1. Introduction

Since its inception in 1990, multiphoton fluorescence microscopy has become one of the major optical imaging modalities in biomedical research. The improved axial depth discrimination as well as image penetration depth with reduced sample damage, this approach provides potential applications in bio-imaging especially for the in vivo case [1–3]. But the high peak intensity of the femtosecond pulsed laser used in multiphoton fluorescence microscopy may result in specimen photo-damage [4,5]. An approach to strengthen multiphoton signal along with reduced specimen photo-damage is to use excitation sources with narrower pulse widths. It was found that the multiphoton signal can be significantly raised with the signal being inversely proportional to the laser pulse widths. McConnell and Riis observed a sevenfold increase in two-photon excited fluorescence yield when laser pulses were compressed from 250 to 35 fs [6]. Then, Xu and Webb examined the impact of pulse duration on multiphoton signal and observed an inverse relationship for pulses longer than 90 fs [7]. However, in using these techniques with narrow pulse width compression, a dispersion compensator is needed to adjust the dispersive effects. Since different microscope objectives may contribute to different degrees of pulse broadening (adjustment on the broadening were made through the use of optical components such as prism pairs or gratings [8,9]), these techniques are neither convenient nor suitable for biologists. Clearly, a device enabling on-specimen compression of excited laser pulse where the compression effect occurs after the light passing through the objective and optical components will be significant to the multiphoton fluorescence microscopy.

PC has been a fascinating research area of modern optics since 1987 [10,11]. In recent years, photonic crystal (PC) structures are being studied for enhancing the excitation field and fluorescence. The most attractive property of PCs is the existence of photonic bandgap (PBG), characterized by the spatial distribution of refractive index. Photons of particular wavelengths are localized and forbidden to propagate through PCs in the PBG region. A narrow spectral windows in the PBG can be created by inserting a third dielectric material as a central defect layer in the PC structure called as defect mode [12,13]. The first fluorescence enhancement in PCs was demonstrated by Ye et al. The improvement in the signal was achieved because of the highly localized excitation field at the defect layer [14]. In the same context, three other groups (Ye, Soboleva, and Lidstone et al.) proposed advance PCs in a total-internal-reflection geometry to enhance the fluorescence openly [15–17]. Besides photon localization, the other property of PC slab is the pulse compression of the excited femtosecond laser pulses. Pulse compression is caused by the strong frequency and spatial dispersion near the edge of PBG when a light propagates through the PC slab [18,19]. The same effect holds true for the tunable, hybrid PC/liquid crystal (LC) structure. The optical properties of a 1D PC/LC were first investigated by Ozaki et al. He applied an external voltage to reveal the wavelength tuning of the extraordinary components in the defect modes [20]. Exploiting different modes of LCs in the PC/LC hybrid structure, different tuning properties were reported [21–23,12,13]. In this work, one-dimensional PC (1D PC) nanostructures were fabricated and utilized for on-chip compression of femtosecond laser pulses. Here a PC/LC hybrid structure was exploited to compress the excited pulse (property of PC) and study the spectral selection property (property of LC).

2. Experiment

Figure 1(a) illustrates the experimental device used. The photonic device is composed of two PC slides sandwiching with LCs. Each PC cell was structured on quartz substrates coated with indium–tin oxide (ITO) and the periodic alternative multilayers. The multilayers include five layers of high reflection index (n_H = 2.18) and four layers of low reflection index (n_L = 1.47). To provide high reflectivity, the optical path length (OPL) of these two materials is set to be equal to one-quarter of the incident light wavelength, i.e., n_Hd_H = n_Ld_L = λ/4, where λ is the center wavelength of the PBG, n_H, n_L, and d_H, d_L are the refractive indices and thicknesses of the high- and low-index materials, respectively. The fluorescence images were obtained using laser scanning microscope system (LSM 510 META, Zeiss, Jena, Germany) coupled to a femtosecond, titanium:sapphire laser (Tsunami, Spectra-Physics, Mountain View, CA). An oil-immersion objective (Nikon, Plan Fluor 40 × NA 1.40, WD = 0.21 mm) was used for focusing the laser source and collection of the emission signal in the epi-illuminated geometry. The laser output wavelength was tuned to 790 nm, pulse width was estimated to be 100 fs with 80 MHz pulse repetition frequency. All acquired optical images were 230 μm × 230 μm in area.

 

Fig. 1 (a) the schematic diagram and the structure of the 1D PC/LC devices, (b) actual pictures and specifications of device, (c) the SEM image of the PC (one side), (d) the transmission spectrum of the pure PC (black) and PC/LC device (red), and (e) the reorientation of the LC molecules and the corresponding refractive indices for TM and TE polarizations.

