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Abstract
In this work, we have demonstrated a stimulated emission (SE)-based pump-probe microscopy with subharmonic fast gate synchronization, which allows over an order of magnitude improvement in signal-to-noise ratio. Critically, the alternative way of modulation is implemented with the highest possible frequency that follows the lasers’ repetition rate. Its working is based on a homemade frequency divider that divides the repetition frequency (76 MHz) of the Ti:sapphire (probe) laser to half of the repetition frequency, 38 MHz, which is used to synchronously drive the pump laser and to provide the reference signal for the ensuing lock-in detection. In this way, SE can be detected with sensitivity reaching the theoretical (shot noise) limits, with a much lower time constant (0.1 ms) for faster image acquisition.
© 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
Nonlinear optics based pump-probe microscopy allows high contrast or label free imaging, such as stimulated emission (SE), stimulated Raman scattering (SRS), two-photon absorption (TPA), and excited state absorption (ESA). Pump-probe is a powerful technique in revealing molecular specificity with high sensitivity, enhanced penetration depth, and improved signal-to-noise ratio for biological and material sciences [1–9]. Specifically, SE is one of the most valuable contrasts, attributing to its transition through real states that enable an effective cross section several orders of magnitudes greater than the ones through virtual state transition. Over the years, SE has been the working mechanism to achieve super-resolution in stimulated emission depletion (STED) microscopy [10], in the context of pump-probe microscopy. Additionally, fluorescence lifetime imaging has been demonstrated through SE-based pump-probe setup with time delay configuration [11–13]. Sub-diffraction resolution imaging can also be achieved with a balanced detector and an annular filter using intensity modulated laser diode [14,15]. All of these approaches critically relied on lock-in detection with intensity modulation either on the pump or the probe beams. From the perspective of signal processing, the lock-in implement usually requires expansive electro-optics and acousto-optic modulators to achieve high frequency modulation, which leads to increased instrumentation complexity [16–18]. In conventional fluorescence SE microscopy, the general approach is to modulate the pump beam at a selected frequency. The SE signal carried by the probe beam is then extracted by demodulating the output of the photodetector placed in the forward propagating direction. However, detecting SE this way may saturate the detector, attributing to the overwhelming power of the probe beam relative to the very low SE signal. It is thus crucial to select a detector with high sensitivity, excellent high frequency response, and very broad dynamic range.
The very high modulation frequency (∼38MHz) has been realized in lock-in detection with intra-cavity wavelength modulation on an optical parametric oscillator [19]. Furthermore, the half repetition frequency technique has been reported earlier in SRS microscopy with two-branch ultrafast fiber source [20]. Additionally, the subharmonic synchronization with half repetition frequency on one of the (pulsed) lasers has been carried out with lock-in detection, achieving simultaneously the shot noise limited sensitivity, high image acquisition rate (in SRS spectral microscopy), and boxcar averaging with low duty cycle [21–25]. Note that raising the modulation frequency in the lock-in detection (ideally >1 MHz) serves two purposes: (1) The image acquisition time is greatly reduced by shortening the pixel dwell time (2) High modulation frequency also effectively minimizes the ubiquitous 1/f noise. In this work, we are exploring the capacity of high modulation frequency (∼38 MHz) in SE microscopy for fluorescence detection, which presents unique considerations as compared with other nonlinear optical modalities that are based on virtual state transition. The exemplification is conducted through ATTO647N fluorescent dye.
