Three-dimensional localisation of fluorescence resonance energy transfer in living cells under two-photon excitation

Three-dimensional (3-D) imaging of fluorescence resonance energy transfer (FRET) in human cells under two-photon excitation was demonstrated in this study. A sample was prepared by expressing a donor and an acceptor in living cells and using an antibody to secure the proximity of contact between the donor and the acceptor. The quenching of fluorescence emission of a donor in the double-labelled cells indicates the presence of FRET that occurred in these living cells. Because of the quadratic relation of the excitation power, 3-D localisation of FRET becomes possible.     

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。     

Cell-permeant cytoplasmic blue fluorophores optimized for in vivo two-photon microscopy with low-power excitation

Because of the spreading of nonlinear microscopies in biology, there is a strong demand for specifically engineered probes in these applications. Herein, we report on the imaging properties in living cells and nude mice brains of recently developed water soluble blue fluorophores that show efficient diffusion through cell membranes and blood-brain barriers. They are characterized by two-photon absorption cross-sections of 100-150 Goeppert-Mayer range in the near IR and fluorescence efficiencies of up to 72% in water. They were found to stain homogeneously the cytoplasm of cultured living cells within minutes. Moreover, their diffusion times and fluorescence characteristics in the cytoplasm suggest a hydrophobic association with intracellular membranes. Their intracellular fluorescent decays were found to be almost mono-exponential, a very favorable feature for fluorescence lifetime imaging. Two photon images of living cells were obtained with a good signal to noise ratio using laser powers in the sub-milliwatt range. This allows continuous imaging without significant photobleaching for tens of minutes. In addition, these fluorophores allowed in vivo three-dimensional two-photon imaging of mice cortex vasculatures and extra vasculature structures, with no sign of toxicity.     

Effects of donor and acceptor's fluorescence lifetimes on the method of applying Förster resonance energy transfer in STED microscopy

Förster resonance energy transfer (FRET) probes being used to improve the resolution of stimulated emission depletion (STED) microscopy are numerically discussed. Besides the FRET efficiency and the excitation intensity, the fluorescence lifetimes of donor and acceptor are found to be another key parameter for the resolution enhancement. Using samples of FRET pairs with shorter donor lifetime and longer acceptor lifetime enhances the nonlinearity of the donor fluorescence, which leads to an increased resolution. The numerical simulation shows that a double resolution improvement of STED microscopy can be achieved by using Cy3–Atto647N samples when compared with that of using standard Cy3‐only samples.     

Real-time cellular uptake of serotonin using fluorescence lifetime imaging with two-photon excitation

The real-time uptake of serotonin, a neurotransmitter, by rat leukemia mast cell line RBL-2H3 and 5-hydroxytryptophan by Chinese hamster V79 cells has been studied by fluorescence lifetime imaging microscopy (FLIM), monitoring ultraviolet (340 nm) fluorescence induced by two-photon subpicosecond 630 nm excitation. Comparison with two-photon excitation with 590 nm photons or by three-photon excitation at 740 nm shows that the use of 630 nm excitation provides optimal signal intensity and lowered background from auto-fluorescence of other cellular components. In intact cells, we observe using FLIM three distinct fluorescence lifetimes of serotonin and 5-hydroxytryptophan according to location. The normal fluorescence lifetimes of both serotonin (3.8 ns) and 5-hydroxytryptophan (3.5 ns) in solution are reduced to approximately 2.5 ns immediately on uptake into the cell cytosol. The lifetime of internalized serotonin in RBL-2H3 cells is further reduced to approximately 2.0 ns when stored within secretory vesicles.     

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.     

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.     

The effects of spherical aberration on multiphoton fluorescence excitation microscopy

Multiphoton fluorescence excitation microscopy is almost invariably conducted with samples whose refractive index differ from that of the objective immersion medium, conditions that cause spherical aberration. Due to the quadratic nature of multiphoton fluorescence excitation, spherical aberration is expected to profoundly affect the depth dependence of fluorescence excitation. In order to determine the effect of refractive index mismatch in multiphoton fluorescence excitation microscopy, we measured signal attenuation, photobleaching rates and resolution degradation with depth in homogeneous samples with minimal light scattering and absorption over a range of refractive indices. These studies demonstrate that signal levels and resolution both rapidly decline with depth into refractive index mismatched samples. Analyses of photobleaching rates indicate that the preponderance of signal attenuation with depth results from decreased rates of fluorescence excitation, even in a system with a descanned emission collection pathway. Similar results were obtained in analyses of fluorescence microspheres embedded in rat kidney tissue, demonstrating that spherical aberration is an important limiting factor in multiphoton fluorescence excitation microscopy of biological samples.     

