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Single femtosecond laser-based coherent anti-Stokes Raman scattering (CARS) microscopy, using a photonic crystal fiber (PCF) pumped in the near-IR to generate a supercontinuum for the Stokes source, is rapidly being adopted as a cost-effective approach. A PCF with two closely-lying zero dispersion wavelengths is a popular choice for the Stokes source, but it is often limited to imaging lipids. A polarization-maintaining PCF with two far-lying zero dispersion wavelengths offers important advantages for polarization CARS microscopy, and for CARS imaging in the fingerprint region. This PCF fiber, though commercially available, has limited use for CARS microscopy in the C-H bond region. The main problem is that the supercontinuum from this fiber is typically noisier than that from a standard PCF with two closely-lying zero dispersion wavelengths. To overcome this, we determined the optimum operating conditions for generating a low-noise supercontinuum out of a PCF with two far-lying zero dispersion wavelengths, in terms of the input parameters of the excitation pulse. We measured the relative intensity noise (RIN) of the Stokes and the corresponding CARS signal as a function of the input laser parameters in this fiber. We showed that the results of CARS imaging using this alternate fiber are comparable to those achieved using the standard fiber, for input laser pulse conditions of low average power, narrow pulse width with slightly positive chirp, and polarization direction parallel to the slow axis of the selected fiber.
© 2014 Optical Society of America
1. Introduction
Coherent anti-Stokes Raman scattering (CARS) microscopy, using a single femtosecond (fs) laser for both the pump and the Stokes beams, has been gaining popularity [1–6]. In this approach, a portion of the fs laser is used as the pump beam, while the Stokes beam is obtained by generating a supercontinuum (SC) by coupling the rest of the fs laser beam to a photonic crystal fiber (PCF), followed by a band-pass filter to select the desired Stokes wavelength.
This single fs-laser approach reduces the complexity of the CARS setup and results in a more compact and cost-effective system. Moreover, the narrowband imaging approach enables fast acquisition time CARS imaging of biological samples at a fixed Raman shift. By combining the spectral focusing technique with the single femtosecond (fs) laser CARS approach, the rapid scanning Raman shift CARS microscopy in the C-H bond and fingerprint regions becomes feasible [7,8].Moreover, by combining this technique with an endoscope/exoscope, the development of a cost-effective, miniaturized, portable multimodal CARS imaging platform for in-vivo and bed-side clinical applications becomes more feasible [9–11].
One of the important issues with the single fs-laser approach, particularly if a fs fiber laser is used, is to find a PCF that can generate a stable, low noise, broad supercontinuum spectrum with low average power from the laser source [12,13].
In our earlier work, we successfully demonstrated the use of a 12 cm PCF with two closely-lying zero dispersion wavelengths (ZDWs) at 775 nm and 945 nm, (NL-1.4-775-945 Crystal Fiber), housed in a hermetically sealed package (FemtoWhite CARS, NKT Photonics), for single fs laser CARS microscopy of lipid-rich biological specimens [2]. This PCF resulted in a highly stable supercontinuum which was used as the Stokes beam in a CARS imaging setup, and subsequently became the standard fiber for performing CARS at the C-H bond region [14]. However, the application of this fiber is often limited to CARS imaging of molecular species with vibration at wavenumbers ≥ 2000 cm−1 Raman shift, and an appropriate choice of pump wavelength around 800 nm [2]. In addition, as it is not a polarization maintaining fiber, it cannot be used for polarization CARS microscopy. Thus, a polarization maintaining PCF that can provide a wider range of Stokes wavelengths to reach the lower Raman shifts in the fingerprint region (~800 cm−1-1800 cm−1), with comparable quality (low noise and high coherence), is highly desirable.