2. Experimental
2.1 The working principle of subharmonic fast gate synchronization
The physical mechanism of SE is shown in Fig. 1(a), as well as the comparison of SE signal detection with lock-in detection via an intensity or amplitude modulation, Fig. 1(b), and subharmonic fast gate synchronization, Fig. 1(c). In intensity modulation, the pump beam, ${I_{pu}}$, with repetition frequency, ${\omega _{rep\; ({pr} )}}$, is modulated at a frequency, ${\omega _m}$, and the demodulation signal embedded in the probe beam, ${I_{pr}}$, is extracted at the same frequency accordingly. For comparison, in subharmonic modulation the synchronization to the pump laser is implemented at the Nyquist frequency [20], i.e., the pump beam is gated at the half of the repetition frequency, $\frac{{{\omega _{rep\; ({pr} )}}}}{2}$, of the probe beam. The subharmonic modulation (gated at half of the repetition rate) serves to achieve very high modulation frequency with automatic synchronization between the pump and the probe pulses, while without attenuation or distortion on the modulation beam. Experimentally, a small part of the probe beam, ∼1 mW, is split to trigger the frequency divider (FD) circuit, which divides the repetition frequency of the probe beam into a designated fraction. The output of FD, set at half repetition frequency, is then connected to the fast gate port of the pump laser driver to achieve subharmonic fast gating modulation.
Fig. 1. (a) Jablonski diagram of pump-probe based SE process. S0 and S1 are the ground and the excited states. The excitation (pump beam), SE (probe beam), and fluorescence emission are represented by blue (
Unlike the intensity modulation with sinusoidal waveform, which has rounded rise and fall time, the pulse selection through subharmonic triggering contributes to sharp and efficient modulation that improves greatly the SNR in the lock-in detection. Note that when the duty cycle is 50%, the on-time and the off-time are equal, which presents the optimal case of modulation, since lock-in detection is essentially synchronized averaging.
2.2 Subharmonic modulation SE microscope setup
The subharmonic fast gating synchronization technique is detailed below.
The experimental setup of fast gating synchronized subharmonic modulation based SE microscopy is depicted in Fig. 2. The pump (excitation) beam at a wavelength λpu = 635 nm is generated by a picosecond pulsed diode laser driver (PDL 800-D, PicoQuant, Germany) that drives a laser head (LDH-D-C-635M, PicoQuant, Germany). The pulse width of the pump beam is approximately 120 ps. A Ti:sapphire laser (Mira 900-F, Coherent Inc., USA) is used as the probe (SE) beam, with a central wavelength, λpr = 780 nm, and the repetition frequency of 76 MHz. The pump beam is temporally synchronized with the probe beam by externally triggering the pump laser driver, with the synchronization signal coupled through the coaxial cable connecting a fast photodiode (TDA 200, PicoQuant, Germany). The pulse width of the probe beam is stretched to approximately 2.2 ps, after passing through the two long dispersive glass rods (SF-6) with a length of 15 cm each, to avoid two-photon excited fluorescence. A small part of the probe beam (∼1 mW) is split into the home-made frequency divider (FD) circuit to generate the synchronization triggering by a piece of cover glass. The main part of the beam is directed into the scanning microscope setup. The pump and the probe beams are aligned and combined within the galvano-mirror scanning system (FV300, Olympus, Japan). The output voltages for fast gating and lock-in amplifier reference are shown is Fig. 3(a). The repetition frequency of the probe beam is divided by the FD circuit into half (38 MHz), which is in turn used to trigger the pump laser driver as well as providing the reference for the high speed lock-in amplifier (HF2LI, Zurich Instrument, Switzerland).
Fig. 2. Experimental setup of subharmonic fast gate synchronized SE microscopy. LIA: Lock-in amplifier; PDL: Pulsed laser driver; M: Mirror; FD: Frequency divider; TDA: Trigger diode assembly; BS: Beam splitter; GR: Glass rod; OL: Objective lens; S: Sample; CL: Condenser lens; L: Lens; F: Filter; PD: Photodiode.
Fig. 3. (a) The oscilloscope signals for fast gate (black line), lock-in reference signal (red line), and the FD response to the probe beam (blue line). (b) The noise power spectrum of the PD circuit without optical input.
A pair of objective lenses (UPlan FLN 20×/0.50 and UPlanSapo 20x/0.75, Olympus Japan) are used to focus the beams and collect the SE signal. A bandpass filter (FF01-769/41-25, Semrock, USA) is placed before the home-made Si PIN photodiode (S3399, Hamamatsu, Japan) circuit to discriminate the pump beam and pass only the probe beam which carries SE signal. The noise power spectrum of the PD circuit is shown Fig. 3(b). The relatively broad spectrum ranging from 28 MHz to 44 MHz indicates a broadband response of the PD circuit. Critically, the circuit is able to discriminate signal at the primary frequency, 76 MHz. The demodulation signal at 38 MHz is detected by the PD circuit. The time constant of the lock-in is set at 0.1 ms for all measurements. The output of the lock-in amplifier is connected to the A/D converter of the galvano-mirror based laser scanning system to reconstruct the SE image.
