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Time alignment optimization of pulsed STED microscopy
Optik 127 (2016) 6610–6617
Contents lists available at ScienceDirect
Optik journal homepage: www.elsevier.de/ijleo
Original research article
Time alignment optimization of pulsed STED microscopy Fei Gao, Yunhai Zhang ∗ , Haomin Yang, Yun Xiao, Tongda Wei, Jian Chang Jiangsu Key Laboratory of Medical Optics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, PR China
a r t i c l e
i n f o
Article history: Received 22 February 2016 Accepted 15 April 2016 Keywords: Time alignment STED Superresolution
a b s t r a c t Stimulated emission depletion (STED) microscopy implemented with both pulsed excitation and STED beams (pulsed STED) is especially suitable in biological superresolution imaging as it reduces photobleaching rates and provides better resolution. Proper time alignment between the excitation and STED pulse can improve resolution. To get the optimal time alignment the fluorescence depletion effect was analysed under different time alignments in theory first. Then, the relationship between the time alignment and the position of two moveable mirrors used for changing the excitation optical path length was established. Finally, the depletion experiments were performed under different time alignments. By both simulation and experimentation, the optimal time alignment was got which was about 210 ps when the widths of excitation and STED pulses were both 120 ps. STED image with 60 nm resolution was achieved based on the optimal time alignment. The method provides a reliable way to optimize the time alignment of pulsed STED microscopy. © 2016 Elsevier GmbH. All rights reserved.
1. Introduction In 1994, Hell and Wichmann [1] first proposed and demonstrated STimulated Emission Depletion (STED) microscopy which got superresolution in far-field fluorescence microscopy using point spread function engineering. Traditional farfield optical microscopy has a limited resolution which is about 250 nm in lateral and about 500 nm in axial when using visible light [2]. However, STED microscopy can achieve sub–100 nm resolution [3–8] by depopulating the excited states of the fluorophores within the doughnut beam profile. Therefore the effective point spread function of the fluorescence is smaller than the diffraction limit. A theoretically unlimited resolution can be obtained which is given by Eq. (1) [9–11]: d ≈ /(2NA
1 + ISTED /IS ),
(1)
where denotes the wavelength of light, NA the numerical aperture of the objective lens, ISTED the maximum intensity of the STED doughnut, and IS a saturation intensity which is characteristic of the dye. However, due to such factors as photobleaching, the intensity of the STED light has a limit. Thus the resolution cannot be improved unlimitedly. Lateral resolution of /25 and axial resolution of /23 has been achieved in STED microscopy by Westphal et al. [12]. So far, combination of pulsed excitation beam and pulsed STED beam (known as pulsed STED), pulsed excitation beam and continuous STED beam, or continuous excitation beam and continuous STED beam can be applied in STED microscopy [6,13]. Compared with the other two, pulsed STED microscopy reduces fluorescence photobleaching rates as pulsed laser
∗ Corresponding author at: Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, PR China. E-mail address: [email protected] (Y. Zhang). http://dx.doi.org/10.1016/j.ijleo.2016.04.094 0030-4026/© 2016 Elsevier GmbH. All rights reserved.
F. Gao et al. / Optik 127 (2016) 6610–6617
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Fig. 1. The energy levels of a typical fluorophore.
has much higher peak intensity than continuous wave laser with the same average power. The time alignment between the excitation pulse and the STED pulse seems to be less than molecular vibrational relaxed time (generally less than 1 ps) to de-excite excited molecules by stimulated emission effectively. However, because the STED pulse has a Gaussian shape in time domain and high intensity, its rising edge has depletion effect. As a result, a proper time alignment can improve the resolution of pulsed STED microscopy [14–16]. In this paper, we simulated the fluorescence depletion effect versus the time alignment, and then presented an experimental method to obtain the optimal time alignment.
