2.1.

DNA Vector Preparation, Transfection, and Cell Culture

The plasmid encoding for ECFP (pECFP-C1) was obtained from Clontech (Clontech, Mountain View, California). pCerulean-C1 was designed by mutating pECFP-C1 at the following residues: S72A, Y145A, and H148D, according to Rizzo ¹⁶ The plasmid encoding the EYFP-Cerulean hybrid protein was constructed as described earlier,¹¹ and the plasmid encoding the EYFP-ECFP hybrid protein was created in the same way. Restriction and sequencing analysis confirmed the correctness of both fusion constructs.

HEK293 cells were transfected using the conventional calcium phosphate precipitation technique and grown on coverslips. For confocal imaging and lifetime measurements, the coverslips were transferred to a microscope chamber that was superfused with phosphate buffered saline solution (PBS) containing (in $\mathrm{mmol}{\mathrm{l}}^{-1}$ ): $\mathrm{NaCl}137$ , $\mathrm{KCl}2.7$ , ${\mathrm{Na}}_2{\mathrm{HPO}}_4$ $6.5$ , ${\mathrm{KH}}_2{\mathrm{PO}}_4$ $1.5$ , $\mathrm{pH}7.4$ .

2.2.

Isolation of Fluorescent Proteins

Fluorescent proteins were expressed as soluble N-terminal hexa-His-tag fusions under control of an isopropyl- $\beta$ -D-1-thiogalactopyranoside (IPTG) inducible promoter (pET16b vector) in E. coli BL21 (DE3). After cell lysis by ultrasonication, proteins were purified by metal chelate affinity chromatography (His-trap HP, Amersham), eluted with $250\mathrm{mM}$ imidazole, and dialyzed against a sodium phosphate buffer ( $50\mathrm{mM}$ ${\mathrm{NaH}}_2{\mathrm{PO}}_4$ , $300\mathrm{mM}$ $\mathrm{NaCl}$ , pH 7.4) and diluted to a final concentration of $20\mu \mathrm{M}$ .

To investigate the effects of irradiation on the isolated proteins, the diluted protein solution was filled into capillaries with an inner diameter of $150\mu \mathrm{m}$ (Hilgenberg, Malsfeld, Germany). The droplets were small enough so that they could be scanned completely by our laser scanning microscope (LSM). To prevent evaporation during the course of the experiment, the capillaries were sealed with modeling clay.

2.3.

Experimental Setup

A scheme of the setup used in this study is shown in Fig. 1a . It consists of a laser scanning microscope (LSM-510, Carl Zeiss $\mathrm{GmbH}$ , Jena, Germany), a polychromator (250is, Chromex Inc., Albuquerque, New Mexico), and a streak camera (C5680 with a M5677 sweep unit, Hamamatsu Photonics Deutschland $\mathrm{GmbH}$ , Herrsching, Germany). The sample was excited by ultrashort light pulses that were generated by a mode-locked Titanium:Sapphire laser (Mira 900, Coherent GmbH, Dieburg, Germany) pumped by a $5\mathrm{W}$ , continuous-wave, frequency-doubled $\mathrm{Nd}:{\mathrm{YVO}}_4$ laser ( $5\mathrm{W}$ Verdi, Coherent). For measurements in cells, a pulse picker (Model 9200, Coherent) was used to decrease the frequency of the laser pulses to $2\mathrm{MHz}$ , the maximum repetition rate of the single-sweep unit of our streak camera. Isolated fluorescent proteins in capillaries were, however, exposed to the full repetition rate of the Titanium:Sapphire laser $\left(78\mathrm{MHz}\right)$ , while streak images were still acquired with a repetition rate of $2\mathrm{MHz}$ . In this way, we could follow up the effects of irradiation in a reasonable period of time despite the relatively large volume of the solution in the capillaries.

