1. Introduction

In recent years, fluorescence lifetime imaging microscopy (FLIM) has been experiencing a rapid increase of attention, because it can provide local environment information of a fluorophore but does not depend on fluorophore concentration. Furthermore, FLIM is minimally affected by tissue absorption and scattering or fluctuations of the excitation intensity [1–4]. Hence, it is widely used in chemistry, biology, and physics in conjunction with traditional fluorescence intensity measurements or spectral measurements. Although FLIM using confocal [5] or multiphoton microscopy [6,7] can achieve a very high spatial resolution, data acquisition speed is not fast enough to study dynamic cellular processes or to be used for in vivo tissue diagnosis during surgical operation. In contrast, wide-field FLIM (WF-FLIM) systems provide higher imaging rates through parallel pixel acquisition [8–11] which is demanded for applications such as imaging of proteins during their interactions in living cells, in vivo tissue diagnosis [12], imaging of rotational mobility of fluorophores [13–15], and assays of microarrays. WF-FLIM can be implemented in frequency or time domain [2]. Time domain WF-FLIM has briskly advanced thanks to the development of pulsed lasers. WF-FLIM is mostly used for planar samples at present. Nevertheless, three-dimensional (3D) imaging is feasible for translucent samples (even in presence of scattering [3]), too, by composing the 3D image from a series of image sections at several focusing depths [16]. In normal WF-FLIM setups, just like in most FLIM systems, optical sections are obtained by mechanically changing the distance between the specimen and the objective lens to refocus at different depths. This brings about two disadvantages: first, neither moving the specimen nor moving the objective (connected with the whole microscope tube) is convenient or fast enough. Secondly, the most important shortcoming for some applications in biology or chemistry is that the motion may significantly disturb the specimen [17]. Since fluorescence lifetime is very sensitive to changes of the local environment of the fluorophore, it is best to avoid moving the sample.

In this paper a three dimensional WF-FLIM system is achieved by combining wide-field FLIM with a remote optical refocusing system that does not require mechanical movement of the specimen for three dimensional imaging [17,18]. We test this system using a fluorescent microsphere as sample. The results show that the design has been implemented successfully.

2. Experimental setup

As shown in Fig. 1 , the experimental setup comprises a system for fluorescence excitation, a detection system, and a system for remote refocusing. Fluorescence was excited by Ti:sapphire femtosecond laser pulses (800 nm, 100 fs, 1 mJ at 1 KHz) that were frequency doubled to 400 nm by a BBO crystal. Compared to the fluorescence lifetime (typically nanoseconds), duration of the exciting pulse is short enough to permit time-resolved fluorescence measurements. The divergence of the excitation laser beam is increased by a concave lens (CL) such that a suitably wide field is excited in the focal plane of the objective lens DO1. A polystyrene microsphere (diameter of 15 μm) with a surface coating of a fluorescent dye (Fluosphheres, Invitrogen Company) was used as test sample. Since polystyrene is transparent in the visible, the microsphere becomes a luminous spherical shell when illuminated by the exciting laser pulse.

 

Fig. 1 Experimental setup for 3D wide-field fluorescence lifetime imaging microscopy. DO1, DO2: dry objective lenses; TL1, TL2, TL3: tube lenses; DM: dichroic mirror; BS: beam splitter, M: reflection mirror mounted on a translation stage (TS); CL: concave lens.

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Two 40x dry objectives (DO1 and DO2) and two tube lenses (TL1 and TL2) form a 4f refocusing system that is designed to satisfy Abbe's sine condition as well as Herschel condition [17,18] and thus simultaneously satisfy both conditions for perfect imaging with wide-angle pencils. So both, the spherical aberration, which is the main aberration of optical imaging systems with wide-angle pencils, and the circular coma are eliminated and a perfect 3D image of the specimen is obtained in the focal region of the objective lens DO2.

For the section detection system, we put a reflection mirror (M) mounted on an axial scanning translation stage (TS) in the focal region of DO2. The mirror M, the objective DO2, the beam splitter BS, and tube lens TL3 compose a conventional microscope. The magnified 2D slices of the perfect 3D image of the sample at different focusing depths are imaged onto the detector by moving mirror M along the optical axis. This avoids moving of the sample, i.e., we achieve a 3D lifetime image while keeping the specimen stationary. Using a small mirror M and a high speed translation stage, 3D lifetime imaging can be achieved at high speed to sufficiently suppress bleaching of the sample. The detector is an intensified CCD (ICCD) camera (PicoStar HR12 Camera System, LAVision) combined with a spectrograph (Spectra Pro 300i Acton). A mirror and two gratings are installed in the spectrograph such that the system can be switched between 3D imaging mode and time-resolved fluorescence spectroscopy mode. The spectrograph, the translation stage (M122.1DG, Physik Instrumente), and the ICCD are controlled by a computer synchronized with the femtosecond laser. A long-pass filter (FEL0500) is placed before the imaging lens TL3 to block the exciting laser pulse.