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3. Results and discussion

Figure 1(b) shows the photograph of the photonic device, which has a thickness of roughly 0.34 mm (each PC coated on quartz substrate has a thickness of 0.17 mm and is sandwiched to make a device) in order to enable the oil-immersion objective with high resolution. Figure 1(c) exhibits the scanning electron microscopy (SEM) image of one side of the PC structure. The multilayers are deposited pacifically which can be observed from SEM image. The thicknesses of high and low reflection index layers were determined to be 68.09 nm and 102.37 nm, respectively, leading to the central wavelength of the stop band at 600 nm and the width of PBG is around 250 nm as shown in Fig. 1(d). If a LC layer is infiltrated into a cell formed by sandwiching two PCs, the periodicity of LC get disrupted and partial defect modes will be generated within the PBG that allow the transmission of photons at specific wavelengths(Fig. 1(d)). When the incoming light is normal to the PC/LC cell with its transverse electric (TE) polarization direction parallel to the LC molecular axis, the OPL is contributed by the sole extraordinary refractive index (n_e) as shown in Fig. 1(e). The appearance of peaks in the PBG of the transmission spectrum thus represents extraordinary defect modes. Upon applying the voltage in TE mode, the molecules reorient and tilted towards the z-axis. The resultant effective index is thus decreased, leading to the shift in the defect modes towards the shorter wavelength in the spectrum (i.e. blue shifting). On the other hand, the transverse magnetic (TM) mode corresponds to n_o. Since the polarization is always perpendicular to the optical axis despite the molecular reorientation, the experienced refractive index for TM wave remains to be n_o upon applying voltage. Therefore, the defect mode does not shift with the voltage for TM wave. Note that TE and TM modes for polarized waves also correspond to the extraordinary and ordinary waves of LC molecular, respectively.

Figure 2(a) represents the transmission spectrum of the 1D PC/LC devices with various applied voltages in experimental. The spectrum is measured for 1 μm thick LC defect layer in 1D PC. It seems that the blue shifting of defect mode with higher voltage is due to decrease in the effective refractive index. Thus, the central wavelength of defect modes can be controlled by applying voltages and hence the fluorescence signal can be filtered selectively. This will be referred to as the “tunable enhancement in fluorescence” (TEF) mode in the following text. In addition, a larger number of defect modes can be obtained by using LCs with higher refractive index or by increasing the thickness of the defect layer. Figures 2(b) and 2(c) demonstrate the transmittance spectra of the 1D PC/LC with 5 and 15 μm thicknesses of the LC defect layers. It can be seen that the thicker cell gap typically 15μm with larger numbers of defect modes, enables the 1D PC/LC structure to be applicable for the use of strong enhancement in fluorescence (SEF) mode as it contains the maximum number of defect modes transmitting fluorescence signal. Note that the photoluminescence spectra of the two fluorescent balls: red (R) and green (G) fluorescent balls as shown in Figs. 2(b) and 2(c) were imaged and tested by the on-chip PC/LC devices based on multiphoton fluorescence microscopy. Figures 2(d) and 2(e) show the Finite Difference Time Domain (FDTD) method simulating electric field energy communicated in the both TE and TM modes based on the PC devices. When the emission fluorescence does not correspond to the wavelength of the defect modes in the device, fluorescence will be confined in the sandwiched sample layer and be absorbed. If the defect modes were tuned to the wavelength of the emission light, the fluorescence will transmit through the device and can be detected by the objective. The FDTD simulated results of the PC/LC device suggest that this tuning can be realized in both TE and TM modes. It is worth to mention that the simulation of the defect mode for different wavelengths (Figs. 2(d) and 2(e)) is in good agreement with the experimental result (Fig. 2(b, c)). Note that the edge of photonic band is used for pulse compression and the photonic defect modes are employed to filter the fluorescence signal.

 

Fig. 2 (a) the transmission spectra of the 1D PC/LC devices at various applying voltages when the defect layer is 1-μm thick, in TEF mode; (b) and (c) are respectively the transmission spectra when the thickness is 5 and 15 μm, and the photoluminescence spectra of the two fluorescent channels are also inserted. 15-μm thick sample operates in the SEF mode; (d) and (e) are FDTD simulations of electromagnetic wave of the used pulse duration 100 fs for TE and TM waves in 5 μm cell gap, respectively.