An available fiber that can satisfy these requirements would be a polarization maintaining PCF, NL-PM-750 (NKT Photonics, Denmark), with the two far-lying ZDWs at 750 nm and 1260 nm (Fig. 1(b)). The fiber is available in a compact module, FemtoWhite 800 (NKT Photonics) with a mark that indicates the direction of the slow polarization axis of the fiber module [15]. Moreover, the hermetically sealed module FemtoWhite 800 provides a convenient and robust solution for fiber handling, as well as coupling excitation light into the PCF with two far-lying ZDWs. A number of groups have successfully used the two far-lying ZDWs fiber to perform narrow band and broadband multiplex CARS imaging in both C-H and fingerprint regions. However, they did not examine the quality of the generated supercontinuum out of the fibre systematically [6, 14–19]. Klarskov et al. [20] investigated the different PCF designs with two ZDWs, to determine the best PCF design for generating the Stokes beam for the CARS microscopy. They did this by comparing the spectral density of the generated SC around 648 nm and 1027 nm of each PCF with an existing standard PCF (NL-1.4-775-945 Crystal Fiber) for CARS microscopy [20]. In their measurements, they assumed that the best fiber is the one that can generate the highest amount of spectral density of polarization maintained SC around 648 nm and 1027 nm to generate CARS signal, while they ignored the amplitude noise and stability of the generated SC in their measurements. Since CARS is a nonlinear four-wave mixing process, it does not depend only on the intensity of the pump and the Stokes beams, but also on their quality in terms of coherence, polarization and intensity stability. As the pump is of high quality, the intensity and relative intensity noise (RIN) of the generated CARS signal can be used to determine the quality of supercontinuum generation.
Fig. 1 (a) Group velocity dispersion parameter (β2) of PCF with two closely-lying ZDWs and (b) PCF with two far-lying ZDWs (Courtesy of Crystal Fiber Inc.), (c) The supercontinuum spectra out of two far ZDWs (black line) and two closely-lying ZDWs(blue dotted line) fibers for a same input power of 530 mW and pulse duration of ~100 fs. Vertical lines indicate the position of two ZDWs. (Intensity scales of two SCs are not exactly the same). (d) CARS spectrum of Oil, ~2850 cm−1, Nitrobenzene, ~1350 cm−1, and Toluene, ~990 cm−1, using the spectral focusing technique, our existing miniaturized fiber delivered CARS exoscope and the two far-lying ZDWs fiber to generate SC. (The exposure times of each CARS spectra were 0.1s, 5s and 1s respectively).
Previous work did not examine the signal quality of the generated Stokes and CARS signal from the FemtoWhite 800 module, nor did it compare the quality of the generated images with those obtained with the FemtoWhite CARS module containing the PCF with two closely-lying ZDWs. Consequently, the FemtoWhite 800 module was not used at its optimal point to generate the highest possible CARS signal intensity with the minimum noise value, in order to obtain high contrast CARS images with short acquisition times for studying dynamic living systems. We determined the optimal operating parameters for the fiber with two far-lying ZDWs (NL-PM-750) inside a FemtoWhite-800 module [15] for CARS microscopy, corresponding to the C-H vibration band at ~2850 cm−1. We also took relative intensity noise (RIN) measurements of the generated Stokes beam and its corresponding CARS signal of an oil sample, and compared the results with those obtained from the standard fiber module containing the PCF with two closely-lying ZDWs [15].
In this paper, after a short introduction on SC generation and the noise amplification mechanism in both fibers, the experimental setup and results are presented. The relative intensity noise (RIN) of the SC out of the two far-lying ZDW fibers and two closely-lying ZDW fibers, and the RIN of the generated CARS signals of a bulk oil sample for different input laser conditions, are demonstrated. Based on the RIN of the Stokes and CARS signals, as well as the CARS signal intensity, the optimum conditions for implementing the two far-lying ZDWs PCF are shown. Finally, to compare the performance of the two fibers for CARS microscopy, the CARS images of a standard 4.5 μm polystyrene bead sample using both fibers are presented.
