A Nonparametric Method for Analysis of Fluorescence Emission in Combined Time and Wavelength Dimensions

We report a method for accurate recovery of tissue intrinsic fluorescence emission characteristics, including fluorescence lifetimes and spectral profiles, from complex two-dimensional (spectro-temporal) emission waveforms. Most algorithms for analysis of fluorescence data address separately the characteristics of either spectral emission or fluorescence relaxation time. We developed a novel nonparametric analytical method that allows for identification and estimation of the intrinsic Fluorescent Impulse Response Kernel (FIRK) simultaneously in time and wavelength dimensions. Modeling of FIRK was based on the characteristics of spectro-temporal fluorescence waveforms. Due to the decaying behavior of the fluorescence, a linear combination of discrete Laguerre functions was used to model the fluorescence response in time. To address the large variability of spectral profiles of distinct fluorophores, a discrete Fourier series expansion was used to model the variation of fluorescence intensity across wavelength. The proposed method was validated on synthetic fluorescence data and data measured from fluorescence lifetime standards and tissue endogenous fluorescent biomolecules. We determined that this method provides a direct recovery of the two-dimensional FIRK and accurate estimation (residual error < 6%) of a broad range of fluorescence lifetimes including the sub-nanosecond range. The FIRK retrieved using this method can further facilitate modeling and recognition of pathological and physiological conditions in tissues.     

Gabor analysis of auditory midbrain receptive fields: spectro-temporal and binaural composition

The spectro-temporal receptive field (STRF) is a model representation of the excitatory and inhibitory integration area of auditory neurons. Recently it has been used to study spectral and temporal aspects of monaural integration in auditory centers. Here we report the properties of monaural STRFs and the relationship between ipsi- and contralateral inputs to neurons of the central nucleus of cat inferior colliculus (ICC) of cats. First, we use an optimal singular-value decomposition method to approximate auditory STRFs as a sum of time-frequency separable Gabor functions. This procedure extracts nine physiologically meaningful parameters. The STRFs of approximately 60% of collicular neurons are well described by a time-frequency separable Gabor STRF model, whereas the remaining neurons exhibited obliquely oriented or multiple excitatory/inhibitory subfields that require a nonseparable Gabor fitting procedure. Parametric analysis reveals distinct spectro-temporal tradeoffs in receptive field size and modulation filtering resolution. Comparisons between an identical model used to study spatio-temporal integration areas of visual neurons further shows that auditory and visual STRFs share numerous structural properties. We then use the Gabor STRF model to compare quantitatively receptive field properties of contra- and ipsilateral inputs to the ICC. We show that most interaural STRF parameters are highly correlated bilaterally. However, the spectral and temporal phases of ipsi- and contralateral STRFs often differ significantly. This suggests that activity originating from each ear share various spectro-temporal response properties such as their temporal delay, bandwidth, and center frequency but have shifted or interleaved patterns of excitation and inhibition. These differences in converging monaural receptive fields expand binaural processing capacity beyond interaural time and intensity aspects and may enable colliculus neurons to detect disparities in the spectro-temporal composition of the binaural input.     

Stability of spectro-temporal tuning over several seconds in primary auditory cortex of the awake ferret

The steady-state spectro-temporal tuning of auditory cortical cells has been studied using a variety of broadband stimuli that characterize neurons by their steady-state responses to long duration stimuli, lasting from about a second to several minutes. Central sensory stations are thought to adapt in their response to stimuli presented over extended periods of time. For instance, we have previously shown that auditory cortical neurons display a second order of adaptation, whereby the rate of their adaptation to the repeated presentation of fixed alternating stimuli decreases with each presentation. The auditory grating (or ripple) method of characterizing central auditory neurons, and its extensions, have proven very effective. But these stimuli are typically used with spectro-temporal content held fixed over time-scales of seconds, introducing the possibility of rapid adaptation while the receptive field is being measured, whereas the neural response used to compute a spectro-temporal receptive field (STRF) assumes stationarity in the neural input/output function. We demonstrate dynamic changes in some parameters during the measurement of the STRF over a period of seconds, even absent of a relevant behavioral task. Specifically, we find in the primary auditory cortex of the awake ferret, small but systematic changes in duration and breadth of tuning of STRFs when comparing the early (0.25-1.75 s) and late (4.5-6 s) segments of the responses to these stimuli.     