2. Theory The energy levels in Fig. 1 shows the excitation and the subsequent emission process of a typical fluorophore. S0 and S1 are the ground and the first excited electronic state, respectively. L0 is a low vibrational level of S0 , and L3 is a higher level of S0 . Similarly, L2 is the relaxed vibrational level of S1 , and L1 is the directly excited level of S1 . The excitation light generates the transition from L0 to L1 , and after vibrational relaxation (from L1 to L2 ), the STED light induces the transition from L2 to L3 by stimulated emission and depletes the excited state before fluorescence takes place. The temporal behaviours of the population probabilities ni (t) of the levels Li (i = 0, 1, 2, 3) of dyes are described by a set of coupled differential equations (Eq. (2)) with initial and normalization condition (Eq. (3)) under the room temperature and isolated system. IEXC is the intensity of excitation beam, and ISTED is the intensity of STED beam. In an excitation and depletion period, IEXC and ISTED are Gaussian distribution in time domain. EXC and STED are the wave frequency of excitation and STED pulses, respectively. 01 and 23 are the cross sections for the absorptions from L0 to L1 and from L3 to L2 , respectively. Typical values for 01 and 23 are 10−16 cm2 . FLUO is the average fluorescence lifetime and VIBR is the average vibrational relaxation time for L1 to L2 and L3 to L0 . FLUO is of the order of 2 ns, VIBR is of the order of 0.1 ps, and the quenching rate Q is typically 108 s−1 . The value of the Planck Constant h is 6.62606957 × 10−34 Js. To simulate the optimal time alignment of the excitation pulse and the STED pulse, it assumes that the widths of the Gaussian shaped excitation and STED pulses are both 120 ps; the peak intensity are both 30MW/cm2 ; and the wavelengths are 488 nm and 592 nm, respectively. So IEXC , ISTED , EXC and STED are given by Eq. (4). IEXC 01 dn0 1 = (n1 − n0 ) + n3 , VIBR dt hEXC IEXC 01 1 dn1 = (n0 − n1 ) − n1 , VIBR dt hEXC dn2 ISTED 23 1 1 = (n3 − n2 ) + n1 − ( + Q )n2 , VIBR FLUO dt hSTED
(2)
ISTED 23 1 1 dn3 = (n2 − n3 ) − n3 + ( + Q )n2 VIBR FLUO dt hSTED
n0 |t=0 = 1, ni |t=0 = 0(i = 1, 2, 3),
3 i=0
ni = 1
(3)
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Fig. 2. (a) (t = −50 ps, 200 ps, 450 ps) and (c) (t = 160 ps, 220 ps, 280 ps) show the relationship between the population probability of L2 (n2 ) and the time (t) under different time alignments (t). (b) (the time alignment range from −100 ps to 450 ps with an interval of 50 ps) and (d) (the time alignment range from −160 ps to 290 ps with a finer interval of 10 ps) show the relationship between the average value of n2 and t.
−
(t − 258 × 10−12 )
IEXC = 30 × e −
ISTED = 30 × e
vEXC = vSTED =
c EXC
=
(120 × 10−12 )
2
2
MW/cm2 ,
(t − 258 × 10−12 − t) (120 × 10−12 ) 3 × 108 m/s 488 × 10−9 m
2
2
MW/cm2 ,
(4)
= 6.147541 × 1014 s−1 ,
c 3 × 108 m/s = 5.067568 × 1014 s−1 = STED 592 × 10−9 m
The population probability of L2 reveals the intensity of spontaneous fluorescence and the depletion efficiency. In order to obtain the optimal time alignment, the rate equations of fluorophore (Eq. (2) and Eq. (3)) were solved by 4-order Runge-Kutta algorithm, and the population probability of L2 (n2 ) versus the time (t) under different time alignments (t) were simulated. Firstly, the time alignment was varied from −100 ps to 450 ps with an interval of 50 ps, and under each time alignment, the population probability of L2 versus the time was simulated. Fig. 2(a) shows some of the curves that n2 as function of t. To directly show the depletion effect, an average of n2 was calculated by Eq. (5): n2 = (n2 (t1 ) + n2 (t2 ) + ... + n2 (tn ))/n, n = 5000, t1 = 0ps, t2 = 1ps, ......, t5000 = 4999ps
,
(5)
where n2 (ti ) is the value of n2 at time ti , and n is the algorithm steps. Fig. 2(b) indicates that less population probability of L2 , i.e., higher depletion efficiency is under the time alignment range from 150 ps to 300 ps. To view the detail of the depletion effect, we varied the time alignment from −160 ps to 290 ps with a finer step of 10 ps, and under each time alignment, the simulation mentioned above was performed again. The results were illuminated in Fig. 2(c) and (d). Fig. 2(d) shows that the least population probability of L2 , i.e., the highest depletion efficiency is under the time alignment around 210 ps. Therefore, the optimal time alignment about 210 ps was achieved by simulation when the width of excitation and STED pulses were both 120 ps.