2. Spectral features of pulse in PCFs with closely-lying and far-lying ZDWs
The supercontinuum generation mechanisms in the PCF with two closely-lying ZDWs and the PCF with two far-lying ZDWs are not the same due to the difference in their dispersion profiles (Fig. 1(a) and 1(b)). This difference in dispersion plays a significant role in the phase matching conditions during the SC generation. When an 800 nm wavelength, low power femtosecond laser pulse propagates a short distance (~12 cm) through the fiber with two closely-lying ZDWs, the supercontinuum is generated by self-phase modulation (SPM) and phase-matched four-wave mixing (FWM) [21]. In this case, the supercontinuum is more independent of the input pulse parameters and is coherent through its entire spectrum. The phase-matched FWM mechanism causes a significant depletion in the anomalous region between the two zeros during the SC propagation in the fiber, causing a very low spectral density between the two ZDWs (Fig. 1(c)).
In the PCF with two far-lying ZDWs, because of the higher magnitude of β2, (group velocity dispersion parameter) in the anomalous dispersion region, and the less phase-matched FWM, the supercontinuum generation is mostly dominated by higher-order noise sensitive soliton fission [21–23]. Moreover, the modulation instability (MI) gain in the anomalous dispersion region is bigger due to the higher value of β2 and consequently, the generated SC out of this fiber is more sensitive to the input pulse parameters and noises. Thus, controlling those parameters can have a significant effect on the quality of the produced SC [22, 24–27].
The balance between group velocity dispersion in the anomalous region and SPM leads to the formation of solitons in two far-lying ZDWs fiber. At higher amplitudes of the input fs pulse, a high-order soliton with a soliton number of N is formed. Higher-order solitons of the nonlinear Schrödinger equation show periodic changes with propagation, although, in vicinity of the zero dispersion wavelengths with a big positive magnitude of third order dispersion and self-steepening effect, the propagation behaviour of the higher ordered solitons will be different. In this situation, a higher-order soliton pulse will split into N pulses after a short propagation distance of Lfiss, and generates N different red-shifted soliton pulses with different group velocities [22–26]. Here Lfiss is the soliton fission length and is defined as:
Where and
LD is the dispersion length, T0 is the temporal width of the input pulse, LNL is the nonlinear length, P0 is the peak power, and γ is the nonlinear coefficient of the core.
Figure 2 shows the generated supercontinuum spectrum out of two far-lying ZDWs fiber, as the input power increases from 28 mW to 319 mW. It can be seen that as the input power to the fiber increases, each generated soliton pulse loses energy by emitting non-solitonic, blue-shifted radiation that is phase-matched to the corresponding pulse, while simultaneously moving to the IR region until reaching stability.
Fig. 2 The measured SC spectrum out of the PCF with two far-lying ZDWs, for low input average laser power. As it is shown, the soliton fission mechanism happens around 34 mW of input power, generating a red-shifted soliton around 830 nm and corresponding blue-shifted non-solitonic peak around 620 nm. At the higher input average power, the presence of several solitons with different red shifted frequencies causes a broad spectrum in the blue and red spectral regions of the generated SC at the input powers more than 160 mW. (Average coupling efficiency, η = 40%).
In the case of higher input average power (P0 > 160 mW) there will be higher order soliton fission processes due to the presence of several solitons with different red shifted frequencies. In addition, there is a broad-spectrum peak in the blue spectral region of the generated SC as a result of the presence of corresponding blue-shifted non-solitonic radiation for each soliton. The intermediate region in the SC spectrum arises because of four wave mixing between the solitons and their corresponding blue-shifted non-solitonic continuum during the SC generation [24, 25] (Fig. 2).
Figure 3 shows the soliton fission length in both fibers as a function of input average power for a 100 fs pulse (input pulse width). As two closely-lying ZDWs fiber has a smaller β2 value compared to two far-lying ZDWs fiber, the soliton fission length is much longer for two closely-lying ZDWs fiber. In fact, at the nominal average power of 300mW and for a fiber length of 12 cm, Lfiss is much shorter than the fiber length for the case of two far-lying ZDWs fiber and much longer than 12 cm for the case of two closely-lying ZDWs fiber. This indicates that the soliton fission mechanism cannot occur in the implemented two closely-lying ZDWs fiber (the fiber with two closely-lying ZDWs) at 300 mW input laser power during the SC generation for the chosen fiber length. Therefore, SPM and phase-matched FWM mechanism are dominant in SC generation in this fiber, leading to a less noisy and more coherent SC [21].