The diagram of the PD circuit is shown in Fig. 4. The PD (D1) is biased at 5 V, and its photocurrent is filtered with a 3rd-order Butterworth bandpass filter centered at 38 MHz and a band elimination filter at 76 MHz. The bandpass filter is composed of inductors (L1, L2, and L3), and capacitors (C1, C2, and C3), and the band elimination filter is composed of L4, C4 and a trimmer capacitor (VC1). Then the photocurrent is led to the load resistor (R1), and its voltage is amplified with AD8099 (U1). Its output is again filtered with another bandpass filter and another band elimination filter, and then amplified with U2. In the design of the circuit, we have taken into account the junction capacitance of the photodiode, which is compensated for by adjusting C1.
Fig. 4. The circuit diagram of the PD circuit. The operation current and voltage are <200 mA and ± 5 V, respectively.
3. Results and discussion
3.1 Detection of SE signal from the fluorescent dye
To evaluate the photodetector saturation, we used aqueous ATTO647N fluorescent dye solution (in deionized water) with various concentration (0.1-1 mM) for SE signal measurement. The power of the pump beam is set at 1.5 mW and the SE signals are detected by varying the probe beam powers. Figure 5(a) shows that the SE signal increases linearly with the probe beam power under various concentrations. The figure also shows that when the power of probe beam is above 28 mW, the SE signal starts to saturate. The power used in our study is also sufficient for biological imaging experiments. Note that the ATTO647N fluorescent dye has been widely used in STED microscopy due to its excellent photo-stability under high laser intensity. Figure 5(b) shows the linear dependence of SE signal on dye concentrations.
Fig. 5. (a) SE signals from samples with varying dye concentrations as a function of the probe beam powers. Photodetector saturation is observed when the power of the probe beam is greater than 28 mW. (b) SE signal as a function of dye concentration, with the pump beam set at 1 mW and the probe beam at 5 mW, respectively.
3.2 Evaluation of shot noise, sensitivity, and signal-to-noise ratio
To measure the shot noise limit, the probe beam is focused on the photodiode with the pump beam completely blocked. The fluctuations of the signal over a period of time is then recorded as a function of varying input optical powers, as shown in Fig. 6. According to the Poisson distribution, the number of the photocarriers is equal to its variance. Therefore, the optical power can be evaluated by taking the variance divided by the slope of the graph which corresponds to a DC photocurrent of 1.64 mA, taking the responsivity (r) of the photodiode being 0.58 (A/W) at the wavelength of 780 nm. The theoretical estimation is derived as the following.
Fig. 6. Signal variance, σ2, as a function of optical powers. The variance and slope of the graph are 3.26×10−10 and 1.15×10−10, respectively.