Tuning for spectro-temporal modulations as a mechanism for auditory discrimination of natural sounds

Vocal communicators discriminate conspecific vocalizations from other sounds and recognize the vocalizations of individuals. To identify neural mechanisms for the discrimination of such natural sounds, we compared the linear spectro-temporal tuning properties of auditory midbrain and forebrain neurons in zebra finches with the statistics of natural sounds, including song. Here, we demonstrate that ensembles of auditory neurons are tuned to auditory features that enhance the acoustic differences between classes of natural sounds, and among the songs of individual birds. Tuning specifically avoids the spectro-temporal modulations that are redundant across natural sounds and therefore provide little information; rather, it overlaps with the temporal modulations that differ most across sounds. By comparing the real tuning and a less selective model of spectro-temporal tuning, we found that the real modulation tuning increases the neural discrimination of different sounds. Additionally, auditory neurons discriminate among zebra finch song segments better than among synthetic sound segments.     

Spectro-temporal response field characterization with dynamic ripples in ferret primary auditory cortex

To understand the neural representation of broadband, dynamic sounds in primary auditory cortex (AI), we characterize responses using the spectro-temporal response field (STRF). The STRF describes, predicts, and fully characterizes the linear dynamics of neurons in response to sounds with rich spectro-temporal envelopes. It is computed from the responses to elementary "ripples," a family of sounds with drifting sinusoidal spectral envelopes. The collection of responses to all elementary ripples is the spectro-temporal transfer function. The complex spectro-temporal envelope of any broadband, dynamic sound can expressed as the linear sum of individual ripples. Previous experiments using ripples with downward drifting spectra suggested that the transfer function is separable, i.e., it is reducible into a product of purely temporal and purely spectral functions. Here we measure the responses to upward and downward drifting ripples, assuming reparability within each direction, to determine if the total bidirectional transfer function is fully separable. In general, the combined transfer function for two directions is not symmetric, and hence units in AI are not, in general, fully separable. Consequently, many AI units have complex response properties such as sensitivity to direction of motion, though most inseparable units are not strongly directionally selective. We show that for most neurons, the lack of full separability stems from differences between the upward and downward spectral cross-sections but not from the temporal cross-sections; this places strong constraints on the neural inputs of these AI units.     

Cross-correlation and joint spectro-temporal receptive field properties in auditory cortex

Recordings were made from the right primary auditory cortex in 17 adult cats using two eight-electrode arrays. We recorded the neural activity under spontaneous firing conditions and during random, multi-frequency stimulation, at 65 dB SPL, from the same units. Multiple single-unit (MSU) recordings (281) were stationary through 900 s of silence and during 900 s of stimulation. The cross-correlograms of 545 MSU pairs with peak lag times within 10 ms from zero lag time were analyzed. Stimulation reduced the correlation in background activity, and as a result, the signal-to-noise ratio of correlated activity in response to the stimulus was enhanced. Reconstructed spectro-temporal receptive fields (STRFs) for coincident spikes showed larger STRF overlaps, suggesting that coincident neural activity serves to sharpen the resolution in the spectro-temporal domain. The cross-correlation for spikes contributing to the STRF depended much stronger on the STRF overlap than the cross-correlation during either silence or for spikes that did not contribute to the STRF (OUT-STRF). Compared with that for firings during silence, the cross-correlation for the OUT-STRF spikes was much reduced despite the unchanged firing rate. This suggests that stimulation breaks up the large neural assembly that exists during long periods of silence into a stimulus related one and maybe several others. As a result, the OUT-STRF spikes of the unit pairs, now likely distributed across several assemblies, are less correlated than during long periods of silence. Thus the ongoing network activity is significantly different from that during stimulation and changes afterng arousal during stimulation.     

