Two-photon excited lifetime imaging of autofluorescence in cells during UV A and NIR photostress

By monitoring coenzyme autofluorescence modifications. as an indicator of cell damage. the cellular response to femtosecond near-infrared (NIR) radiation (two-photon absorption) was compared with exposure to low-power UV A radiation (one-photon absorption). Excitation radiation from a tunable Ti-sapphire laser. focused through highnumerical- aperture microscope optics. provided diffractionlimited mlcrobeams of an adjustable peak power. Laser scanning NIR microscopy was used to detect spatially the intracellular distribution of fluorescent coenzymes by fluorescence intensity imaging as well as fluorescence lifetime imaging (_T-mapping). Upon the onset of UV or NIR exposure. Chinese hamster ovary cells exhibited blue/green autofluorescence witq a mean lifetime of 2·2 ns. which was attributed to NAD(P)H in mitochondria. Exposure to 365 nm radiation from a high-pressure mercury lamp (1 m W. 300 J cm^-2 ) resulted in oxidative stress correlated with increased autofluorescence intensity. onset of nuclear fluorescence. and a fluorescence lifetime decrease. The cellular response to femtosecond NIR micro beams depended significantly on peak power. Peak powers above a threshold value of about 0·5kW (average power: 6mW). 0·55kW (7mW) and 0·8kW (lOmW) at 730nm. 760nm and 800nm. respectively. resulted in the onset of short-lived luminescence with higher intensity (100x) than the intracellular NAD(P)H fluorescence. This luminescence. accompanied by destruction of cellular morphology. was localized and occurred in the mitochondrial region. In contrast. beams at a power of less than 0·5 kW allowed nondestructive fluorophore detection with high spatial and temporal resolution without modification of cellular redox state or cell morphology.     

荧光共振能量转移显微术及其新进展

本文叙述荧光共振能量转移显微术及荧光寿命成像显微术的原理、方法及特点。同时介绍利用荧光共振能量转移显微术研究信号分子Rac蛋白在3T3成纤维细胞内的定位及活化过程,以及利用荧光共振能量转移—荧光寿命成像显微术研究转录因子CAATT/增强子结合蛋白α在小鼠垂体细胞内的二聚化现象。     

Measurement of nanosecond time-resolved fluorescence with a directly gated interline CCD camera

CCD cameras coupled optically to gated image intensifiers have been used for fast time‐resolved measurements for some years. Image intensifiers have disadvantages, however, and for some applications it would be better if the image sensor could be gated directly at high speed. Control of the ‘charge drain’ function on an interline‐transfer CCD allows the sensor to be switched rapidly from an insensitive state. The temporal and spatial properties of the charge drain are explored in the present paper and it is shown that nanosecond time resolution with acceptable spatial uniformity can be achieved for a small commercial sensor. A fluorescence lifetime imaging system is demonstrated, based on a repetitively pulsed laser excitation source synchronized to the CCD control circuitry via a programmable delay unit.     

Photo-physical properties of Ca2+-indicator dyes suitable for two-photon fluorescence-lifetime recordings

We analyzed the suitability of various Ca²⁺-indicator dyes for quantitative two-photon fluorescence-lifetime imaging. Although fura-2, fluo-3, BTC and calcein did not show useful Ca²⁺-dependent lifetime changes, calcium orange, calcium green-1, oregon green-2 and -5N, as well as magnesium green allowed to quantify the Ca²⁺-free and Ca²⁺-bound dye fractions by a double-exponential lifetime analysis. For the latter dyes, we derived calibration formalisms that correct for lifetime distortions by dye impurities and Ca²⁺-dependent extinction coefficients.     

Simultaneous confocal lifetime imaging of multiple fluorophores using the intensity-modulated multiple-wavelength scanning (IMS) technique

We demonstrate the simultaneous recording of confocal lifetime images of multiple fluorophores. The confocal microscope used in the study combines intensity-modulated laser illumination, lock-in detection and spectral separation of the fluorescent light. A theoretical investigation is presented that describes how the signal-to-noise ratio (SNR) depends on various factors such as modulation frequency, degree of modulation and number of detected photons. Theory predicts that, compared with ordinary intensity images, lifetime images will have a SNR that is, at best, approximately four times lower. Experimental results are presented that confirm this prediction.     

Confocal pH imaging of microscopic specimens using fluorescence lifetimes and phase fluorometry: influence of parameter choice on system performance

We investigate the performance of confocal pH imaging when using phase fluorometry and fluorophores with pH-dependent lifetimes. In these experiments, the specimen is illuminated by a laser beam, whose intensity is sinusoidally modulated. The lifetime-dependent phase shift in the fluorescent signal is detected by a lock-in amplifier, and converted into a pH value through a calibration procedure. A theoretical investigation is made of how the different system parameters will influence the results concerning sensitivity and noise. Experiments carried out with the fluorophore SNAFL-2 support these theoretical predictions. It is found that, under realistic experimental conditions, we can expect a pH change of 0.1 units to be easily detected in an 8-bit digital image. However, the pixel-to-pixel root mean square noise is often of the order of one pH unit. This comparatively high level of noise has its origin in photon quantum noise. pH measurements on living cells show a systematic deviation from expected values. This discrepancy appears to be the result of fluorophore interaction with various cell constituents, and is the subject of further investigation.     

