Frequency-domain Fluorescence Lifetime Imaging Microscopy System Research Articles

Widefield multifrequency fluorescence lifetime imaging using a two-tap complementary metal-oxide semiconductor camera with lateral electric field charge modulators.

Widefield frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) measures the fluorescence lifetime of entire images in a fast and efficient manner. We report a widefield FD-FLIM system based on a complementary metal-oxide semiconductor camera equipped with two-tap true correlated double sampling lock-in pixels and lateral electric field charge modulators. Owing to the fast intrinsic response and modulation of the camera, our system allows parallel multifrequency FLIM in one measurement via fast Fourier transform. We demonstrate that at a fundamental frequency of 20 MHz, 31-harmonics can be measured with 64 phase images per laser repetition period. As a proof of principle, we analyzed cells transfected with Cerulean and with a construct of Cerulean-Venus that shows Förster Resonance Energy Transfer at different modulation frequencies. We also tracked the temperature change of living cells via the fluorescence lifetime of Rhodamine B at different frequencies. These results indicate that our widefield multifrequency FD-FLIM system is a valuable tool in the biomedical field.

Widefield frequency domain fluorescence lifetime imaging microscopy (FD-FLIM) for accurate measurement of oxygen gradients within microfluidic devices.

An oxygen gradient is a key variable influencing various biological activities in vivo, such as tissue repair and tumor growth. To study the phenomenon, in vitro cell studies using microfluidic devices capable of generating oxygen gradients have been developed recently. However, it is challenging to accurately measure the gradient profiles in devices. The traditional fluorescence intensity-based method suffers from the difficulty of accurate measurement due to background fluorescence artefacts. In addition, it is hard to obtain accurate calibration conditions because of the difficulties to achieve a fully depleted and saturated oxygen concentrations in the devices. To overcome these difficulties, a widefield frequency domain fluorescence imaging microscopy (FD-FLIM) system was constructed and utilized to accurately measure oxygen gradient profiles in a microfluidic device in this paper. Since lifetime-based measurements do not solely depend on intensity variations, oxygen calibration processes are amiable and the measured oxygen concentrations can be more accurate. The performance of the FD-FLIM system was validated by comparing the experimental and simulation results in microfluidic devices with different geometries. The experimental results show that the oxygen gradients generated from the chemical reaction method can provide more hypoxic oxygen conditions compared to the gradients created by the gas flowing method. Owing to the advantages provided by the widefield microscopy technique, the image acquisition time can be significantly reduced resulting in less photobleaching for time-lapsed imaging applications. Consequently, the measurement technique developed in this paper is an efficient tool, which can greatly help scientists to better study biological activities under various oxygen conditions.

Modulated electron-multiplied fluorescence lifetime imaging microscope: all-solid-state camera for fluorescence lifetime imaging

We have built an all-solid-state camera that is directly modulated at the pixel level for frequency-domain fluorescence lifetime imaging microscopy (FLIM) measurements. This novel camera eliminates the need for an image intensifier through the use of an application-specific charge coupled device design in a frequency-domain FLIM system. The first stage of evaluation for the camera has been carried out. Camera characteristics such as noise distribution, dark current influence, camera gain, sampling density, sensitivity, linearity of photometric response, and optical transfer function have been studied through experiments. We are able to do lifetime measurement using our modulated, electron-multiplied fluorescence lifetime imaging microscope (MEM-FLIM) camera for various objects, e.g., fluorescein solution, fixed green fluorescent protein (GFP) cells, and GFP-actin stained live cells. A detailed comparison of a conventional microchannel plate (MCP)-based FLIM system and the MEM-FLIM system is presented. The MEM-FLIM camera shows higher resolution and a better image quality. The MEM-FLIM camera provides a new opportunity for performing frequency-domain FLIM.

Monitoring biosensor activity in living cells with fluorescence lifetime imaging microscopy.

Live-cell microscopy is now routinely used to monitor the activities of the genetically encoded biosensor proteins that are designed to directly measure specific cell signaling events inside cells, tissues, or organisms. Most fluorescent biosensor proteins rely on Förster resonance energy transfer (FRET) to report conformational changes in the protein that occur in response to signaling events, and this is commonly measured with intensity-based ratiometric imaging methods. An alternative method for monitoring the activities of the FRET-based biosensor proteins is fluorescence lifetime imaging microscopy (FLIM). FLIM measurements are made in the time domain, and are not affected by factors that commonly limit intensity measurements. In this review, we describe the use of the digital frequency domain (FD) FLIM method for the analysis of FRET signals. We illustrate the methods necessary for the calibration of the FD FLIM system, and demonstrate the analysis of data obtained from cells expressing “FRET standard” fusion proteins. We then use the FLIM-FRET approach to monitor the changes in activities of two different biosensor proteins in specific regions of single living cells. Importantly, the factors required for the accurate determination and reproducibility of lifetime measurements are described in detail.

Photon budget analysis for fluorescence lifetime imaging microscopy

We have constructed a mathematical model to analyze the photon efficiency of frequency-domain fluorescence lifetime imaging microscopy (FLIM). The power of the light source needed for illumination in a FLIM system and the signal-to-noise ratio of the detector have led us to a photon "budget." These measures are relevant to many fluorescence microscope users and the results are not restricted to FLIM but applicable to widefield fluorescence microscopy in general. Limitations in photon numbers, however, are more of an issue with FLIM compared to other less quantitative types of imaging. By modeling a typical experimental configuration, examples are given for fluorophores whose absorption peaks span the visible spectrum from Fura-2 to Cy5. We have performed experiments to validate the assumptions and parameters used in our mathematical model. The influence of fluorophore concentration on the intensity of the fluorescence emission light and the Poisson distribution assumption of the detected fluorescence emission light have been validated. The experimental results agree well with the mathematical model. This photon budget is important in order to characterize the constraints involved in current fluorescent microscope systems that are used for lifetime as well as intensity measurements and to design and fabricate new systems.

Optimized protocol of a frequency domain fluorescence lifetime imaging microscope for FRET measurements

Frequency-domain fluorescence lifetime imaging microscopy (FLIM) has become a commonly used technique to measure lifetimes in biological systems. However, lifetime measurements are strongly dependent on numerous experimental parameters. Here, we describe a complete calibration and characterization of a FLIM system and suggest parameter optimization for minimizing measurement errors during acquisition. We used standard fluorescent molecules and reference biological samples, exhibiting both single and multiple lifetime components, to calibrate and evaluate our frequency domain FLIM system. We identify several sources of lifetime precision degradation that may occur in FLIM measurements. Following a rigorous calibration of the system and a careful optimization of the acquisition parameters, we demonstrate fluorescence lifetime measurements accuracy and reliability. In addition, we show its potential on living cells by visualizing FRET in CHO cells. The proposed calibration and optimization protocol is suitable for the measurement of multiple lifetime components sample and is applicable to any frequency domain FLIM system. Using this method on our FLIM microscope enabled us to obtain the best fluorescence lifetime precision accessible with such a system.

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.

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.