Gender differences in hemispheric asymmetry of syllable processing: left-lateralized magnetic N100 varies with syllable categorization in females

The present study used magnetic source imaging to examine gender differences in the functional hemispheric asymmetry of auditory processing. The auditory evoked N100m was examined in male and female subjects in response to natural syllables with varying consonant and vowel as well as nonspeech noise. In an additional task subjects had to categorize different syllables from the first 35 ms of syllables, that is, the plosive and the formant transition. Syllable-evoked N100m activity was larger in the left than in the right hemisphere in female but not in male subjects. This gender-specific hemispheric asymmetry was speech specific, that is, absent when processing meaningless noise. Only in females did the degree of left-lateralization predict successful syllable categorization from short syllable bursts: Results suggest gender-specific differences in spectro-temporal analysis of speech.     

Nonlinearity of coding in primary auditory cortex of the awake ferret

Neural computation in sensory systems is often modeled as a linear system. This first order approximation is computed by reverse correlating a stimulus with the spike train it evokes. The spectro-temporal receptive field (STRF) is a generalization of this procedure which characterizes processing in the auditory pathway in both frequency and time. While the STRF performs well in predicting the overall course of the response to a novel stimulus, it is unable to account for aspects of the neural output which are inherently nonlinear (e.g. discrete events and non-negative spike rates). We measured the STRFs of neurons in the primary auditory cortex (AI) of the awake ferret using spectro-temporally modulated auditory gratings, or ripples. We quantified the degree of nonlinearity of these neurons by comparing their responses to the responses predicted from their respective STRFs. The responses of most cells in AI exhibited a squaring, nonlinear relation to the stimuli used to evoke them. Thus, the nonlinearity of these cells was nontrivial, that is it was not solely the result of spike rate rectification or saturation. By modeling the nonlinearity as a polynomial static output function, the predictive power of the STRF was significantly improved.     

Mitochondrial Inner Membrane Electrophysiology Assessed by Rhodamine-123 Transport and Fluorescence

Rhodamine-123 is widely used to make dynamic measurements of mitochondrial membrane potential both in vitro and in situ. Yet data interpretation is difficult due to a lack of quantitative understanding of how membrane potential and measured fluorescence are related. To develop such understanding, a model for dye transport across the mitochondrial inner membrane and partition into the membrane was developed. The model accounts for experimentally measured dye self-quenching and was integrated into a model of mitochondrial electrophysiology to estimate transients in mitochondrial membrane potential from kinetic fluorescence measurements. Our analysis indicates that (i) R123 fluorescence peaks at concentrations near 50 μM due to self-quenching; (ii) measured fluorescence intensity and membrane potential are related by a non-linear calibration curve sensitive to certain experimental details, including total concentration of dye and mitochondria in suspensions; and (iii) the time courses of membrane potential and electron transport fluxes following a perturbation (i.e. addition of ADP) significantly differ from observed transients in fluorescence intensity. These findings are consistent with the model predictions that mitochondria display a characteristic time of response to changes in substrate concentration of less than 0.1 s, corresponding to the time scale over which the rate of ATP synthesis changes to meet changes in ADP concentration.     

Phosphorescence decay time measurements using intensity correlation spectroscopy

In this paper, we report on phosphorescence measurements for oxygen dynamics in cells by means of a correlation method, which is an expansion of the fluorescence correlation spectroscopy. The intensity correlation function of the emission excited by a pulsed light source was measured. With changing the pulse timing, both the fluorescence correlation function and the decay time of phosphorescence could be analyzed. This method was applied for the analysis of the oxygen dynamics in HeLa cells stained by Pd(II)-porphine. The decay function consisted of two exponential components, which might be attributed to free and protein-bound forms of Pd(II)-porphine in the cell, respectively. The relative change of the oxygen concentration under normal and uncoupled respiration conditions was also measured. The simplicity of this method is a great advantage in the biological applications. Although the current system we used was limited in the temporal resolution, the method is in principle applicable to faster decay time measurements down to the nano-second range of the fluorescence decay times.     