Fig. 3 Soliton fission length vs. input average power before coupling to the fiber. The intersection of vertical dotted line at 300 mW and soliton fission length (Lfiss) curve indicates the value of the fission length for each fiber type. (Coupling efficiency = 40%, laser pulse width = 100 fs).
3. Method: RIN measurements and CARS imaging
3.1 RIN measurement
In order to measure the stability of intensity, polarization and coherence of the Stokes beam, the RIN of the Stokes beam and the CARS signal of an oil sample were measured.
Here we define the stability of polarization as the stability of the linear polarization direction of the Stokes beam and the coherence stability as the phase degradation and timing jitter of the generated Stokes pulses [28].
As the CARS mechanism is coherent and nonlinear, and as the RIN of the Ti-sapphire laser beam (−113 dB/HZ) is much lower than the RIN of the Stokes beam (range between ~-97 dB/Hz and ~-79 dB/Hz), the measured RIN of the CARS signal is a good indicator of the coherence, polarization and intensity stability of the Stokes beam. The RIN of the Stokes beam was measured by Fourier-analysis of the electrical signal from a photodetector using an electrical spectrum analyser (ESA) in the range of 0-81 MHz with a resolution bandwidth of 1 KHz and 100 Hz [27]. For each measurement, the signal power at 80 MHz was compared to the noise figure at a fixed point in the frequency range. For the RIN measurement of the CARS signal, the beam-scanning mirror was turned off. Then the CARS signal, generated by temporally and spatially overlapped Stokes and pump beams from a very small volume focused inside a drop of an oil sample, was collected and sent to the Photomultiplier tube (PMT, Hamamatsu H7422P-40). The corresponding electrical signal out of the PMT was amplified and sent to the ESA for RIN analysis.
3.2 CARS imaging
The pump and Stokes wavelengths were selected for CARS imaging of the lipid density corresponding to the molecular vibrations of C-H bonds in the range of 2800-3000 cm−1 Raman shift. The input pulse at 800 nm after adjusting power, chirp and polarization direction was coupled to the PCF with two far/closely-lying ZDWs to generate the Stokes beam. The pump and Stokes beams were collinearly combined and spatio-temporally overlapped inside the sample by using a dichroic mirror, delay line and our home-built scanning microscope [2]. The average powers (measured by optical power meter) of the pump and the Stokes beams at the sample were about 11 mW and ~2 mW respectively.
3.3 Experimental setup
To measure the relative intensity noise (RIN) of the generated supercontinuum around 1040 nm and RIN of the generated CARS signal using the IR portion of SC, Stokes beam, (λC = 1040 nm, full width at half maximum, FWHM, = 60 nm) as well as to obtain the CARS image of the lipid rich biological samples, the following setup was used (Fig. 4).
Fig. 4 Experimental setup: (1) Ti:sapphire laser, (2) Faraday isolator, (3) half wave plate, (4) beam splitter, (5) prism compressor, (6) microscope objective lens (40x), (7) PCF with two far/closely ZDWs, (8) collimating aspheric lens of 5mm focal length, (9) band pass filter, (10) flip mirror for routing to RIN measurement setup, (11) dichroic mirror for overlapping Stokes beam on the pump beam, (12) X-Y scanning mirrors, (13) focusing objective lens, (14) short pass filter, (15) PMT amplifier and discriminator, (16) diffraction grating mirror and (17) fast photodetector.
By using a double path prism compressor, the chirp and pulse temporal width of fs laser pulses in the Stokes arm were adjusted. An intensity autocorrelator was used to measure the pulse width of the fs laser pulse before coupling to the PCF fiber(s). By using a half wave plate before the coupling lens (6), the direction of the polarization of the laser beam was controlled. This light was coupled into a module, FemtoWhite 800 (FemtoWhite CARS) containing two far-lying ZDWs fiber (two closely-lying ZDWs fiber).
