreasing temperature. A synthetic phospholipid system of dioleoyl phosphatidylcholine was investigated because of its low phase transition temperature and showed a very definite intensity change at -25 degrees C. trans-Parinaric acid in membrane systems probes an environment of intermediate polarity; this was found from the excitation and emission spectra and from fluorescence decay.     

Optical clearing for improved contrast in second harmonic generation imaging of skeletal muscle

Using second harmonic generation (SHG) imaging microscopy, we have examined the effect of optical clearing with glycerol to achieve greater penetration into specimens of skeletal muscle tissue. We find that treatment with 50% glycerol results in a 2.5-fold increase in achievable SHG imaging depth. Signal processing analyses using fast Fourier transform and continuous wavelet transforms show quantitatively that the periodicity of the sarcomere structure is unaltered by the clearing process and that image quality deep in the tissue is improved with clearing. Comparison of the SHG angular polarization dependence also shows no change in the supramolecular organization of acto-myosin complexes. By contrast, identical treatment of mouse tendon (collagen based) resulted in a strong decrease in SHG response. We suggest that the primary mechanism of optical clearing in muscle with glycerol treatment results from the reduction of cytoplasmic protein concentration and concomitant decrease in the secondary inner filter effect on the SHG signal. The lack of glycerol concentration dependence on the imaging depth indicates that refractive index matching plays only a minor role in the optical clearing of muscle. SHG and optical clearing may provide an ideal mechanism to study physiology in highly scattering skeletal or cardiac muscle tissue with significantly improved depth of penetration and achievable imaging depth.     

Adenylate Cyclases in the Vertebrate Retina: Distribution and Characteristics in Rabbit and Ground Squirrel

Adenylate cyclase activity and the effects of EGTA, 5'-guanylylimidodiphosphate (GPP(NH)P), and dopamine were measured in microdissected layers of rod-dominant (rabbit) and cone-dominant (ground squirrel) retinas, The distribution of basal enzyme activity was similar in both species, with the highest levels found in the inner plexiform and photoreceptor cell inner segment layers, EGTA inhibited adenylate cyclase in the inner retina of both species and stimulated activity in rabbit outer and inner segment layers, but had no effect in these layers from ground squirrel. Enzyme activity was stimulated in all regions by GPP(NH)P, except in the outer segments of the photoreceptors. Dopamine stimulated the enzyme in the outer and inner plexiform and inner nuclear layers in rabbit, but only in the inner plexiform layer in ground squirrel. These data demonstrate that the enzymatic characteristics of adenylate cyclase vary extensively from region to region in vertebrate retina and suggest that cyclic AMP may have multiple roles in this tissue. A model for the distribution of the different forms of adenylate cyclase in retina is proposed.     

Beam and tissue factors affecting Cherenkov image intensity for quantitative entrance and exit dosimetry on human tissue

This study's goal was to determine how Cherenkov radiation emission observed in radiotherapy is affected by predictable factors expected in patient imaging. Factors such as tissue optical properties, radiation beam properties, thickness of tissues, entrance/exit geometry, curved surface effects, curvature and imaging angles were investigated through Monte Carlo simulations. The largest physical cause of variation of the correlation ratio between of Cherenkov emission and dose was the entrance/exit geometry (?50%). The largest human tissue effect was from different optical properties (?45%). Beyond these, clinical beam energy varies the correlation ratio significantly (?20% for X‐ray beams), followed by curved surfaces (?15% for X‐ray beams and ?8% for electron beams), and finally, the effect of field size (?5% for X‐ray beams). Other investigated factors which caused variations less than 5% were tissue thicknesses and source to surface distance. The effect of non‐Lambertian emission was negligible for imaging angles smaller than 60 degrees. The spectrum of Cherenkov emission tends to blue‐shift along the curved surface. A simple normalization approach based on the reflectance image was experimentally validated by imaging a range of tissue phantoms, as a first order correction for different tissue optical properties.

     

Ionic Dependence of Membrane Potential and Glutamate Receptor-Linked Responses in Synaptoneurosomes as Measured with a Cyanine Dye, DiS-C2-(5)

Membrane potentials of particles present in a subcellular brain preparation, called synaptoneurosomes, have been monitored by measurement of changes in the absorbance of a cyanine dye, DiS-C2-5. The membrane potential of the particles seems to be dependent on both Cl- and K+ diffusion potentials, as judged from dependence of the absorbance changes on the K+ equilibrium potential across the membrane in the presence of Ba2+ or when Cl- was replaced with gluconate. The apparent high Cl- permeability of the membrane preparation was reduced in the presence of picrotoxin, a finding suggesting endogenous activation of receptor-linked Cl- channels. Glutamate and kainate caused depolarization of the membranes present in the preparation. This effect was only seen if K+ channels had been blocked in the presence of Ba2+ or 4-aminopyridine. No responses were observed with other glutamate receptor agonists (quisqualate or N-methyl-D-aspartate). The membrane potential of particles present in conventional synaptosomal preparations neither had a high Cl- permeability nor reacted to glutamate or kainate in the present conditions. The results suggest that synaptoneurosome preparations may be used for functional studies on postsynaptic neurotransmitter receptor-linked membrane potential changes with optical probes of membrane potential.     