活体染料CFDA-SE在淋巴细胞增殖研究中的应用

目的 :探讨活体染料CFDA SE在淋巴细胞增殖研究中的应用价值。方法 :利用CFDA SE染色、荧光抗体标记和流式细胞术 ,检测淋巴细胞及其亚群在多克隆刺激剂作用下荧光强度的变化 ,并应用相关软件分析其增殖情况。结果 :PDB ion omycin或ConA刺激 4 8h后均出现淋巴细胞分裂 ,表现为CFSE荧光强度的系列减半。环孢菌素A(CsA)可抑制ConA促进淋巴细胞增殖的作用 ,CFSE荧光无减半。ConA刺激4 8h后 ,出现CD4 T细胞和CD8 T细胞增殖不同步的现象 ,72h后这一现象更加明显。经ModFitTM 软件拟合后 ,得到的各项增殖指标显示 ,ConA对CD8 T细胞的促增殖作用强于对CD4 T细胞的作用。结论 :CFDA SE染色结合荧光抗体标记和流式细胞术 ,是分析淋巴细胞增殖的有力工具     

光敏剂浓度和给药时间对荧光诊断效果的影响

目的 为探索光敏剂浓度和给药时间对蓝光诱导的荧光诊断的影响,以提高荧光诊断的准确度.方法 常规方法制备荷瘤小鼠,分别给荷瘤小鼠静脉注射不同浓度（0～70 mg/kg体质量）的光敏剂-血卟啉衍生物（HpD）,并在给药后不同时间（0～48 h）,以前置395～415 nm滤光片的碘镓灯为激发光源,蓝光诱导载体荧光,通过图像采集系统采集荧光图像,取活组织做病理切片检查.结果 HpD浓度为40mg/kg体质量,给药后18 h,可以较为精确地反映肿瘤组织的浸润范围,荧光诊断准确度高于其他实验组;同时假阳性率低于其它实验组,差异有统计学意义.结论 荧光诊断的效果与光敏剂的浓度和观察时间密切相关;优化2者参数,可提高荧光诊断的准确度并降低假阳性率,对临床肿瘤手术方案的制定有重要参考价值.     

Principal component analysis of dynamic fluorescence tomography in measurement space

Challenges remain in resolving metabolic processes of drugs within small animals using a fluorescence tomographic image. In our previous work, using principal component analysis (PCA), we detected functional structures with different kinetic behaviors, where PCA was applied in fluorescence tomographic sequence (i.e. in the image space). As a result, all measurement data had to be reconstructed before performing PCA, which imposed a large computational burden. In this paper, we propose a new approach and apply PCA directly to fluorescence projection sequence (i.e. in the measurement space). Utilizing the compression property of PCA, it is possible to resolve regions with different kinetics by reconstructing only a few principal components. Hence, the computational cost can be significantly reduced. To evaluate the performance of the new method, numerical simulation and a phantom experiment are performed on a hybrid fluorescence and x-ray computed tomography imaging system. The results demonstrate that the proposed method greatly reduces the computational time compared with the previous method, while keeping a similar resolving capability.     