Multiphoton microscopic imaging of adipose tissue based on second-harmonic generation and two-photon excited fluorescence

The fresh adipose tissue was investigated by the use of multiphoton microscopy (MPM) based on two-photon excited fluorescence and second-harmonic generation (SHG). Microstructure of collagen and adipose cells in the adipose tissue is clearly imaged at a subcellular level with the excitation light wavelengths of 850 and 730 nm, respectively. The emission spectrum of collagen SHG signal and NADH and FAD fluorescence signal can also be obtained, which can be used to quantify the content of collagen and adipose cells and reflect the degree of pathological changes when comparing normal tissue with abnormal adipose tissue in the same condition. The results indicate that MPM has the potential to be applied to investigate the adipose tissue and can be used in the research field of lipid and connective tissues.     

Chromatic two-photon excitation fluorescence imaging

We report on a chromatic axial scanning method for two-photon excitation fluorescence imaging. Effective axial scanning is achieved by incorporating a Fresnel lens in the system, which has large chromatic aberration and can therefore focus the excitation beam to different axial positions depending on its wavelength. We experimentally demonstrated this technique and used it to image the cross-section of fluorescent microspheres.     

Imaging FRET standards by steady-state fluorescence and lifetime methods

Imaging fluorescence resonance energy transfer (FRET) between molecules labeled with fluorescent proteins is emerging as a powerful tool to study changes in ions, ligands, and molecular interactions in their physiological cellular environment. Different methods use either steady-state fluorescence properties or lifetime to quantify the FRET rate. In addition, some provide the absolute FRET efficiency whereas others are simply a relative index very much influenced by the actual settings and instrumentation used, which makes the interpretation of a given FRET rate very difficult. The use and exchange of FRET standards in laboratories using these techniques would help to overcome this drawback. We report here the construction and systematic evaluation of FRET standard probes of varying FRET efficiencies. The standards for intramolecular FRET were protein fusions of the cyan and yellow variants of A. victoria green fluorescent protein (ECFP and citrine) joined by short linkers or larger protein spacers, or ECFP tagged with a tetracysteine motif and labeled with the biarsenical fluorochrome, FlAsH. Negative and positive controls of intermolecular FRET were also used. We compared these FRET standards with up to four FRET quantification methods: ratioing of acceptor to donor emission, donor intensity recovery upon acceptor photobleach, sensitized emission after spectral unmixing of raw images, and fluorescence lifetime imaging (FLIM). The latter was obtained with a frequency-domain setup able to provide high quality lifetime images in less than a second, and is thus very well suited for live cell studies. The FRET rates or indexes of the standards were in good agreement regardless of the method used. For the CFP-tetraCys/FlAsH pair, the rate calculated from CFP quenching was faster than that obtained by FLIM.     

Compact multiphoton/single photon laser scanning microscope for spectral imaging and fluorescence lifetime imaging

An inverted fluorescence microscope was upgraded into a compact three-dimensional laser scanning microscope (LSM) of 65 x 62 x 48 cm dimensions by means of a fast kHz galvoscanner unit, a piezodriven z-stage, and a picosecond (ps) 50 MHz laser diode at 405 nm. In addition, compact turn-key near infrared femtosecond lasers have been employed to perform multiphoton fluorescence and second harmonic generation (SHG) microscopy. To expand the features of the compact LSM, a time-correlated single photon counting unit as well as a Sagnac interferometer have been added to realize fluorescence lifetime imaging (FLIM) and spectral imaging. Using this unique five-dimensional microscope, TauMap, single-photon excited (SPE), and two-photon excited (TPE) cellular fluorescence as well as intratissue autofluorescence of water plant leaves have been investigated with submicron spatial resolution, <270 ps temporal resolution, and 10 nm spectral resolution.     

Single- and two-photon spectral imaging of intrinsic fluorescence of transformed human hepatocytes

Autofluorescence (AF) originating from the cytoplasmic region of mammalian cells has been thoroughly investigated; however, AF from plasma membranes of viable intact cells is less well known, and has been mentioned only in a few older publications. Herein, we report results describing single- and two-photon spectral properties of a strong yellowish-green AF confined to the plasma-membrane region of transformed human hepatocytes (HepG2) grown in vitro as small three-dimensional aggregates or as monolayers. The excitation-emission characteristics of the membrane AF indicate that it may originate from a flavin derivative. Furthermore, the AF was closely associated with the plasma membranes of HepG2 cells, and its presence and intensity were dependent on cell metabolic state, membrane integrity and presence of reducing agents. This AF could be detected both in live intact cells and in formaldehyde-fixed cells.     

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.     

Wavelength division scanning for two-photon excitation fluorescence imaging

We investigate wavelength division scanning for two‐photon excitation fluorescence imaging. Two‐photon imaging using lateral wavelength division scanning is demonstrated. In addition, we theoretically analyse the spatial and temporal properties of a femtosecond laser beam focused by a Fresnel lens and investigate the feasibility of axial scanning using wavelength division.     

