Frequency-domain Fluorescence Lifetime Imaging Microscopy Research Articles

Combination of a spinning disc confocal unit with frequency‐domain fluorescence lifetime imaging microscopy

Wide-field frequency-domain fluorescence lifetime imaging microscopy (FLIM) is an established technique to determine fluorescence lifetimes. Disadvantage of wide-field imaging is that measurements are compromised by out-of-focus blur. Conventional scanning confocal typically means long acquisition times and more photo bleaching. An alternative is spinning-disc confocal whereby samples are scanned simultaneously by thousands of pinholes, resulting in a virtually instantaneous image with more than tenfold reduced photo bleaching. A spinning disc unit was integrated into an existing FLIM system. Measurements were made of fluorescent beads with a lifetime of 2.2 ns against a 5.3 ns fluorescent background outside the focal plane. In addition, living HeLa cells were imaged with different lifetimes in the cytosol and the plasma membrane. In spinning-disc mode, a lifetime of the beads of 2.8 ns was measured, whereas in wide field a lifetime of 4.1 ns was measured. Lifetime contrast within living HeLa cells could be resolved with the spinning-disc unit, where this was impossible in wide field. Integration of a spinning-disc unit into a frequency-domain FLIM instrument considerably reduces artifacts, while maintaining the advantages of wide field. For FLIM on objects with 3D lifetime structure, spinning-disc is by far preferable over wide-field measurements.

Calibration of a wide‐field frequency‐domain fluorescence lifetime microscopy system using light emitting diodes as light sources

High brightness light emitting diodes are an inexpensive and versatile light source for wide-field frequency-domain fluorescence lifetime imaging microscopy. In this paper a full calibration of an LED based fluorescence lifetime imaging microscopy system is presented for the first time. A radio-frequency generator was used for simultaneous modulation of light emitting diode (LED) intensity and the gain of an intensified charge coupled device (CCD) camera. A homodyne detection scheme was employed to measure the demodulation and phase shift of the emitted fluorescence, from which phase and modulation lifetimes were determined at each image pixel. The system was characterized both in terms of its sensitivity to measure short lifetimes (500 ps to 4 ns), and its capability to distinguish image features with small lifetime differences. Calibration measurements were performed in quenched solutions containing Rhodamine 6G dye and the results compared to several independent measurements performed with other measurement methodologies, including time correlated single photon counting, time gated detection, and acousto optical modulator (AOM) based modulation of excitation sources. Results are presented from measurements and simulations. The effects of limited signal-to-noise ratios, baseline drifts and calibration errors are discussed in detail. The implications of limited modulation bandwidth of high brightness, large area LED devices ( approximately 40 MHz for devices used here) are presented. The results show that phase lifetime measurements are robust down to sub ns levels, whereas modulation lifetimes are prone to errors even at large signal-to-noise ratios. Strategies for optimizing measurement fidelity are discussed. Application of the fluorescence lifetime imaging microscopy system is illustrated with examples from studies of molecular mixing in microfluidic devices and targeted drug delivery research.

The application of frequency-domain Fluorescence Lifetime Imaging Microscopy as a quantitative analytical tool for microfluidic devices

We describe the application of wide-field frequency domain Fluorescence Lifetime Imaging Microscopy (FLIM) to imaging in microfluidic devices. FLIM is performed using low cost, intensity modulated Light Emitting Diodes (LEDs) for illumination. The use of lifetime imaging for quantitative analysis within such devices is demonstrated by mapping the molecular diffusion of iodide ions across a microchannel.

Innovating lifetime microscopy: a compact and simple tool for life sciences, screening, and diagnostics

Fluorescence lifetime imaging microscopy (FLIM) allows the investigation of the physicochemical environment of fluorochromes and protein-protein interaction mapping by Forster resonance energy transfer (FRET) in living cells. However, simpler and cheaper solutions are required before this powerful analytical technique finds a broader application in the life sciences. Wide-field frequency-domain FLIM represents a solution whose application is currently limited by the need for multichannel-plate image intensifiers. We recently showed the feasibility of using a charge-coupled device/complementory metal-oxide semiconductor (CCD/CMOS) hybrid lock-in imager, originally developed for 3-D vision, as an add-on device for lifetime measurements on existing wide-field microscopes. In the present work, the performance of the setup is validated by comparison with well-established wide-field frequency-domain FLIM measurements. Furthermore, we combine the lock-in imager with solid-state light sources. This results in a simple, inexpensive, and compact FLIM system, operating at a video rate and capable of single-shot acquisition by virtue of the unique parallel retrieval of two phase-dependent images. This novel FLIM setup is used for cellular and FRET imaging, and for high-throughput and fast imaging applications. The all-solid-state design bridges the technological gap that limits the use of FLIM in areas such as drug discovery and medical diagnostics.

Suppression of photobleaching-induced artifacts in frequency-domain FLIM by permutation of the recording order.