The generated SC out of each module (two closely/far-lying ZDWs fibers) was collimated and passed to a band pass filter (Chroma, central wavelength 1040 nm, FWHM = 60 nm). The filtered SC (here called “Stokes beam”) was directed toward a grating mirror to have a narrower bandwidth Stokes beam (FWHM = 10 nm) for RIN measurement. The RIN of narrower bandwidth Stokes was measured by using a fast photodetector (Newport, 818-BB-21A) and an electrical spectrum analyser (ESA, Agilent E4411B).
4. Results
4.1 Comparing the RIN of two far-lying and two closely-lying ZDWs PCF
In order to compare coherence, intensity and polarization stability of the SC generated by the two far-lying ZDWs fiber with that of the SC of two closely-lying ZDWs fiber the RIN of the Stokes beams, and corresponding CARS signals of an oil sample, from both modules were measured.
MI process and higher–ordered soliton fission mechanism are the two main sources of instabilities during the SC generation in the two far-lying ZDWs fiber [21, 22, 26]. Consequently, by reducing the impact of MI and higher–ordered soliton fission, a more stable generated SC can be achieved. Figure 5 depicts the RIN of Stokes beam as a function of input fs laser pulse width for both fiber types. The positive pulse width means positive chirp while negative pulse width means negative chirp. The input average power was 300 mW with an average coupling efficiency ~40%. As Fig. 5 shows, the Stokes beam of two far-lying ZDWs fiber has a minimum RIN of ~-95 dB/Hz at around + 100 fs input pulse width, which is 10 dB less than its value at 200 fs. This is mainly due to the shorter soliton fission length, for a 100 fs pulse compared to the longer input pulses. This reduces the MI process chance to play a significant role in perturbing the pulse break up process during the supercontinuum generation, leading to higher intensity stability (less RIN) [26].
Fig. 5 The RIN of the Stokes beam, using fibers with two far and two closely-lying ZDWs. The error bars are the variance of the RIN values at each pulse width. The input average power was 300 mW with an average coupling efficiency 40%. The red dashed line shows the value of the RIN of fs laser beam at the 100 fs, which is taken as the minimum theoretical RIN of the generated Stokes beam out of the both fibers. Lower left Corner: the RF power spectrum of the Stokes beam out of two far-lying ZDWs PCF (Resolution bandwidth = 1000 Hz).
In addition, by decreasing the pulse width of the input laser, the number of the noise sensitive higher–ordered soliton fission will decrease, resulting in a less noisy supercontinuum [26].
As Fig. 5 shows, the RIN value of the Stokes beam out of two closely-lying ZDWs fiber is minimal when the fs laser pulse is close to transform limited. In two closely-lying ZDWs fiber, for short transform-limited pulses, or pulses with a small positive chirp that undergo initial compression, the spectral broadening due to self-phase modulation (SPM) is more rapid than for pulses with a large negative/positive chirp [27]. This leads to an overall reduction in noise amplification for transformed limited pulses because of rapid falling of the pulse peak power inside the fiber core, causing a reduced total RIN value [27]. In other words for the short transform-limited pulses, the spectral broadening due to SPM will happen earlier, giving less chance for MI amplification during the pulse propagation in the fiber core.
To determine the polarization noise (instability) as well as the coherence of the Stokes beam out of both fibers, the RIN of the generated CARS signals from both fibers were measured. Figure 6 shows the RIN of CARS signals for a 300 mW average power laser coupled to both PCF fibers as a function of pulse width.
Fig. 6 The RIN of the CARS signal of an oil sample, using fibers with two far and two closely lying ZDWs for 300 mW average power of the laser coupled to both PCF fibers. The error bars are the variance of the RIN values at each pulse width (Negative sign indicate the negative chirp).
As Fig. 6 depicts, the RIN value of CARS of the two closely-lying ZDWs fiber is less than that of the two far-lying ZDWs fiber around 100 fs, which indicates that the Stokes beam of the two closely-lying ZDWs fiber is still less noisy at 300 mW. This result is in line with that of Hilligsoe et al. [21].