The thermal noise of the load resistance R = 100 Ω and first operational amplifier (AD8099) are given by ${v_R} = \frac{{1.3\; nV}}{{\surd Hz}}$ and ${v_A} = \frac{{0.9\; nV}}{{\surd Hz}}$. Therefore, the total thermal noise is ${v_T} = \surd ({v_R}^2 + {v_A}^2) = \frac{{1.6\; nV}}{{\surd Hz}}$. For comparison, the theoretical shot noise current can be estimated by Eq. (1),
where ${v_T}$ is the total thermal noise voltage, R = 100 Ω is the load resistance, and q = 1.6×10−19 C is the elementary charge. The experimental value, 1.64 mA, is only slightly higher than the theoretical one, found to be 0.8 mA according to Eq. (1), indicating excellent sensitivity approximating the shot noise limit and there may be other sources of noise and loss of signal. Note that the phase mismatch of the repetition frequency with external modulation could be another source of noise. For comparison, subharmonic synchronization, the on-off time of modulation signal is perfectly correlated to reference signal for the lock-in detection and the shot noise is confined only in the in-phase component [21].The SNR of SE signal mainly depends on several parameters such as time constant of the lock-in, power of the probe beam, repetition frequency of the laser and thickness of the sample. We can derive the signal-to-noise (SNR), as given by the following equations:
The probe or SE beam induced SE signal per duty cycle can be expressed as [26]:
3.3 Subharmonic SE imaging
The subharmonic modulation based technique for SE imaging is also demonstrated on a labeled biological sample. The blood vessel within the brain tissue slice, with a thickness of 15 µm, from a normal mouse is labeled by anti-CD34 conjugated ATTO 647N-Streptavidin. The pump and the probe beam powers are set at 1 mW and 5 mW, respectively. Figures 7(a) and 7(b), taken independently and approximately 5 minutes apart (with some lateral offset), show the confocal and the subharmonic SE images of a blood vessel within the slice. Note that the SE image (Fig. 7(b)) exhibits higher definition and resolution when compared with the confocal one (Fig. 7(a)), which is attributed to the 2-photon process of SE being less affected by scattering. Additionally, the confocal pinhole alignment is more susceptible than optimizing the overlapping of the pump and the probe beams collinearly. The comparison is similar between confocal microscopy and two-photon microscopy.
Fig. 7. (a) Confocal image versus (b) subharmonic SE image of a blood vessel. The SE image is recorded at 512×512 pixels with a scale bar of 50 µm. The time constant of the lock-in amplifier is set at 0.1 ms. Accordingly, the pixel dwell time is also set at 100 µs, to match the lock-in time constant.
4. Conclusion and outlook
In summary, we present the implement of subharmonic fast gating synchronization technique in SE microscopy for fluorescence detection. Using subharmonic synchronization, the maximum modulation frequency (38 MHz) on the pump laser is achieved without expansive external modulator. In addition, the home-made PD circuit with built-in bandpass filter and the high dynamic range photodetector is advantageous in achieving shot noise limited sensitivity. Imaging of a biological sample is also demonstrated, showing the sensitivity is comparable with confocal microscopy and the image definition is improved. The high modulation frequency approach thus allows greatly reduced pixel dwell times and high speed imaging.
Specifically, we have adapted subharmonic modulation for the applications in SE microscopy that takes place through transitions via real states. Notably, the major difference between SRS and SE is that SRS takes place through virtual states while SE is through real states. The transition of SE has much greater cross sections (∼106) and comes with a measurable lifetime. It is not possible to use gain switched diode laser (with pulse width in the sub-nanosecond regime) in SRS, as in the case of SE. The transition through virtual states in SRS also excludes the possibility of synchronizing the pump and the probe pulses electronically. In another word, the very long lifetime and very large cross section in SE allows far more flexibility than SRS. A unique property of a florescence dye molecule is its lifetime. However, conducting fluorescence lifetime imaging microscopy can be costly using time-correlated single-photon counting (TCSPC) or a gated camera. The pump-probe configuration with controlled delay provides an advantageous alternative [12].
On the other side, due to the length of the fluorescence lifetime, the highest repetition frequency is limited accordingly, i.e., the period of laser pulse repetition cannot be less than the fluorescence lifetime.
Funding
Core Research for Evolutional Science and Technology (JPMJCR1872); Japan Society for the Promotion of Science (JP18K18847); Ministry of Science and Technology, Taiwan (MOST 105-2112-M-010-001-MY3, MOST 108-2112-M-010-001).
Acknowledgments
The authors would like to thank Mr. Khalil Ur Rehman and Mr. Che-Lun Hsu of National Yang-Ming University for their support on some part of this work. We are also grateful to Dr. Yuh-Chiang Shen of National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taiwan for preparing the biological sample.
Disclosures
The authors declare no conflicts of interest.
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), red (
), and green (
) arrows, respectively. The working principles of lock-in detection of SE signal with (b) an intensity or amplitude modulation and (c) subharmonic synchronization at half repetition frequency. The SE signal