3. Results

For 3D imaging, the sections in focus are imaged onto the ICCD by a mirror that is switched instead of the grating into the beam path of the spectrograph. Figure 2 (i) shows several fluorescence intensity image sections of the sample. The gating time of the ICCD is 200 picosecond. The image sections (a) to (h) were obtained by only moving the mirror M, i.e., without moving the sample at all. The spatial resolution is about 1 micrometer as can be deduced from section image (e). The limitation is due to the available numerical aperture of the objective lenses (0.65 NA).

 

Fig. 2 (i) 3D fluorescence intensity image sections of a fluorescent microsphere as test sample with the mirror M on different positions along the optical axis. The gating time of the ICCD is 200 ps. (ii) 3D image of the sample reconstructed from these sections; (iii) 3D lifetime image of the sample reconstructed from 2D lifetime image sections with the mirror M on different positions.

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A 3D image was reconstructed from the image sections of Fig. 2(i) and is shown in Fig. 2(ii). The 3D fluorescence intensity picture reveals an inhomogeneous distribution due to an inhomogeneous coverage of the fluorescence material on the polystyrene microsphere surface. Fluorescence lifetime image sections at different positions of the mirror M were used to reconstruct the 3D lifetime image of the microsphere (Fig. 2(iii)). Comparing Fig. 2(ii) with Fig. 2(iii) we can see that the lifetime image eliminates the effect of inhomogeneous distribution of the fluorescence material. Because of the visual angle it is usually hard to tell from the 3D profiles whether the fluorescent microsphere is a dye-covered polystyrene sphere (with dye molecules only on the surface) or a dye/polystyrene sphere (i.e., with the dye interspersed in the bulk). In our case one can however clearly recognize a ring-shaped structure in several section images of Fig. 2 (i). So, despite of the fact that the non-homogeneity of the polystyrene sphere causes some scattering of the fluorescence light in the sphere and thus reduces somewhat the signal-to-noise ratio, one can definitively infer that the sample is a dye-covered polystyrene sphere rather than a dye-filled sphere. As the fluorescence distribution of the sample forms a spherical shell, the circular fluorescence rings become thicker and thicker as the mirror moves away from the middle position of the microsphere along the optical axis.

Of cause also normal 2D fluorescence lifetime images can be recorded. Figure 3(a) shows lifetime images of section (e) in Fig. 2 (i). Figure 3(b) shows its lifetime curve.

 

Fig. 3 (a) 2D fluorescence lifetime images of section (e) in Fig. 2 (i). (b) Decay curve of the fluorescence intensity; squares are experimental data points, solid curve is a single exponential fit to the data. The decay time is 4.1 ns.

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Since focusing of each section occurs in a different plane, one usually should correct for a time error of the fluorescence lifetime signal. Reference [19] gives the following expression for the defocus coefficient of the system:

 

Fig. 4 Fluorescence lifetime error caused by the refocusing system along the optical axis. z is the distance along optical axis from the focus of the objective lens OD2.

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The time error is only some femtoseconds and thus short enough to be ignored in comparison with the fluorescence lifetime.

Time-resolved spectroscopy is widely used in the study of dynamic processes in many branches of physics, chemistry, and biology for its high spectral and temporal resolution. Replacing the mirror with a grating in the spectrograph, we obtained time-resolved fluorescence spectra of the sample (a proprietary dye from Invitrogen Company), as shown in Fig. 5 .

 

Fig. 5 Time-resolved fluorescence spectra of the sample. The fluorescence intensity is displayed in a pseudo-color mode as a function of both wavelength (horizontally) and time after excitation (vertically).

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The spectral range of the filter for 3D WF-FLIM before imaging was determined by time-resolved spectroscopy. After obtaining the 3D image, we can choose an area of interest in the image for further studies at the high spectral resolution of time-resolved spectroscopy.

4. Conclusion

In summary, we built up a 3D wide-field lifetime imaging microscopy system combined with remote optical refocusing. We demonstrated its excellent ability for 3D lifetime imaging using a fluorescence microsphere as test sample. The system can easily be switched between 3D WF-FLIM and time-resolved fluorescence spectroscopy. We expect this system to be a powerful tool for many applications in biology, chemistry, and medical science.