4.2 Determining the optimum operating point for the fiber with two far- lying ZDWs
In order to reduce the total RIN of the Stokes beam out of two far-lying ZDWs fiber, the average input power of the fs laser beam coupled to the fiber was reduced [27]. By reducing the average power of the coupled fs laser to two far-lying ZDWs fiber, the average power of the generated Stokes beam will be generally reduced. However its stability will increase because of the reduction in noise amplification during the SC generation [27–31]. Therefore, there will be a point of optimum fs laser average power, coupled to two far-lying ZDWs fiber that can generate the highest stable CARS signal intensity with a minimum RIN value.
The polarization direction of the coupled laser beam can also play an important role in the polarization stability of the generated SC and consequently the Stokes beam [31]. Since the intensity of a CARS signal depends on the polarization direction of the Stokes and the pump beam, the polarization direction of the laser beam at 800 nm with slightly positively chirped ~100 fs pulses coupled to the fiber was also considered to find the optimum operation point for this fiber. The average power of the input laser was changed for two different polarization angle (direction) states: one in parallel with slow axis of two far-lying ZDWs fiber (0 ° angle) and the other with a 45° angle with respect to the slow axis of the fiber (45° angle).
Figure 7 shows the RIN of the Stokes beam out of two far-lying ZDWs fiber as a function of input laser average power before coupling to the PCF, for two different polarization states of the input laser. As Fig. 7 shows, the RIN of the 0° and 45° angles Stokes beams do not change much between an input average power range of 500 mW to 200 mW. However, they experience a significant reduction for input powers less than 200 mW, mostly because of exponential reduction of the MI process by reducing the input power [26]. This reduction seems more prominent for the 45° angle. Launching at 0° means that all the power is in the “x” direction of the fiber rather than divided between the “x” and the “y” as in the case of the 45° launching angle. This will result in higher order noise sensitive soliton fission and slightly more intensity RIN [26].
Fig. 7 The RIN of the Stokes beams generated by two different polarization angles of fs laser beam vs. input average power of the fs laser coupled to the PCF with two far-lying ZDWs, with a constant pulse width of ~100 fs. Error bars were negligible compared to the size of data points.
The RIN of the CARS captures the intensity stability, the polarization stability and the coherence of the Stokes beam. To separate the various effects we measure the RIN of the Stokes and generated CARS signal.
The RIN of the Stokes is a good indicator of the intensity stability of the Stokes beam. Since the fast photodetector is blind to the polarization variation of the Stokes beam, to detect the polarization instability of the 45° angle Stokes beam, compared to the slow axis direction of the two far ZDWs PCF, the RIN of the 45° angle Stokes beam was measured once with a polarizer in front of the photodetector (Fig. 4 (#17)) and once without the polarizer. The RIN difference in the two measurements shows the polarization instability of the 45° angle Stokes beam. As it is shown in the Fig. 8, the RIN of 45° angle Stokes beam increases by about 4 dB/Hz when the polarization instability of the 45° angle Stokes beam is considered.
Fig. 8 The RIN of the Stokes beams generated by two different polarization angles of 45° and 0° angles of fs laser coupled to the two far-lying ZDWs fiber, (The measured RIN value variance was ~+/− 0.54 dB/Hz).
Figure 9 shows the RIN of the CARS signal generated by the Stokes beam as a function of the average input laser power. As it is demonstrated, the RIN of the CARS signal generated by the Stokes beam produced by a 45° angle input laser beam (here it is called “45° Stokes”) was constant for all average input powers. This indicates that despite the significant reduction in the RIN intensity of the Stokes beam around 100-200 mW average input laser power as seen in Fig. 7, the 45° Stokes beam is still noisy and its polarization stability does not improve. However, for the Stokes beam generated by 0° input laser polarization direction (here it is called “0° Stokes”) there is a significant reduction in its intensity, coherence and polarization noises (RIN) for input laser average powers less than 200 mW. This can be explained as follows. At a 45° angle and in the absence of cross-phase modulation (XPM), the two polarization components in the “x” (slow axis of the fiber) and “y” (fast axis of the fiber) directions evolve very independently and separately from each other due to their different group velocities [26]. Nevertheless during the SC generation in the early stage, the “x” component gets more energy and compresses more than the “y” component because of interplay between SPM and XPM. With further propagation of a pulse in the “x” direction, the higher-order soliton fission happens earlier because of having a larger β2. This process also happens in the “y” direction, but in a more dramatic way, causing the polarization state of the SC out of the two far-lying ZDWs fiber becomes less stable [31]. Consequently, the 45° angle Stokes beam has less polarization stability.