Fig. 1

(a) Setup: A Titanium:Sapphire laser that is pumped by a $5\text{-}\mathrm{W}$ , frequency-doubled $\mathrm{Nd}:{\mathrm{YVO}}_4$ laser is used as pulsed excitation source. For experiments in cells (green lines), the repetition rate is decreased by a pulse picker to the maximal repetition rate $\left(2\mathrm{MHz}\right)$ of the streak camera. For experiments in capillaries, the pulse picker is bypassed (red lines), and the sample is exposed to the full repetition rate of the Titanium:Sapphire laser. The second harmonic is generated by a BBO crystal and directed into the scan head of the laser scanning microscope. Emitted fluorescence light is collected by the water immersion objective, fed back through the scan head, and guided to one of the confocal channels. Here, the emitted light is coupled into an optical fiber and directed to a polychromator, where it is dispersed along the horizontal axis according to its wavelength. The dispersed spectrum is focused onto the entrance slit of the streak camera. (b) Operating principle of a streak camera: The light pulse to be measured is focused onto the photocathode of the streak tube, where photons are converted to electrons. The photoelectrons are accelerated by the accelerating electrode, pass through a pair of deflection plates, are multiplied in a micro-channel plate (MCP), and hit the phosphor screen of the streak tube, where they are converted to an optical image, the so-called streak image. At the instant the photoelectrons pass through the deflection electrodes, a voltage ramp is applied so that the electrons are swept from top to bottom. Electrons generated at earlier times arrive at the phosphor screen in a position close to the top of the screen, while those electrons that are generated at later times arrive at a position close to the bottom of the screen. Hence, the time at which a photon left the sample can be determined by the vertical position of the photoelectron in the streak image. The horizontal position of the photoelectron depends on the wavelength of the incident light pulse, since a spectrograph was used to focus the spectrum onto the photocathode. The streak image is read out by a CCD camera and transferred to a computer. From the streak image, the fluorescence spectrum can be obtained by summing up fluorescence intensities along the time axis (vertical axis) and plotting the resulting intensities versus the wavelength (horizontal axis). Accordingly, the fluorescence decay curve can be obtained by summing the fluorescence intensities in the wavelength region of interest and plotting the resulting intensities versus the time axis. (Color online only.) [Figure reprinted with permission from Nature Biotechnology (Ref. 15).]

The second harmonic $\left(430\mathrm{nm}\right)$ of the pulse-picked or unmodified laser beam was generated by a $\beta$ -barium borate (BBO) crystal. The beam was directed to the scan head of the confocal microscope. Cells were imaged with a $40\times$ C-Apochromat water immersion objective (NA 1.20, Zeiss); capillaries were imaged with a $10\times$ C-Apochromat water immersion objective (NA 0.45, Zeiss).

To measure the laser power in the focal plane, the diffuser and the sensor cell of a commercial power meter head (FieldMaster with LM2 head, Coherent) were removed and attached to a coverslip, which could be mounted in the same type of chamber we used for the confocal measurements of living cells. The active area of the sensor was large enough to collect all the light exiting the pupil of the objective. Comparison with a calibrated power meter showed that the power measurement with the modified power head was accurate.

For experiments in cells, the intensity of the laser beam $\left(2\mathrm{MHz}\right)$ was attenuated by a neutral density filter so that the power in the focal plane was $1.0\mu \mathrm{W}$ . This beam was used to scan the sample continuously. The pixel dwell time was $6.4\mu \mathrm{s}$ . These settings correspond to settings that are used for fluorescence lifetime imaging experiments. For experiments in capillaries, the intensity of the laser beam $\left(78\mathrm{MHz}\right)$ was adjusted with the neutral density filter so that the power in the focal plane was $100\mu \mathrm{W}$ . The region of interest (ROI) that was chosen for bleaching had a height of 10 pixels $\left(18\mu \mathrm{m}\right)$ and a length that was equal to the full width of an image (512 pixel, $921.4\mu \mathrm{m}$ ). The ROI was kept constant in all experiments and always positioned in the middle of the capillary so that all experiments are comparable to each other. The droplets were small enough so that the full length of the droplet was always scanned and exposed to the Titanium:Sapphire laser beam, which excludes the possibility that the measurement was biased by axial diffusion of ECFP or Cerulean from unexposed parts of the capillaries [Fig. 3a]. The scanning speed was adjusted such that the pixel dwell time was $6.4\mu \mathrm{s}$ .