Direct modulation of the effective sensitivity of a CCD detector: a new approach to time-resolved fluorescence imaging

In this paper a novel approach to frequency‐domain fluorescence lifetime imaging (FLIM) is described. In a CCD camera a single pixel is defined by a charge pattern on a group of electrodes. By modulation of the pattern of voltages defining the pixel structure it is possible to modulate the sensitivity of the CCD at radio frequency. The modulation enhances the noise performance of the CCD, in contrast to the deterioration in performance seen when an intensifier stage is similarly modulated. The new technology has potential applications to a wide range of assays as well as in conventional FLIM applications. Unlike intensifier‐based systems, the directly modulated CCD is physically small, inexpensive, robust and offers superior resolution and noise performance.     

Fluorescence lifetime imaging microscopy using near-infrared contrast agents

Although single-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to image molecular processes using a wide range of excitation wavelengths, the captured emission of this technique is confined to the visible spectrum. Here, we explore the feasibility of utilizing near-infrared (NIR) fluorescent molecular probes with emission >700 nm for FLIM of live cells. The confocal microscope is equipped with a 785 nm laser diode, a red-enhanced photomultiplier tube, and a time-correlated single photon counting card. We demonstrate that our system reports the lifetime distributions of NIR fluorescent dyes, cypate and DTTCI, in cells. In cells labelled separately or jointly with these dyes, NIR FLIM successfully distinguishes their lifetimes, providing a method to sort different cell populations. In addition, lifetime distributions of cells co-incubated with these dyes allow estimate of the dyes' relative concentrations in complex cellular microenvironments. With the heightened interest in fluorescence lifetime-based small animal imaging using NIR fluorophores, this technique further serves as a bridge between in vitro spectroscopic characterization of new fluorophore lifetimes and in vivo tissue imaging.     

Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation

A scanning microscope utilizing two-photon excitation in combination with fluorescence lifetime contrast is presented. The microscope makes use of a tunable femtosecond titanium:sapphire laser enabling the two-photon excitation of a broad range of fluorescent molecules, including UV probes. Importantly, the penetration depth of the two-photon exciting (infra)red light is substantially greater than for the corresponding single-photon wavelength while photobleaching is significantly reduced. The time structure of the Ti:Sa laser can be employed in a straightforward way for the realization of fluorescence lifetime imaging. The fluorescence lifetime is sensitive to the local environment of the fluorescent molecule. This behaviour can be used for example to quantify concentrations of ions, such as pH and Ca²⁺, or pO₂ and pCO₂. In the set-up presented here the fluorescence lifetime imaging is accomplished by time-gated single photon counting. The performance and optical properties of the microscope are investigated by a number of test measurements on fluorescent test beads. Point-spread functions calculated from measurements on 230-nm beads using an iterative restoration procedure compare well with theoretical expectations. Lifetime imaging experiments on a test target containing two different types of test bead in a fluorescent buffer all with different lifetimes (2.15 ns, 2.56 ns and 3.34 ns) show excellent quantitative agreement with reference values obtained from time correlated single photon counting measurements. Moreover, the standard deviation in the results can be wholly ascribed to the photon statistics. Measurements of acridine orange stained biofilms are presented as an example of the potential of two-photon excitation combined with fluorescence lifetime contrast. Fluorescence lifetime and intensity images were recorded over the whole sample depth of 100 μm. Fluorescence intensity imaging is seriously hampered by the rapid decrease of the fluorescence signal as a function of the depth into the sample. Fluorescence lifetime imaging on the other hand is not affected by the decrease of the fluorescence intensity.     

Double-pulse fluorescence lifetime measurements

It is demonstrated that fluorescence lifetimes in the nanosecond and picosecond time-scale range can be observed with the recently proposed double-pulse fluorescence lifetime imaging technique (Müller et al. , 1995, Double-pulse fluorescence lifetime imaging in confocal microscopy. J. Microsc 177, 171–179).
A laser source with an optical parametric amplifier (OPA) system is used to obtain short pulse durations needed for high time resolution, wavelength tunability for selective excitation of specific fluorophores and high pulse energies to obtain (partial) saturation of the optical transition.
It is shown that fluorescence lifetimes can be determined correctly also with nonuniform saturation conditions over the observation area.
A correction scheme for the effect on the measurements of laser power fluctuations, which are inherently present in OPA systems, is presented. Measurements on bulk solutions of Rhodamine B and Rhodamine 6G in different solvents confirm the experimental feasibility of accessing short fluorescence lifetimes with this technique.
Because signal detection does not require fast electronics, the technique can be readily used for fluorescence lifetime imaging in confocal microscopy, especially when using bilateral scanning and cooled CCD detection.     