F. Gao et al. / Optik 127 (2016) 6610–6617
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Fig. 3. (a) shows the scheme and (b) is a photograph of the experimental setup. The supercontinuum laser is split into two beams using a dichroic beam splitter (D1) from which the excitation and STED wavelengths are extracted by means of two band pass filters (BP1 and BP2). Both beams are expanded and collimated by beam expanders (L1, L2 and L3, L4), and coupled into the main light path using two dichroic beam splitters (D2 and D3). D2 is installed in a kinematic mirror mount with two piezo adjusters to align the excitation and STED beams accurately. Two moveable mirrors (M2 and M3) are installed on an optical delay line to adjust the time alignment between the excitation pulse and the STED pulse. The doughnut shape is produced by using a vortex-phase plate (VPP). Both excitation light path and depletion light path have a half-wave plate (AHWP1 and AHWP2) for adjusting the polarization, and a quarterwave plate (AQWP) generating circular polarization. P1, P2: polarizer; BP3: band pass detection filter; L5: pinhole lens; SM: scan mirrors of galvanometer type; SL: scan lens; TL: tube lens; OL: 100× objective lens.
3. Experimental method After simulation, we presented an experimental method to obtain the optimal time alignment of a customized pulsed STED microscopy system. Fig. 3(a) shows the scheme, and Fig. 3(b) is a photograph of the experimental setup. A commercial
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Fig. 4. The excitation pulse and the STED pulse displayed on the oscilloscope. (a) x1 = 0 mm, t1 = −716.80 ps; (b) x2 = 220 mm, t2 = 750.40 ps.
supercontinuum fiber laser (SC-450-PP-HE, Fianium, UK) was used to provide both excitation and STED pulses by using a dichroic beam splitter. The excitation wavelength of 488 nm and the STED wavelength of 592 nm were extracted by two band pass filters, respectively. Since originating from the same light source, the excitation and STED pulses are inherently synchronized. Thus, the time alignment can be adjusted by changing the excitation optical path lengths with a proper difference compared with the depletion optical path. The supercontinuum laser has the total power of 2W and the width of the pulses is 120 ps, which are appropriate for STED. A 1 MHz repetition rate was used to limit photobleaching of the fluorophore. The key points of the time alignment optimization method were as follows: (1) Two moveable mirrors installed on an optical delay line (ODL220-FS/M, Thorlabs, USA) were used to change the excitation optical path length. The relationship between the time alignment and the position of the moveable mirrors was determined quantificationally. In Fig. 3(a), the difference of the excitation path length and the STED path length is S = 2 × (S1 -S2 ). S was increased while S2 was decreased when the moveable mirrors were moved from the position of 0 mm to 220 mm. Next, the relationship between the time alignment and the position of the moveable mirrors was determined quantificationally. The excitation and STED beams were collected into a same fast photodiode (G4176-03, 30 ps rise time, Hamamatsu, Japan). The photodiode’s output was connected to a bias tee (5541A, Pulse Labs, USA) to remove the DC component, and then fed into a fast oscilloscope. The two pulse trains can be observed approaching together when the moveable mirrors along the optical delay line are properly moved. Then the time alignment can be read out from the display of the oscilloscope. When the mirrors were moved to the position of x1 = 0 mm and x2 = 220 mm, the time alignment read from the display were t1 = −716.80 ps and t2 = 750.40 ps respectively, as shown in Fig. 4. It is noted that the oscilloscope cannot resolve the peaks of the excitation and STED pulses if they are too close. The relationship between the time alignment (t) and the position of the moveable mirrors (x) is linear, so it could be described by Eq. (6): t =
t1 − t2 t2 × x1 − t1 × x2 ×x+ = 6.67x − 716.80 x1 − x2 x1 − x2
(6)
(2) The depletion experiments at different positions of the moveable mirrors were performed and the depletion efficiency was calculated. The optimal time alignment was corresponding to the position of maximum depletion efficiency. The depletion experiments needed to be performed using Gaussian shaped STED beam. Therefore the blank area of the vortex-phase plate was placed to the STED path instead of taking it away, so that the STED path length was almost unchanged. The mirrors were moved to different positions, and at each position, 40 nm fluorescent beads were imaged in excitation mode (excitation beam on, STED beam off), depletion mode (excitation beam on, STED beam on) and excitation mode (excitation beam on, STED beam off) in sequence. The imaging was performed at an excitation/STED wavelength pair of 488 nm/592 nm. The laser power at the back aperture of the objective lens was ∼1 W for excitation and ∼100 W for STED. I1 was used to denote the total summed pixel value of the first excitation mode image, and I2 and I3 were used to denote that of the depletion mode and the second excitation mode images. The depletion efficiency could be described as 1-I2 /I1 , and the higher depletion efficiency meant the better time alignment. The experimental data was presented in Table 1.