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In order to measure the pulse widths of excitation on-specimen, an optical autocorrelator was exploited in microscopy system. This allows us to detect the autocorrelation signal at the focal plane of the objective (Fig. 3(a)). The output of a Ti:sapphire laser is sent through a 50% beam splitter, and one arms of the beam passes through a variable-delay line device. A high-NA objective is mounted on a variable-height translation stage. The two-photon fluorescence is monitored on the backscattering direction, reflecting off the dichroic mirror and passing a filter to block scattered light at wavelength of Ti:sapphire. Measurements of the two-photon fluorescence signals are detected by a photomultiplier tube. The interferometric autocorrelation of a chirp-free, transform-limited optical pulse is characterized by a fully symmetric curve, with a peak-to-background ratio of the constructive interferences of 8:1. The autocorrelation signal traces are shown in Figs. 3(b) and 3(c). It shows a proper 8:1 ratio and slight asymmetry. A slight asymmetry of the interference spectrum confirms that the pulses are not in perfect Gaussian shape. For the analysis of the traces, the time scale of the autocorrelation was calibrated by taking the Fourier transform of the data. The envelope of the constructive interferences is subsequently fitted to the interferometric autocorrelation function of Gaussian input pulses. The original pulse width of the commercial Ti-Sapphire laser is about 100 fs. However, after passing through the optical components and objective of the microscope, the pulse width was measured to be 270 fs (see Fig. 3(b)). By increasing excitation power, the pulse width decreases in a nonlinear fashion. The shortest achievable pulse duration was around 30 fs (Fig. 3(d)). A reduction by a factor of nine was achieved. According to the past research, as the pulse duration time compress to one-ninth, the two-photon fluorescence signal is enhanced nine times [6]. When a PC slab is attached in the back of the sample, the forward fluorescence light is reflected back and detected by the objective, making the fluorescence signal detection even more efficient with a signal gain up to 15-fold.

 

Fig. 3 (a) the system design of homemade optical autocorrector in microscopy. HW stands for half-wave plates, LP stands for linear polarizers, QW stands for quarter-wave plates, M stands for mirrors, B stands for a beam splitter, L stands for lenses, O stands for the objective, D stands for dichroic mirrors, and F stands for filters; (b) and (c) are the autocorrection traces without and with the devices applied 80 mW; (d) pulse duration versus applied powers.

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The bio-images obtained from multiphoton fluorescence microscopy system were process with the software IDL to compose all channel of photomultiplier tubes [24]. Figure 4(a) exhibits the red fluorescent balls images at different excitation power, with and without the PC/LC device under 0 V. The spectral bandwidth of defect mode at 0 V is 20 nm (Fig. 2(a)). It is obvious that the intensity of fluorescent images is stronger with the PC/LC device. Figure 4(b) shows the green fluorescent balls images with and without the PC/LC device under 10 V which has a result similar to red fluorescent balls. Quantifying the image by the software Image J [24], an 8 ± 1-fold enhancement was observed in the red channel. 10 ± 1-fold enhancement was observed in the green channel by applying voltage of 10 V. The spectral bandwidth of defect mode under 10 V is 25 nm (Fig. 2(a)). It is worth to mention that in SEF mode, 15 ± 1-fold increment was observed at the expense of sacrificing the tunability of the TEF mode. The effect of photo-damage was also investigated with and without the PC/LC device under high excitation power. Figure 4(c) illustrates the images of the red channel at different exposure time at high excitation power the operating power 40 mW with device and 150 mW without device were applied to achieve the similar fluorescence intensity from the fluorescent microspheres. When the PC/LC device was not used, photo-damage becomes apparent once the exposure time reached to or beyond 1.5 hrs. If the proposed device was used, the photo-damage can be reduced, as lower excitation power is needed to achieve the same signal intensity. Thus, PC/LC device can efficiently reduce the operation power and photo-damage. Figure 4(d) demonstrated the same effect in the green fluorescent balls under 10 V in TEF mode. Comparing to the past PC techniques [14–17], this novel PC/LC device is much more powerful and well applicable for bio-imaging and other imaging devices. Moreover, this device for pulse width compression doesn’t require any dispersion correction, and is therefore easy to use for biologists.

 

Fig. 4 (a) the red and (b) the green fluorescent ball images at different excitation power with (left) and without (right) the PC/LC. The images of (c) the red and (d) the green fluorescent images at different illumination time with and without the PC/LC. The PC/LC device was operating in TEF mode.

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4. Conclusions

A novel photonic device with two modes: tunable and strong fluorescence enhanced modes, as a device in multiphoton microscopy with low photo-damage was first demonstrated. The device with LCs as a defect layer infiltrated within 1D PCs is proposed. The device utilizes the pulse compression effect of PCs and the electrical tunability of LCs. The images that we observed shows a maximum gain of 15-fold without extra compensation. With the unique properties of the PC/LC structure, new possibilities and applications for the fluorescence enhanced bio-images are expected.