Photobleaching can lead to significant errors in frequency-domain fluorescence lifetime imaging microscopy (FLIM). Existing correction methods for photobleaching require additional recordings and processing time and can result in additional noise. A method is introduced that suppresses the effects of photobleaching without the need for extra recordings or processing. Existing bleach correction methods and the method introduced in this report whereby the recording order of the phases is permuted were compared using numerical simulations. Certain orders were found to make measurements virtually insensitive to photobleaching. At 12 recordings, errors in measured phase and modulation depth decreased by a factor 512 and 393, respectively, compared to recordings using sequential recording order. The optimal order is independent of modulation depth, phase, and extent of photobleaching. Thus, the same order can be used for practically all situations. Application of the method in FLIM measurements of EYFP-transfected HeLa cells was found effectively to suppress photobleaching induced artifacts. In view of the ease of implementation, its inherent robustness, and the possibility to still apply existing correction methods afterward, there is no good reason not to use the permuted recording order presented in this report instead of a sequential order.

PhiFLIM: a new method to avoid aliasing in frequency-domain fluorescence lifetime imaging microscopy.

In conventional wide-field frequency-domain fluorescence lifetime imaging microscopy (FLIM), excitation light is intensity-modulated at megahertz frequencies. Emitted fluorescence is recorded by a CCD camera through an image intensifier, which is modulated at the same frequency. From images recorded at various phase differences between excitation and intensifier gain modulation, the phase and modulation depth of the emitted light is obtained. The fluorescence lifetime is determined from the delay and the decrease in modulation depth of the emission relative to the excitation. A minimum of three images is required, but in this case measurements become susceptible to aliasing caused by the presence of higher harmonics. Taking more images to avoid this is not always possible owing to phototoxicity or movement. A method is introduced, phiFLIM, requiring only three recordings that is not susceptible to aliasing. The phase difference between the excitation and the intensifier is scanned over the entire 360 degrees range following a predefined phase profile, during which the image produced by the intensifier is integrated onto the CCD camera, yielding a single image. Three different images are produced following this procedure, each with a different phase profile. Measurements were performed with a conventional wide-field frequency-domain FLIM system based on an acousto-optic modulator for modulation of the excitation and a microchannel-plate image intensifier coupled to a CCD camera for the detection. By analysis of the harmonic content of measured signals it was found that the third harmonic was effectively the highest present. Using the conventional method with three recordings, phase errors due to aliasing of up to +/- 29 degrees and modulation depth errors of up to 30% were found. Errors in lifetimes of YFP-transfected HeLa cells were as high as 100%. With phiFLIM, using the same specimen and settings, systematic errors due to aliasing did not occur.

Fluorescence lifetime‐resolved pH imaging of living cells

The regulation and maintenance of intracellular pH are critical to diverse metabolic functions of the living cells. Fluorescence time-resolved techniques and instrumentations have advanced rapidly and enabled the imaging of intracellular pH based on the fluorescence lifetimes. The frequency-domain fluorescence lifetime imaging microscopy (FLIM) and fluorophores displaying appropriate pH-dependent lifetime sensitivities were used to determine the temporal and spatial pH distributions in the cytosol and vesicular compartment lysosomes. We found that cytosolic pH levels are different in 3T3 fibroblasts, Chinese hamster ovary (CHO) cells, and MCF-7 cells when using the pH probe carboxy-SNAFL2. We also tracked the transient cytosolic pH changes in the living CHO cells after treatments with proton pump inhibitors, ion exchanger inhibitors, and weak base and acid. The intracellular lysosomal pH was determined with the acidic lifetime probes DM-NERF dextrans, OG-514 carboxylic acid dextrans, and LysoSensor DND-160. Our results showed that the resting lysosomal pH value obtained from the 3T3 fibroblasts was between 4.5 and 4.9. The increase of lysosomal pH induced by the treatments with proton pump inhibitor and ionophores also were observed in our FLIM measurements. Our lifetime-based pH imaging data suggested that FLIM can measure the intracellular pH of the resting cells and follow the pH fluctuations inside the cells after environmental perturbations. To improve the z-axis resolution to the intracellular lifetime-resolved images, we are investigating the implementation of the pseudo-confocal capability to our current FLIM apparatus.