Fig. 3

Analysis of streak images obtained from isolated ECFP and Cerulean. (a) Confocal image of capillaries filled with purified ECFP (upper panel) and Cerulean solution (lower panel). (b) Streak images obtained from the capillaries shown in (a). (c) Fluorescence emission spectra of ECFP (upper panel) and Cerulean (lower panel) obtained in $5\text{-}\min$ time intervals (from top to bottom: 0 to $5\min$ : dark blue; 5 to $10\min$ : light blue; 10 to $15\min$ : cyan; 15 to $20\min$ green; 20 to $25\min$ : orange; 25 to $30\min$ : red). (d) Time course of the fluorescence intensity of ECFP (upper panel) and Cerulean (lower panel) in the wavelength range from 465 to $495\mathrm{nm}$ . (e) Fluorescence decay curves of ECFP (upper panel) and Cerulean (lower panel) in the wavelength range from $465\mathrm{nm}$ to $495\mathrm{nm}$ obtained in $5\text{-}\min$ time intervals. Colors are assigned as described earlier. All data have been processed as described in Fig. 2. (Color online only.)

In both in vivo and in vitro experiments, the fluorescence emitted by the sample was collected by the water immersion objective, fed back through the scan head, and guided to one of the confocal channels. Here, the emitted light was coupled into an optical fiber and directed to a polychromator. The polychromator spread the incident light along the horizontal axis and focused it on the entrance slit of the streak camera. The operation principle of the streak camera is explained in more detail in Fig. 1b.^{15, 17} In brief, incoming photons hit the photocathode, where they are converted to photoelectrons. The photoelectrons are accelerated by the accelerating electrode, pass through a pair of deflection plates, are multiplied in a multichannel plate (MCP) and hit a phosphor screen, where they are reconverted to an optical image, the so-called streak image. At the instant the photoelectrons pass through the deflection electrodes, a voltage ramp is applied so that the electrons are “swept” from top to bottom. Electrons leaving the photocathode at earlier times arrive at the phosphor screen in a position close to the top, whereas those electrons that leave the photocathode at a later time arrive at a position closer to the bottom. Thus, the vertical position of a spot depends on the time at which the photoelectron has left the photocathode; the horizontal position depends on the wavelength of the incident photon, because a spectrograph was used to focus the spectrum onto the streak-camera entrance slit. The streak images are read out by a CCD camera and transferred in $111\text{-}\mathrm{ms}$ intervals to a computer, where they are accumulated to yield the final photon counting streak images. In parallel, detected photons were recorded in dynamic photon counting (dpc) files, in which photons are saved with a time tag allowing for a detailed time-dependent analysis of both the spectrum and the fluorescence decays during the measurement period.

2.4.

Data Analysis

To visualize and evaluate changes, which occur during irradiation, streak images were reconstructed in $5\text{-}\min$ time intervals from the dpc files. From these streak images, fluorescence spectra and fluorescence decays were extracted, as shown in Fig. 1b. Fluorescence decays were evaluated using the “iterative reconvolution method” and a modified Gaussian-Newtonian algorithm to approximate the parameters of a mono- or biexponential model. All steps of the analysis were performed using our own software, which was developed using MATLAB (MathWorks, Natick, Massachusetts). The goodness of the fit was judged by the value of the reduced ${\chi }_v^2$ .