Filter cubes with built-in ultrabright light-emitting diodes as exchangeable excitation light sources in fluorescence microscopy

The use of ultrabright light‐emitting diodes as a potential substitute for conventional excitation light sources in fluorescence microscopy is demonstrated. We integrated ultrabright light‐emitting diodes in the filter block of a conventional fluorescence microscope together with a collimating Fresnel lens, a holographic diffuser and emission filters. This setup enabled convenient changes between different excitation light sources and resulted in high excitation efficiencies. Quantitative comparison of image intensities of test samples revealed that light‐emitting diodes yielded intensities in the range of a mercury arc lamp depending on the wavelength. The use of ultrabright light‐emitting diodes also enabled luminescence lifetime imaging without the need for image intensification.     

Frequency domain lifetime and spectral imaging microscopy

In the femtoliter observation volume of a two-photon microscope, multiple fluorophores can be present and complex photophysics can take place. Combined detection of the fluorescence emission spectra and lifetimes can provide deeper insight into specimen properties than these two imaging modalities taken separately. Therefore, we have developed a detection scheme based on a frequency-modulated multichannel photomultiplier, which measures simultaneously the spectrum and the lifetime of the emitted fluorescence. Experimentally, the efficiency of the frequency domain lifetime measurement was compared to a time domain set-up. The performance of this spectrally and lifetime-resolved microscope was evaluated on reference specimens and living cells labeled with three different stains targeting the membrane, the mitochondria, and the nucleus.     

Spectra and lifetimes of fluorescence resonance energy transfer fluorophores under two-photon excitation

We show two-photon spectra and lifetimes acquired using conventional confocal microscopes equipped with an ultra-short pulsed laser and a time-gated intensified charge coupled device. We report on the two-photon spectra and lifetimes of Alexa350, enhanced green fluorescent protein (EGFP), EGFP-CD46, and Cy3 labelled antibodies. Cellular and extracellular EGFP two-photon spectra and lifetimes are compared.     

Time-domain whole-field fluorescence lifetime imaging with optical sectioning

A whole-field time-domain fluorescence lifetime imaging (FLIM) microscope with the capability to perform optical sectioning is described. The excitation source is a mode-locked Ti:Sapphire laser that is regeneratively amplified and frequency doubled to 415 nm. Time-gated fluorescence intensity images at increasing delays after excitation are acquired using a gated microchannel plate image intensifier combined with an intensified CCD camera. By fitting a single or multiple exponential decay to each pixel in the field of view of the time-gated images, 2-D FLIM maps are obtained for each component of the fluorescence lifetime. This FLIM instrument was demonstrated to exhibit a temporal discrimination of better than 10 ps. It has been applied to chemically specific imaging, quantitative imaging of concentration ratios of mixed fluorophores and quantitative imaging of perturbations to fluorophore environment. Initially, standard fluorescent dyes were studied and then this FLIM microscope was applied to the imaging of biological tissue, successfully contrasting different tissues and different states of tissue using autofluorescence. To demonstrate the potential for real-world applications, the FLIM microscope has been configured using potentially compact, portable and low cost all-solid-state diode-pumped laser technology. Whole-field FLIM with optical sectioning (3D FLIM) has been realized using a structured illumination technique.     

基于PCA的时间分辨油荧光光谱分析及优化

激光诱导荧光技术可广泛应用于油污染的监测中,然而普通的油荧光光谱技术只能实现油污染监测的粗分类,无法区分原油与燃料油的荧光特征。本文基于主成分分析方法(PCA)的时间分辨油荧光分类方法,实验测量了20种油样本的时间分辨荧光光谱特征,给出了对应的荧光寿命和时间分辨油荧光光谱的时序特征。在此基础上,利用前三个主成分构成的三维特征矢量空间,通过分析不同采集时刻下油样本矢量间相关距离的变化,对油样本的时间分辨荧光光谱进行聚类分析。为了体现油荧光变化的时序性,引入矢量距离的离散度参量,提出基于PCA进行时间分辨油荧光光谱分析的优化方法。实验结果表明,基于时间分辨油荧光光谱识别可实现原油与燃料油的光谱时序特征区分,具备良好的油荧光分类效果。     

Frequency-domain fluorescence microscopy with the LED as a light source

We describe a frequency-domain lifetime fluorometer based on a microscope and a modulated light-emitting diode (LED) excitation source (370/460 nm), which operates in the frequency range 120 Hz–250 MHz. We collected multifrequency phase and modulation fluorescence responses from cellular areas as small as 10–15 µm in diameter. We also collected fluorescence lifetime data from cells stained by a lipophilic coumarin sensitized europium fluorophore, Coum-Eu, with a millisecond lifetime, and Ru(bpy)₂phe-C₁₂, with microsecond lifetime. Nanosecond lifetimes from native nuclei stained with SYTO 14 and SYTO 16 probes were measured as well. We demonstrate that a simple LED excitation source can, for many applications, successfully replace complex and expensive laser systems, which have been used for cellular frequency-domain lifetime measurements. As the LEDs are very stable with low noise, it will be possible to image even smaller sample areas using brighter LEDs. With availability of modulated LEDs emitting at several wavelengths covering almost the entire visible spectrum it is easy to assemble a system for the fluorophore of choice. The ability to select an excitation source for a given fluorophore and low price make such an excitation source even more practical.