Dynamic Fluorescence Anisotropy Imaging Microscopy inthe Frequency Domain (rFLIM)

We describe a novel variant of fluorescence lifetime imaging microscopy (FLIM), denoted anisotropy-FLIM or rFLIM, which enables the wide-field measurement of the anisotropy decay of fluorophores on a pixel-by-pixel basis. We adapted existing frequency-domain FLIM technology for rFLIM by introducing linear polarizers in the excitation and emission paths. The phase delay and intensity ratios (AC and DC) between the polarized components of the fluorescence signal are recorded, leading to estimations of rotational correlation times and limiting anisotropies. Theory is developed that allows all the parameters of the hindered rotator model to be extracted from measurements carried out at a single modulation frequency. Two-dimensional image detection with a sensitive CCD camera provides wide-field imaging of dynamic depolarization with parallel interrogation of different compartments of a complex biological structure such as a cell. The concepts and technique of rFLIM are illustrated with a fluorophore-solvent (fluorescein-glycerol) system as a model for isotropic rotational dynamics and with bacteria expressing enhanced green fluorescent protein (EGFP) exhibiting depolarization due to homotransfer of electronic excitation energy (emFRET). The frequency-domain formalism was extended to cover the phenomenon of emFRET and yielded data consistent with a concentration depolarization mechanism resulting from the high intracellular concentration of EGFP. These investigations establish rFLIM as a powerful tool for cellular imaging based on rotational dynamics and molecular proximity.

Global Analysis of Fluorescence Lifetime Imaging Microscopy Data

Global analysis techniques are described for frequency domain fluorescence lifetime imaging microscopy (FLIM) data. These algorithms exploit the prior knowledge that only a limited number of fluorescent molecule species whose lifetimes do not vary spatially are present in the sample. Two approaches to implementing the lifetime invariance constraint are described. In the lifetime invariant fit method, each image in the lifetime image sequence is spatially averaged to obtain an improved signal-to-noise ratio. The lifetime estimations from these averaged data are used to recover the fractional contribution to the steady-state fluorescence on a pixel-by-pixel basis for each species. The second, superior, approach uses a global analysis technique that simultaneously fits the fractional contributions in all pixels and the spatially invariant lifetimes. In frequency domain FLIM the maximum number of lifetimes that can be fit with the global analysis method is twice the number of lifetimes that can be fit with conventional approaches. As a result, it is possible to discern two lifetimes with a single-frequency FLIM setup. The algorithms were tested on simulated data and then applied to separate the cellular distributions of coexpressed green fluorescent proteins in living cells.

Fluorescence lifetime imaging of nuclear DNA: effect of fluorescence resonance energy transfer.

DNA fluorescence dyes have been used to study DNA dynamics, chromatin structure, and cell cycle analysis. However, most microscopic fluorescence studies of DNA use only steady-state measurements and do not take advantage of the additional information content of the time-resolved fluorescence. In this paper, we combine fluorescence imaging of DNA with time-resolved measurements to examine the proximity of donors and acceptors bound to chromatin. We used frequency-domain fluorescence lifetime imaging microscopy to study the spatial distribution of DNA-bound donors and acceptors in fixed 3T3 nuclei. Over 50 cell nuclei were imaged in the presence of an AT-specific donor, Hoechst 33258 (Ho), and a GC-specific acceptor, 7-aminoactinomycin D (7-AAD). The intensity images of Ho alone showed a spatially irregular distribution due to the various concentrations of DNA or AT-rich DNA throughout the nuclei. The lifetime imaging of the Ho-stained nuclei was typically flat. Addition of 7-AAD decreased the fluorescence intensity and lifetime of the Ho-stained DNA. The spatially dependent phase and modulation values of Ho in the presence of 7-AAD showed that the Ho decay becomes nonexponential, as is expected for a resonance energy transfer (RET) with multiple acceptors located over a range of distances. In approximately 40 nuclei, the intensity and lifetime decrease was spatially homogeneous. In approximately 10 nuclei, addition of 7-AAD resulted in a spatially nonhomogeneous decrease in intensity and lifetime. The RET efficiency was higher in G(2)/M than in G(0/1) phase cells. Because RET efficiency depends on the average distance between Ho and 7-AAD, data suggest that the heterogeneity of lifetimes and spatial variation of the RET efficiency are caused by the presence of highly condensed regions of DNA in nuclei.

Construction and characterization of a frequency-domain fluorescence lifetime imaging microscopy system

The construction of a homodyne frequency domain fluorescence lifetime imaging microscope is described. The system consists of (i) an intensity-modulated laser excitation source, (ii) an epifluorescence microscope, (iii) a gain-modulated microchannel plate (MCP) image intensifier, and (iv) a slow-scan CCD camera. The phase and modulation homogeneity of the MCP image intensifier were determined at frequencies of 40, 100, 160, and 240 MHz. The detected modulation depths were 65, 52, 32, and 23%, respectively, and were highly homogeneously distributed. The phasedistribution image revealed iris effects at frequencies of 160 and 240 MHz but was homogeneous at lower frequencies. Lifetime imaging of a solution of the fluorescent flavoprotein lipoamide dehydrogenase demonstrated (i) the accuracy of the determined lifetimes (< 60 ps), (ii) the time resolution of the instrument (< 50 ps), and (iii) the average precision for single pixel fluorescence lifetimes (50 ps is feasible). The imaging of tiny fluorescent microspheres revealed that even in a volume of 0.3 x 10-15 L, the standard error in the lifetimes can be as low as 79 ps. The spatial resolution of the instrument is estimated to be < 400 nm in the object plane at a 100 x magnification.