Since, in most cases, a biexponential model had to be chosen to fit the data adequately, we decided to fit all data to a biexponential model to better quantify the acceleration of the fluorescence decay upon irradiation. To facilitate the direct comparison of the data, amplitude-weighted mean lifetimes $\left({\tau }_m\right)$ were calculated from the parameters of the biexponential fit according to:

The FRET efficiency is defined as the fraction of energy absorbed by the donor that is transferred to the acceptor. It is given by the ratio between the energy transfer rate and the total fluorescence decay rate of the donor. It can be calculated by comparing the fluorescence intensity $\left(I_{DA}\right)$ or fluorescence lifetime $\left({\tau }_{DA}\right)$ of the donor in presence of an acceptor with the fluorescence intensity $\left(I_D\right)$ or fluorescence lifetime $\left({\tau }_D\right)$ of the donor in absence of an acceptor:

In an ideal scenario, the donor fluorophore employed in FRET experiments exhibits a monoexponential fluorescence decay. Then, in the presence of an interaction partner labeled with an appropriate acceptor fluorophore, one would observe a biexponential fluorescence decay, in which the fast lifetime component $\left({\tau }_f\right)$ could be attributed to the fraction of interacting donor molecules whose lifetime is quenched by FRET. The slow lifetime component $\left({\tau }_s\right)$ would be caused by free, noninteracting donor molecules and should be close to the control values obtained in absence of the acceptor.^{11, 19} In this case, the FRET efficiency of a donor-acceptor pair would have been calculated by relating the fast lifetime component of the biexponential fit to the donor fluorescence lifetime obtained in control measurements.

In this study, however, we show that fluorescence lifetimes of ECFP and Cerulean decrease upon prolonged irradiation and exhibit a pronounced biexponential fluorescence decay. To better describe the fluorescence decay and quantify the area under the decay curve, we used the amplitude weighted mean lifetimes $\left({\tau }_m\right)$ for calculating the “apparent” FRET efficiency. This approach is justified in so far as we do not investigate a mixed system of interacting and noninteracting donor molecules: Cells express either the donor molecules alone or hybrid proteins, in which donor and acceptor are covalently linked to each other. Thus, only one population of donor molecules exists. The one and only aim of this approach is to demonstrate the errors one can make when the observed decrease in the fluorescence lifetime is erroneously attributed to FRET.

3.1.

Streak Camera Measurements of ECFP and Cerulean

To investigate the changes in the photophysical properties of ECFP and Cerulean induced by irradiation, we expressed ECFP and Cerulean in HEK293 cells and scanned the cells continuously on the stage of a confocal microscope with the pulse-picked $\left(2\mathrm{MHz}\right)$ and frequency-doubled laser beam $(\lambda =430\mathrm{nm})$ . The intensity was attenuated by a neutral density filter so that the power in the focal plane was $1.0\mu \mathrm{W}$ . These settings correspond to settings that are used for fluorescence lifetime imaging experiments. Figure 2b shows streak images of ECFP (upper panel) and Cerulean (lower panel) that have been obtained during an acquisition period of $25\min$ . In addition to the streak images, so-called dynamic photon counting (dpc) files were recorded, in which each detected photon is saved with a time tag along with its wavelength and time coordinates. This information can be used to follow up in an offline analysis the total number of emitted photons as a function of time, or to recover streak images, fluorescence spectra, or fluorescence decays in any time intervals of interest. Figures 2c and 2e show an overlay of spectra and fluorescence decays, respectively, that have been reconstructed from the dpc data in $5\text{-}\min$ intervals. The overlay of the fluorescence spectra of ECFP [Fig. 2c, upper panel] shows that the amplitude of the fluorescence spectra decreased during the course of the experiment. Superposition of the normalized fluorescence decays [Fig. 2e, upper panel] shows that the fluorescence decay also underwent changes during the course of the experiment. It was accelerated upon prolonged irradiation. In the example shown, the mean lifetime decreased by 19% from ${\tau }_m\left(5\min \right)=2.29\mathrm{ns}$ to ${\tau }_m\left(10\min \right)=1.85\mathrm{ns}$ and further down to ${\tau }_m\left(25\min \right)=1.44\mathrm{ns}$ . In a series of eight experiments, the mean fluorescence lifetime decreased from $\overline{{\tau }_m}\left(5\min \right)=(2.33\pm 0.19)\mathrm{ns}$ to $\overline{{\tau }_m}\left(25\min \right)=(1.28\pm 0.22)\mathrm{ns}$ .