Fig. 9 The RIN of the CARS signal using PCF with two far-lying ZDWs, generated by two different Stokes beams of two different input laser polarization angles (0° and 45° to the slow axis of the PCF). Average input laser power was ~100 mW and pulse width is 100 fs.
In Fig. 9, the ~-85 dB/Hz RIN of the CARS signal with an average power of 100 mW is comparable with the RIN of the CARS signal (~-84 dB) generated by the Stokes beam out of two closely-lying ZDWs fiber with an average power of 300 mW, which was selected as our standard CARS signal in all our measurements (Fig. 6). This indicates an intensity and polarization-stable Stokes beam out of two far-lying ZDWs fiber at this selected operating condition. In summary, launching a low power fs pulse at 0° will generate a more stable polarized SC, although it has a slightly more intensity RIN value compared to the 45° angle scenario for the same input power laser pulse.
The CARS intensity is proportional to the Stokes intensity, I Stokes, multiplied by the square of the intensity of the pump, I Pump, (Eq. (2) below). As CARS is a nonlinear process, it also depends on the polarization and coherence of both the pump and the Stokes beams [32]. Because the CARS signal intensity strongly depends on the polarization direction of the Stokes beam (ideally it should be parallel to the pump polarization direction), any polarization instability in the Stokes beam will generate less CARS signal and more noise that leads to an enhancement in the RIN value of the generated CARS signal (Fig. 9).
Figure 10 shows a comparison between the CARS signal intensity of an oil sample at 0° Stokes input angle for different average input powers. All the measured polarization, intensity and pulse width of the pump beam at the sample were constant since we used the same portion of the fs laser beam as our pump. As we have a constant and stable pump and identical experimental setup during all measurements, any changes in the intensity of the CARS signal can be attributed to either the power or quality (i.e. coherence, phase and polarization stability) of the Stokes beam at the sample. By comparing the intensity of the CARS signal and its corresponding Stokes beam power at the sample, we can determine the quality of the Stokes beam.
Fig. 10 The 0° Stokes power (~100 fs pulse width) at the focal point of the CARS microscope, at the oil sample, and the intensity of its generated CARS signal, as a function of the average laser power coupled to the PCF with two far-lying ZDWs. The averaged coupling efficiency = 40% (measurement error was less than 5%).
As shown in Fig. 10, by increasing the average input power (P0) coupled to two far-lying ZDWs fiber, the Stokes power at the sample increases. However, the CARS signal intensity does not follow a similar trend especially with low power, because of a substantial improvement of the Stokes signal quality by reducing the input laser power (P0) coupled to two far-lying ZDWs fiber. For example at P0 = 100 mW the Stokes power at the sample was half of the value of the Stokes power at P0 = 400 mW, while its corresponding CARS signal intensity was double, i.e. the Stokes quality was much better at P0 = 100 mW compared to 400 mW despite a 50% reduction in the Stokes power intensity at the sample. Unfortunately, it was found that by reducing P0 to values less than 100 mW, the Stokes power reduced significantly and rapidly, therefore, it was not sufficient to generate a strong and detectable CARS signal for performing CARS imaging of the biological samples. Consequently the P0 = 100 mW, with 0° polarization direction and ~100 fs pulse width with slightly positively chirped pulses, was chosen as the optimum operation point for using two far-lying ZDWs fiber to perform CARS imaging of lipid rich biological samples.