A synergic simulation-optimization approach for analyzing biomolecular dynamics in living organisms

A synergic duo simulation–optimization approach was developed and implemented to study protein–substrate dynamics and binding kinetics in living organisms. The forward problem is a system of several coupled nonlinear partial differential equations which, with a given set of kinetics and diffusion parameters, can provide not only the commonly used bleached area-averaged time series in fluorescence microscopy experiments but more informative full biomolecular/drug space–time series and can be successfully used to study dynamics of both Dirac and Gaussian fluorescence-labeled biomacromolecules in vivo. The incomplete Cholesky preconditioner was coupled with the finite difference discretization scheme and an adaptive time-stepping strategy to solve the forward problem. The proposed approach was validated with analytical as well as reference solutions and used to simulate dynamics of GFP-tagged glucocorticoid receptor (GFP-GR) in mouse cancer cell during a fluorescence recovery after photobleaching experiment. Model analysis indicates that the commonly practiced bleach spot-averaged time series is not an efficient approach to extract physiological information from the fluorescence microscopy protocols. It was recommended that experimental biophysicists should use full space–time series, resulting from experimental protocols, to study dynamics of biomacromolecules and drugs in living organisms. It was also concluded that in parameterization of biological mass transfer processes, setting the norm of the gradient of the penalty function at the solution to zero is not an efficient stopping rule to end the inverse algorithm. Theoreticians should use multi-criteria stopping rules to quantify model parameters by optimization.     

Drug effects analysis on cells using a high throughput microfluidic chip

Usually cell-based assay is performed using titer plates. Because of the large library of chemical compounds, robust and rapid methods are required to find, refine and test a potential drug candidate in an efficient manner. In this article, the drug effects analysis on human breast cancer cells with a droplet microfluidic chip is reported. Each droplet serves as a nanoliter-volume titer plate and contains a human breast cancer cell MDA-MB-231, Cytochalasin D drug solution and cell viability indicator such as Calcein AM, which emits cytoplasmic green fluorescence. The drug effects on each cell are monitored in real time using a fluorescence microscope and by analyzing the fluorescence image of each cell. Clear change of the cell shape and size has been observed after the drug treatment, which is similar to that of conventional petri dish technique, suggesting this approach is a potential viable technical platform for drug effect analysis and for high throughput drug screen and discovery.     

SYBR Green I-induced fluorescence in cultured immune cells: a comparison with Acridine Orange

Using fluorescence microscopy, we explored the ability of cultured immune cells to take up aqueous SYBR Green I (SGI). SYBR Green I, a highly sensitive fluorescent nucleic acid stain, which preferentially binds to dsDNA over ssDNA or RNA with little background fluorescence from unbound molecules. A time course study over 2h using final dilutions of SGI of 1:10,000 and 1:100,000 at 22 and 37 degrees C, revealed the dye quickly entered the cells, stained mitochondrial DNA then nuclear DNA, and SGI-induced green fluorescence increased over time. As staining progressed, heterochromatin appeared as more intense green fluorescent lines, patches and circles against the lower fluorescence of the nucleoplasm. The lower fluorescence from the nucleoplasm indicated SGI also bound to areas of euchromatin. Similar progressive uptake experiments were carried out with the permeant DNA dye Acridine Orange (AO) to provide insight into staining patterns and mode of uptake. Statistical analysis of cells prestained with SGI then tested with Trypan Blue for changes in membrane permeability, revealed no significant difference between controls and treatment for each temperature. It appears that SGI does not compromise cells for up to 2 h following initial exposure.     

Correlation of quantitative light-induced fluorescence and optical coherence tomography applied for detection and quantification of early dental caries

Fluorescence loss in enamel following demineralization has been correlated with the amount of mineral lost during the demineralization. The correlation between fluorescence loss measured by quantitative light-induced fluorescence (QLF) and the reflectivity loss measured by a versatile en face optical coherence tomography (OCT) system was investigated in a demineralization process to produce artificial dental caries. We used an OCT system that can collect A-scans (reflectivity versus depth), B-scans (longitudinal images), and C-scans (en face images). The power to the sample was 250 microW, the wavelength lambda = 850 nm, and the depth resolution in air 16 microm. A-scans, which show the profile of the reflectivity versus the depth of penetration into the tooth tissue, were used for quantitative analysis of the reflectivity loss. The results have shown that both the fluorescence radiance and reflectivity of the enamel decrease with increasing de- mineralization time. A linear correlation was observed between the percentage of fluorescence loss measured by QLF and the percentage of reflectivity loss measured by OCT. It was concluded that the decrease in reflectivity of the enamel during demineralization, measured by OCT, could be related to the amount of mineral lost during the de- mineralization process.     