Fig. 2

Analysis of streak images obtained from HEK293 cells expressing ECFP and Cerulean. (a) Confocal image of HEK293 cells expressing ECFP (upper panel) and Cerulean (lower panel). (b) Streak images obtained from the cells shown in (a). Each dot in the streak image represents one photon that was detected during the measurement. The $x$ coordinate is determined by its wavelength, and the $y$ coordinate depends on the time after the excitation pulse. Counts per pixel are encoded by color ranging from black (no counts) through green and yellow to red (high count rate). (c) Fluorescence emission spectra of ECFP (upper panel) and Cerulean (lower panel) obtained in $5\text{-}\min$ time intervals (from top to bottom: 0 to $5\min$ : dark blue; 5 to $10\min$ : light blue; 10 to $15\min$ : cyan; 15 to $20\min$ : green; 20 to $25\min$ : orange). The fluorescence emission spectra have been reconstructed from the respective dpc files. All spectra have been normalized with respect to the peak of the respective emission spectrum. (d) Time course of the fluorescence intensity of ECFP (upper panel) and Cerulean (lower panel) in the wavelength range from $465\mathrm{nm}$ to $495\mathrm{nm}$ . Counts in the wavelength range from $465\mathrm{nm}$ to $495\mathrm{nm}$ have been binned in $1\text{-}\min$ intervals and normalized with respect to the highest count rate at the beginning of the recording. (e) Fit of the fluorescence decay curves of ECFP (upper panel) and Cerulean (lower panel) in the wavelength range from 465 to $495\mathrm{nm}$ obtained in $5\text{-}\min$ time intervals (from top to bottom: 0 to $5\min$ : dark blue; 5 to $10\min$ : light blue; 10 to $15\min$ : cyan; 15 to $20\min$ : green; 20 to $25\min$ : orange). The fluorescence decay curves have been reconstructed from the respective dpc files. To facilitate comparison of the decay kinetics, all curves have been normalized with respect to their maximum. (Color online only.)

Cerulean (Fig. 2, lower row) showed a similar behavior, although the changes in the fluorescence spectra [Fig. 2c, lower row] were not so pronounced as in the ECFP spectra. Also, the fluorescence decay curves were accelerated upon prolonged irradiation. In the example shown, the mean lifetime decreased by 22% from ${\tau }_m\left(5\min \right)=2.96\mathrm{ns}$ to ${\tau }_m\left(10\min \right)=2.32\mathrm{ns}$ and further down to ${\tau }_m\left(25\min \right)=1.71\mathrm{ns}$ . In a series of nine experiments, the mean fluorescence lifetime decreased from $\overline{{\tau }_m}\left(5\min \right)=(2.66\pm 0.13)\mathrm{ns}$ to $\overline{{\tau }_m}\left(25\min \right)=(1.52\pm 0.25)\mathrm{ns}$ .