Fluorescence resonance energy transfer detected by scanning near-field optical microscopy

Fluorescence resonance energy transfer (FRET) between excited fluorescent donor and acceptor molecules occurs via the Förster mechanism over a range of 1–10 nm. Because of the strong (sixth power) distance dependence of the signal, FRET has been used to assess the proximity of molecules in biological systems. We used a scanning near-field optical microscope (SNOM) operated in the shared-aperture mode using uncoated glass fibre tips to detect FRET between dye molecules embedded in polyvinyl alcohol films and bound to cell surfaces. FRET was detected by selective photobleaching of donor and acceptor fluorophores. We also present preliminary results on pixel-by-pixel energy transfer efficiency measurements using SNOM.     

Harmonic optical microscopy and fluorescence lifetime imaging platform for multimodal imaging

In this work, we proposed and built a multimodal optical setup that extends a commercially available confocal microscope (Olympus VF300) to include nonlinear second harmonic generation (SHG) and third harmonic generation (THG) optical (NLO) microscopy and fluorescence lifetime imaging microscopy (FLIM). We explored all the flexibility offered by this commercial confocal microscope to include the nonlinear microscopy capabilities. The setup allows image acquisition with confocal, brightfield, NLO/multiphoton and FLIM imaging. Simultaneously, two‐photon excited fluorescence (TPEF) and SHG are well established in the biomedical imaging area, because one can use the same ultrafast laser and detectors set to acquire both signals simultaneously. Because the integration with FLIM requires a separated modulus, there are fewer reports of TPEF+SHG+FLIM in the literature. The lack of reports of a TPEF+SHG+THG+FLIM system is mainly due to difficulties with THG because the present NLO laser sources generate THG in an UV wavelength range incompatible with microscope optics. In this article, we report the development of an easy‐to‐operate platform capable to perform two‐photon fluorescence (TPFE), SHG, THG, and FLIM using a single 80 MHz femtosecond Ti:sapphire laser source. We described the modifications over the confocal system necessary to implement this integration and verified the presence of SHG and THG signals by several physical evidences. Finally, we demonstrated the use of this integrated system by acquiring images of vegetables and epithelial cancer biological samples. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.     

Robust approaches to quantitative ratiometric FRET imaging of CFP/YFP fluorophores under confocal microscopy

Ratiometric quantification of CFP/YFP FRET enables live-cell time-series detection of molecular interactions, without the need for acceptor photobleaching or specialized equipment for determining fluorescence lifetime. Although popular in widefield applications, its implementation on a confocal microscope, which would enable sub-cellular resolution, has met with limited success. Here, we characterize sources of optical variability (unique to the confocal context) that diminish the accuracy and reproducibility of ratiometric FRET determination and devise practical remedies. Remarkably, we find that the most popular configuration, which pairs an oil objective with a small pinhole aperture, results in intractable variability that could not be adequately corrected through any calibration procedure. By quantitatively comparing several imaging configurations and calibration procedures, we find that significant improvements can be achieved by combining a water objective and increased pinhole aperture with a uniform-dye calibration procedure. The combination of these methods permitted remarkably consistent quantification of sub-cellular FRET in live cells. Notably, this methodology can be readily implemented on a standard confocal instrument, and the dye calibration procedure yields a time savings over traditional live-cell calibration methods. In all, identification of key technical challenges and practical compensating solutions promise robust sub-cellular ratiometric FRET imaging under confocal microscopy.     

Effects of aberrations and specimen structure in conventional, confocal and two-photon fluorescence microscopy

Specimen-induced aberrations cause a reduction in signal levels and resolution in fluorescence microscopy. Aberrations also affect the image contrast achieved by these microscopes. We model the effects of aberrations on the fluorescence signals acquired from different specimen structures, such as point-like, linear, planar and volume structures, when imaged by conventional, confocal and two-photon microscopes. From this we derive the image contrast obtained when observing combinations of such structures. We show that the effect of aberrations on the visibility of fine features depends upon the specimen morphology and that the contrast is less significantly affected in microscopes exhibiting optical sectioning. For example, we show that point objects become indistinguishable from background fluorescence in the presence of aberrations, particularly when imaged in a conventional fluorescence microscope. This demonstrates the significant advantage of using confocal or two-photon microscopes over conventional instruments when aberrations are present.     

Scanning near-field optical microscopy using semiconductor nanocrystals as a local fluorescence and fluorescence resonance energy transfer source

Local fluorescence probes based on CdSe semiconductor nanocrystals were prepared and tested by recording scanning near‐field optical microscopy (SNOM) images of calibration samples and fluorescence resonance energy transfer SNOM (FRET SNOM) images of acceptor dye molecules inhomogeneously deposited onto a glass substrate. Thousands of nanocrystals contribute to the signal when this probe is used as a local fluorescence source while only tens of those (the most apical) are involved in imaging for the FRET SNOM operation mode. The dip‐coating method used to make the probe enables diminishing the number of active fluorescent nanocrystals easily. Prospects to realize FRET SNOM based on a single fluorescence centre using such an approach are briefly described.