5. CARS imaging results
In order to show the impact of the optimization of the parameters of the laser input coupled to the two far-lying ZDWs fiber, CARS images of the 4.5 μm polystyrene beads sample were obtained (Fig. 11) under the same imaging conditions but at two different (optimized and non-optimized) input conditions for the fiber. As seen in Fig. 11 (b), at the condition of 230 fs pulse width and average input power of ~300 mw coupled into the two far-lying ZDWs fiber, the CARS image is more pixelated. This is attributed to an increase of the RIN of the CARS signal and is consistent with the ~3.6 dB/Hz worsening of the RIN seen earlier in Figs. 6 and 9, going from the optimized to non-optimized input condition for the two far-lying ZDWs fiber.
Fig. 11 The CARS image of the 4.5 μm polystyrene bead sample using two far-lying ZDWs fiber under similar imaging conditions, but with optimized (a) and non-optimized (b) input conditions of the fiber. (a) Pumping the PCF with 100 fs laser and average input power ~100 mW and (b) pumping the PCF with a 230 fs laser beam and average input power ~300 mW coupled into the fiber. Frame size is 256x 256 pixels with ~5.5 s acquisition time per frame.
To demonstrate the ability of using two far-lying ZDWs fiber at optimum operating conditions to perform high contrast and low noise CARS imaging of lipid density of biological samples, we performed CARS imaging of myelin structures of a fixed nerve sample (Fig. 12).
Fig. 12 CARS images (256 x 256 pixels) of myelin from fixed unstained mouse nerve using two far-lying ZDWs fiber module under optimum operating condition.
To compare the performance of two far-lying ZDWs fiber with the standard two closely-lying ZDWs fiber, the CARS image of a 4.5 μm polystyrene beads (with a strong Raman peak at 3070 cm−1) sample was performed.
Two far-lying ZDWs fiber was operated at its optimum operation condition (average power 100 mW, 100 fs pulse width and 0° polarization direction) while input fs laser beam parameters to the two closely-lying ZDWs fiber was 300 mW, 100 fs pulse width and 0° polarization direction. As it is shown in Fig. 13, there is no significant difference between the CARS image of the 4.5 μm bead sample using either fiber.
Fig. 13 (Above) (a) CARS images of 4.5 μm polystyrene bead sample using two far-lying ZDWs fiber operating at its optimum point and (b) two closely-lying ZDWs fiber operating at 300 mW average power input fs laser using our existing miniaturized fiber delivered CARS exoscope. (Below) (c) The intensity profile of a selected bead image, in above images ((a) and (b)), using both fibers.
6. Conclusion
In this paper, we investigated the conditions under which a module consisting of 12 cm of polarization-maintaining PCF with two far-lying ZDWs, can be used to perform a high quality and stable polarization CARS imaging of lipid rich density biological samples. The RIN of Stokes and CARS signals were measured as functions of the chirp, temporal duration, polarization and average power of the input femtosecond laser coupled to the PCF with two far-lying ZDWs. We showed that the generated Stokes from the PCF with two far-lying ZDWs often has a higher RIN and lower quality than the Stokes beam obtained from the module consisting of 12 cm of a PCF with two closely-lying ZDWs. However, a comparable high quality CARS signal is obtained from the fiber with two far-lying ZDWs, when the coupled laser has pulse duration around 100 fs, a slightly positive chirp and minimum possible average power (around 100 mW), as well as a polarization direction parallel to the slow axis of the PCF. The low fs laser power requirement for the two far-lying ZDWs (100mW versus 300mW) provides the opportunity to use this fiber with low power sources, such as femtosecond fiber lasers, to perform single fs laser CARS microscopy/endoscopy.
To demonstrate that the quality of the CARS signal using both fibers is comparable at optimal conditions, a CARS imaging of a 4.5 μm polystyrene bead sample was performed using both fibers. This work could be extended to determine the optimum operating conditions for CARS microscopy in the fingerprint region.
Acknowledgments
We express our special thanks to Dr. K. Khan, who read the first manuscript very thoroughly and gave us his valuable feedbacks and Dr. Hossein Aleyasin for helping in the preparation of mouse nerve biological samples.
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