These results could be confirmed for ECFP and Cerulean that were expressed as $N$ -terminal His-tag fusions in E. coli and purified by nickel affinity chromatography. The purified proteins were diluted with PBS to a final concentration of $20\mu \mathrm{M}$ . A small aliquot of this solution was filled into a capillary with an inner diameter of $150\mu \mathrm{m}$ and imaged on the stage of our confocal microscope. To better observe the effects of irradiation despite the bigger volume, the sample was exposed to the full repetition rate of the Titanium:Sapphire laser. The intensity was adjusted so that the power in the focal plane was $100\mu \mathrm{W}$ . The pixel dwell time was set to $6.4\mu \mathrm{s}$ . Again, ECFP exhibited a decrease in both fluorescence intensity and fluorescence lifetime upon irradiation (Fig. 3 , upper row). Here, the mean fluorescence lifetime decreased by 23% from ${\tau }_m\left(5\min \right)=2.20\mathrm{ns}$ to ${\tau }_m\left(30\min \right)=1.70\mathrm{ns}$ after $30\text{-}\min$ exposure. In a series of eight experiments, the mean fluorescence lifetime decreased from $\overline{{\tau }_m}\left(5\min \right)=(2.19\pm 0.11)\mathrm{ns}$ to $\overline{{\tau }_m}\left(30\min \right)=(1.64\pm 0.06)\mathrm{ns}$ .

Under the same conditions, Cerulean (Fig. 3, lower row) exhibits similar changes. In the example shown, the mean fluorescence lifetime changed by 23% from ${\tau }_m\left(5\min \right)=2.89\mathrm{ns}$ at the beginning to ${\tau }_m\left(30\min \right)=2.23\mathrm{ns}$ at the end of the experiment. In a series of nine experiments, the mean fluorescence lifetime decreased from $\overline{{\tau }_m}\left(5\min \right)=(2.86\pm 0.09)\mathrm{ns}$ to $\overline{{\tau }_m}\left(30\min \right)=(2.19\pm 0.18)\mathrm{ns}$ .

3.2.

Streak Camera Measurements of Hybrid Proteins

The fluorescence decays measured in cells or in the capillary show that ECFP and Cerulean are not ideal donor molecules in experiments, where FRET efficiency is monitored over a longer observation period: In intensity-based estimates of the FRET efficiency, the decrease in fluorescence intensity would be erroneously attributed to FRET. Likewise, the decrease in the ECFP lifetime might invalidate measurements of the donor fluorescence lifetime. To demonstrate how an estimate of FRET can be biased by prolonged irradiation, we constructed an EYFP–Cerulean hybrid protein, in which the donor fluorophore (e.g., ECFP or Cerulean) and the acceptor fluorophore (e.g., EYFP) were closely linked by a short sequence of 10 amino acids (see Sec. 2.1). Figure 4b shows the streak images obtained from Hek293 cells expressing these hybrid proteins. Comparison with the respective streak images from cells expressing only the donor molecules [Fig. 2b] shows that the donor (ECFP or Cerulean) fluorescence signal in the wavelength range between 465 and $495\mathrm{nm}$ is decreased, whereas the acceptor (EYFP) fluorescence between 510 and $540\mathrm{nm}$ is increased. This is also demonstrated by the spectrum [Fig. 4c] and the fluorescence decays [Fig. 4e] that were reconstructed from the respective dpc files. Compared to the spectrum of ECFP or Cerulean, the emission peak of the fluorescence spectrum of the EYFP–ECFP and the EYFP–Cerulean hybrid protein was shifted toward acceptor emission wavelengths, and the fluorescence decay of the ECFP and Cerulean moiety in the hybrid protein was accelerated. Compared to ECFP, whose lifetime was determined to be $(2.33\pm 0.19)\mathrm{ns}$ in living cells, the fluorescence lifetime of the EYFP–CFP hybrid protein was accelerated to ${\tau }_m\left(5\min \right)=1.59\mathrm{ns}$ . Upon irradiation, the donor fluorescence lifetime decreased further to ${\tau }_m\left(25\min \right)=0.98\mathrm{ns}$ . In a series of seven experiments, the mean fluorescence lifetime decreased from $\overline{{\tau }_m}\left(5\min \right)=(1.45\pm 0.11)\mathrm{ns}$ to $\overline{{\tau }_m}\left(25\min \right)=(0.98\pm 0.08)\mathrm{ns}$ . When it is assumed that donor and acceptor fluorophores are separated by a single distance, a FRET efficiency of 37% can be calculated with Eq. 3 from the initial value. During the course of the experiment, however, the lifetime decreased to ${\tau }_m\left(25\min \right)=0.98\mathrm{ns}$ . Based on the latter value, a FRET efficiency of 58% would be calculated, which is beyond the maximal FRET efficiency that should be expected for an ECFP–EYFP FRET pair: GFP and its variants consist of an 11-stranded $\beta$ -barrel, which has the shape of a cylinder with a length of $4.2\mathrm{nm}$ and a diameter of $2.4\mathrm{nm}$ .^{14, 20} Since the fluorophore is located almost in the geometric center of the cylinder,^{14, 20} the minimal distance between the fluorophores that is physically possible is about $5\mathrm{nm}$ . This is close to the Förster distance of $4.9\mathrm{nm}$ for the ECFP and EYFP pair.²¹ Thus, with a stoichiometry of 1:1 between donor and acceptor molecules, FRET efficiencies above 50% should not be observed.