Intravital two-photon microscopy: focus on speed and time resolved imaging modalities

Summary: Initially used mainly in the neurosciences, two-photon microscopy has become a powerful tool for the analysis of immunological processes. Here, we describe currently available two-photon microscopy techniques with a focus on novel approaches that allow very high image acquisition rates compared with state-of-the-art systems. This improvement is achieved through a parallelization of the excitation process: multiple beams scan the sample simultaneously, and the fluorescence is collected with sensitive charge-coupled device (CCD)-based line or field detectors. The new technique's performance is compared with conventional single beam laser-scanning systems that detect signals by means of photomultipliers. We also discuss the use of time- and polarization-resolved fluorescence detection, especially fluorescence lifetime imaging (FLIM), which goes beyond simple detection of cells and tissue structures and allows insight into cellular physiology. We focus on the analysis of endogenous fluorophores such as NAD(P)H as a way to analyze the redox status in cells with subcellular resolution. Here, high-speed imaging setups in combination with novel ways of data analysis allow the generation of FLIM data sets almost in real time. The implications of this technology for the analysis of immune reactions and other cellular processes are discussed.     

氧化应激诱导HepG2肝癌细胞凋亡的研究(英)

目的：直接暴露细胞于活性氧能诱导发生凋亡，本文研究氧化应激诱导HepG2肝癌细胞的死亡及其机制。方法：暴露细胞于2 mmol/L过氧化氢产生氧化应激，用DNA凝胶电泳检测细胞凋亡，用荧光染色法检测细胞线粒体膜电位变化，Western blotting检测细胞浆中细胞色素c变化，fluorometric assay kit检测caspase活性变化。结果：氧化应激作用于HepG2细胞后12 h开始发生凋亡；氧化应激作用后4 h，细胞线粒体膜电位明显下降；胞浆中细胞色素c浓度呈时间依赖性增高；氧化应激作用8 h、12 h后细胞内caspase-3、caspase-9活性分别升高6.7及3.6倍，但caspase-8活性无变化。结论：氧化应激能诱导HepG2肝癌细胞发生凋亡，其途径与线粒体通路及caspase激活有关。     

Compact point-detection fluorescence spectroscopy system for quantifying intrinsic fluorescence redox ratio in brain cancer diagnostics

We report the development of a compact point-detection fluorescence spectroscopy system and two data analysis methods to quantify the intrinsic fluorescence redox ratio and diagnose brain cancer in an orthotopic brain tumor rat model. Our system employs one compact cw diode laser (407 nm) to excite two primary endogenous fluorophores, reduced nicotinamide adenine dinucleotide, and flavin adenine dinucleotide. The spectra were first analyzed using a spectral filtering modulation method developed previously to derive the intrinsic fluorescence redox ratio, which has the advantages of insensitivity to optical coupling and rapid data acquisition and analysis. This method represents a convenient and rapid alternative for achieving intrinsic fluorescence-based redox measurements as compared to those complicated model-based methods. It is worth noting that the method can also extract total hemoglobin concentration at the same time but only if the emission path length of fluorescence light, which depends on the illumination and collection geometry of the optical probe, is long enough so that the effect of absorption on fluorescence intensity due to hemoglobin is significant. Then a multivariate method was used to statistically classify normal tissues and tumors. Although the first method offers quantitative tissue metabolism information, the second method provides high overall classification accuracy. The two methods provide complementary capabilities for understanding cancer development and noninvasively diagnosing brain cancer. The results of our study suggest that this portable system can be potentially used to demarcate the elusive boundary between a brain tumor and the surrounding normal tissue during surgical resection.