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Table 1 The data of the depletion experiments. x (mm)
t (ps)
Vx, Vy (volts)
1-I2 /I1
I3 /I1
120 125 130 135 140 145 150 155 160 165
83.6 116.95 150.3 183.65 217 250.35 283.7 317.05 350.4 383.75
90,80 95,80 100,80 100,85 100,80 100,85 95,85 90,85 90,80 90,85
0.3106 0.392 0.4803 0.4842 0.511 0.494 0.491 0.4631 0.4676 0.4504
0.9936 0.9986 0.9962 0.9953 1.0058 0.9955 0.9926 1.0049 1.0018 0.9977
Fig. 5. The depletion efficiency as function of the position of the moveable mirrors and the time alignment between the excitation pulse and the STED pulse.
In STED microscopy, the excitation beam and the STED beam should be combined together, so that their focus point by the objective could be precisely overlapped. The measurement above might be influenced by the alignment precision of the excitation and STED beams and the fluorescence photobleaching. In order to exclude the possible influence by the fluorescence photobleaching, a lower intensity of the excitation and STED beams should be used. The I3 to I1 ratios (as I3 /I1 listed in Table 1) were very closed to 1 and this indicated that there was almost no photobleaching during the experiments. Some ratios value were larger than 1 because of noise during the image process. Since the movement of the moveable mirrors might cause drifts of the excitation beam, we must align the excitation beam and the STED beam accurately at each position of the moveable mirrors. The alignment experiments were performed through imaging the excitation and STED point spread functions by measuring the scatter light from 60 nm gold nanoparticles. To avoid frequently switching between the depletion experiments and the alignment experiments, we adopted a dichroic mirror which was installed in a kinematic mirror mount with two piezo adjusters. We combined the excitation beam and the STED beam precisely at each position of the moveable mirrors and recorded the corresponding piezo voltages (as Vx, Vy listed in Table 1). Since the piezo adjusters have extremely accurate repeatability, when the moveable mirrors position was changed, the excitation beam and the STED beam can be rapidly combined together with high precision by reloading the piezo voltages recorded before. Therefore, we could perform the above mentioned depletion experiments uninterruptedly. Fig. 5 shows that there is maximum depletion efficiency when the moveable mirrors are at the position of 140 mm, and the corresponding time alignment is 217 ps. So the optimal time alignment of the developed pulsed STED microscopy system is 217 ps that accords with the simulation result. Fig. 6 shows the excitation image before depletion, the depletion image and the excitation image after depletion under the optimal time alignment.
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Fig. 6. The excitation image before depletion (a), the depletion image (b), and the excitation image after depletion (c) under the optimal time alignment. The scale bar represents 1 m.
Fig. 7. Comparison between confocal (a) and STED (b) images of fluorescent beads. The confocal cross section along the traces indicated by the arrows fits to Gaussion and The STED cross section along the traces indicated by the arrows fits to Lorentzian (c). The scale bar represents 1 m.
4. Results and discussion To test the optimal time alignment, a sample of 40 nm fluorescent beads (Yellow-Green FluoSpheres, 40 nm, Molecular Probes, USA) immobilized on a coverslip was first imaged in confocal mode and then in STED mode. When imaged in STED mode, the effective region of the vortex phase plate was inserted into the STED light path. The imaging was performed at an excitation/STED wavelength pair of 488 nm/592 nm. The laser power at the back aperture of the objective lens was ∼1 W for excitation and ∼1 mW for STED. Images were generated by raster scanning the sample over the beams, using a piezoelectric stage (P733.3DD, Physik Instrumente (PI) GmbH & Co. KG, Germany) which has sub-nanometer accuracy. Imaging area was 250 × 250 pixels with 20 nm pixel size and 250 s pixel dwell time. The fluorescence photons were detected by an avalanche photodiode module (SPCM-AQRH-16-FC, Perkin Elmer, Canada) which was connected to a DAQ card (PCIe-6361, National Instruments, USA). Fig. 7 presents images recorded in the confocal mode and the STED mode. Closely spaced nanospheres cannot be resolved in the confocal image, but are clearly discernible in the STED image. The STED cross section indicated by the arrows was fit to a Lorentzian, for which there is precedent in the literature [17,18], and it yielded a FWHM of 70 nm. Taking into account the actual bead diameter the STED image has about 60 nm resolution. In Fig. 8, a microtubule network was imaged both in confocal and STED mode. Images were generated by raster scanning the beams over the sample, using a pair of x-y galvanometers (6215H, Cambridge Technology, USA) with 28 nm pixel size and 500 × 500 pixels. It can be seen that detailed structures and closely spaced fibers are revealed in the STED image while blurred in the confocal image. The significant improvement of resolution in the STED mode verifies the effectiveness of above time alignment optimization method. 5. Conclusions As the STED pulse has the Gaussian shape in time domain and high intensity, its rising edge has depletion effect. As a result, a proper time alignment between the excitation and STED pulse can improve the resolution of pulsed STED microscopy. The optimal time alignment about 210 ps was obtained by simulation when the pulse width of the excitation and STED pulses were both 120 ps. An experimental method was presented to obtain the optimal time alignment of pulsed STED microscopy after simulation. Through the depletion experimental method, the 217 ps optimal time alignment was got for a customized STED microscopy system which was similar to the simulation result 210 ps. Superresolution image with resolution of 60 nm was achieved in the STED microscopy using the optimal time alignment. The simulation and experimental methods in this paper provide a reliable way to optimize the time alignment of pulsed STED microscopy.