Fig. 4

Analysis of streak images obtained from HEK293 cells expressing the EYFP-ECFP and EYFP-Cerulean hybrid protein. (a) Confocal image of HEK293 cells expressing the EYFP-ECFP hybrid protein (upper panel) and the EYFP-Cerulean hybrid protein (lower panel). (b) Streak images obtained from the cells shown in (a). The comparison with the streak images of ECFP and Cerulean [Fig. 2b] shows that the fluorescence signal in the wavelength range between 465 and $495\mathrm{nm}$ is decreased, whereas the acceptor (EYFP) fluorescence in the wavelength range between 510 and $540\mathrm{nm}$ is increased considerably. (c) Fluorescence emission spectra of the EYFP-ECFP hybrid (upper panel) and the EYFP-Cerulean hybrid protein (lower panel) obtained in $5\text{-}\min$ time intervals. Compared to the fluorescence spectra of HEK293 cells expressing ECFP or Cerulean [Fig. 2c] alone, the peak of the fluorescence spectrum is shifted toward acceptor emission wavelengths. (d) Time course of the fluorescence intensity of the EYFP-ECFP hybrid protein (upper panel) and the EYFP-Cerulean hybrid protein (lower panel) in the wavelength range from 465 to $495\mathrm{nm}$ . (e) Fluorescence decay curves of the EYFP-ECFP hybrid protein (upper panel) and the EYFP-Cerulean hybrid protein (lower panel) obtained in $5\text{-}\min$ time intervals in the wavelength range from 465 to $495\mathrm{nm}$ . Compared to the fluorescence decays observed in HEK293 cells expressing ECFP or Cerulean [Fig. 2e], the fluorescence decay is accelerated. All data have been processed as described in Fig. 2. Colors are assigned accordingly. (Color online only.)

Similar results are observed for the analog hybrid-protein containing the Cerulean fluorophore: Compared to Cerulean alone, whose lifetime was determined to be $(2.66\pm 0.13)\mathrm{ns}$ in living cells, the fluorescence lifetime of the EYFP–Cerulean hybrid protein was decreased to ${\tau }_m\left(5\min \right)=1.75\mathrm{ns}$ and ${\tau }_m\left(25\min \right)=0.95\mathrm{ns}$ . In a series of eight experiments, fluorescence lifetimes decreased from $\overline{{\tau }_m}\left(5\min \right)=(1.71\pm 0.07)\mathrm{ns}$ to $\overline{{\tau }_m}\left(25\min \right)=(1.01\pm 0.09)\mathrm{ns}$ . The first value would correspond to a FRET efficiency of 35%, which is almost identical to the FRET efficiency calculated in the case of the EYFP–ECFP hybrid protein. After irradiation, a FRET efficiency of 62% would have been calculated.