Optimal Design for U-bent Fiber-optic LSPR Sensor Probes

The refractive index (RI) sensitivity of a localized surface plasmon resonance (LSPR)-based fiber-optic probes is dependent on surface coverage of gold nanoparticles (GNP), fiber core diameter, and probe geometry. For U-bent LSPR fiber-optic probes, which demonstrated an order higher absorption sensitivity over straight probes, bend diameter and probe length may also have a significant influence on the sensitivity. This study on U-bent fiber-optic LSPR probes is aimed at optimizing these parameters to obtain highest possible RI sensitivity. RI sensitivity increases linearly as a function of surface coverage of GNP in the range of 2–22 %. U-bent fiber-optic probes made of 200-, 400-, and 600-μm fiber core diameter show optimum bend diameter value as ～1.4 mm. In addition, RI sensitivity is almost the same irrespective of fiber core diameter demonstrating flexibility in choice of the fiber and ease in optical coupling. The length of the probe preceding and succeeding the bend region has significantly less influence on RI sensitivity allowing miniaturization of these probes. In addition to these experimental studies, we present a theoretical analysis to understand the relative contribution of evanescent wave absorbance of GNP and refractive losses in the fiber due to GNP, towards the RI sensitivity.     

Calculation of optical signal using three-dimensional bidomain/diffusion model reveals distortion of the transmembrane potential

Optical mapping experiments allow investigators to view the effects of electrical currents on the transmembrane potential, V_m, as a shock is applied to the heart. One important consideration is whether the optical signal accurately represents V_m. We have combined the bidomain equations along with the photon diffusion equation to study the excitation and emission of photons during optical mapping of cardiac tissue. Our results show that this bidomain/diffusion model predicts an optical signal that is much smaller than V_m near a stimulating electrode, a result consistent with experimental observations. Yet, this model, which incorporates the effect of lateral averaging, also reveals an optical signal that overestimates V_m at distances >1 mm away from the electrode. Although V_m falls off with distance r from the electrode as exp(−r/λ)/r, the optical signal decays as a simple exponential, exp(−r/λ). Moreover, regions of hyperpolarization adjacent to a cathode are emphasized in the optical signal compared to the region of depolarization under the cathode. Imaging methods utilizing optical mapping techniques will need to account for these distortions to accurately reconstruct V_m.     

Subzero temperature study of the inner mitochondrial membrane and related phospholipid membrane systems with the fluorescent probe, trans-parinaric acid

The fluorescence intensity of trans-parinaric acid as a function of the temperature indicates a phase transition in bovine heart mitochondrial inner membranes below 0°C. The comparison of the dye fluorescence intensity in intact inner mitochondrial membranes and in vesicles from extracted phospholipids of mitochondria revealed a similar intensity increase with decreasing temperature. A synthetic phospholipid system of dioleoyl phosphatidylcholine was investigated because of its low phase transition temperature and showed a very definite intensity change at ?25°C. trans-Parinaric acid in membrane systems probes an environment of intermediate polarity; this was found from the excitation and emission spectra and from fluorescence decay.     

Evanescent wave absorbance based fiber optic biosensor for label-free detection of E. coli at 280 nm wavelength

A novel label-free technique for the detection of pathogens based on evanescent wave absorbance (EWA) changes at 280 nm from a U-bent optical fiber sensor is demonstrated. Bending a decladded fiber into a U-shaped structure enhances the penetration depth of evanescent waves and hence sensitivity of the probe. We show that the enhanced EWA response from such U-bent probes, caused by the inherent optical absorbance properties of bacterial cells or biomolecules specifically bound to the sensor surface, can be exploited for the detection of pathogens. A portable optical set-up with a UV light emitting diode, a spectrometer and U-bent fiber optic probe of 200 μm core diameter, 0.75 mm bend radius and effective probe length of 1cm demonstrated an ability to detect less than 1000 cfu/ml.     

去血红素细胞色素c的制备及其结构研究

在酸性条件下用硫酸银断裂马心细胞色素ｃ（以下简称ｃｙｔ．ｃ）的肽链与血红素相连的硫醚键,通过酸性丙酮抽提,硫基乙醇处理及超速离心等步骤纯化得去血红素的ｃｙｔ．ｃ（以下简称Ａｐｏ－ｃｙｔ．ｃ）。Ａｐｏ－ｃｙｔ．ｃ与天然ｃｙｔ．ｃ相比,其酸性电泳迁移率明显降低,紫外－可见光谱在１９０～２２０ｎｍ处吸收上升,荧光光谱的最大发射峰波长产生红移,同时ＣＤ谱中α螺旋的特征峰完全消失,这说明在ｃｙｔ．ｃ去血红素的过程中,蛋白质已由原来的紧密球状结构变成了较为松散、伸展的无规卷曲构象。因此,血红素对ｃｙｔ．ｃ天然构象的维持有着重要作用。