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Fig. 8. The confocal (a) and the STED (b) images of the microtubule network. All images are raw data. The scale bar represents 1 m.
Acknowledgments This work is supported by the Program for the National key scientific and research equipment development Foundation of China under Grant No. ZDYZ2013-1. References [1] S.W. Hell, J. Wichmann, Breaking the diffraction resolution limit by stimulated-emission: stimulated-emission-depletion fluorescence microscopy, Opt. Lett. 19 (1994) 780–782. [2] E. Abbe, Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung, Arch. Mikrosk. Anat. 9 (1873) 413–418. [3] T.A. Klar, S.W. Hell, Subdiffraction resolution in far-field fluorescence microscopy, Opt. Lett. 19 (1999) 954–956. [4] T.A. Klar, E. Engel, S.W. Hell, Breaking Abbe’s diffraction resolution limit in fluorescence microscopy with stimulated emission depletion beams of various shapes, Phys. Rev. E 64 (2001) 066613-1––066613-9. [5] D. Wildanger, E. Rittweger, L. Kastrup, S.W. Hell, STED microscopy with a supercontinuum laser source, Opt. Express 16 (2008) 9614–9621. [6] G. Moneron, R. Medda, B. Hein, A. Giske, V. Westphal, S.W. Hell, Fast STED microscopy with continuous wave fiber lasers, Opt. Express 18 (2010) 1302–1309. [7] S. Schrof, T. Staudt, E. Rittweger, N. Wittenmayer, T. Dresbach, J. Engelhardt, S.W. Hell, STED nanoscopy with mass-produced laser diodes, Opt. Express 19 (2011) 8066–8072. [8] D. McBride, C. Su, J. Kameoka, S. Vitha, A low cost and versatile STED superresolution fluorescent microscope, Mod. Instrum. 2 (2013) 41–48. [9] V. Westphal, S.W. Hell, Nanoscale resolution in the focal plane of an optical microscope, Phys. Rev. Lett. 94 (2005) 143903-1–143903-4. [10] S.W. Hell, Microscopy and its focal switch, Nat. Methods 6 (2009) 24–32. [11] B. Harke, J. Keller, C.K. Ullal, V. Westphal, A. Schönle, S.W. Hell, Resolution scaling in STED microscopy, Opt. Express 16 (2008) 4154–4162. [12] V. Westphal, L. Kastrup, S.W. Hell, Lateral resolution of 28 nm (/25) in far field fluorescence microscopy, Appl. Phys. B 77 (2003) 377–380. [13] Y. Wang, C. Kuang, Z. Gu, Y. Xu, S. Li, X. Hao, X. Liu, Time-gated stimulated emission depletion nanoscopy, Opt. Eng. 52 (2013) 093107-1–093107-8. [14] J. Yu, J. Yuan, X. Fang, Y. Li, Effects of excitation and depletion process on resolution of stimulated emission depletion microscope, Guangxue Xuebao 30 (2010), s100405-1–s100405-7. [15] M. Schrader, F. Meinecke, K. Bahlmann, M. Kroug, C. Cremer, E. Soini, S.W. Hell, Monitoring the exited state of a fluorophore in a microscope by stimulated emission, Bioimaging 3 (1995) 147–153. [16] S. Galiani, B. Harke, G. Vicidomini, G. Lignani, F. Benfenati, A. Diaspro, P. Bianchini, Strategies to maximize the performance of a STED microscope, Opt. Express 20 (2012) 7362–7374. [17] G. Vicidomini, G. Moneron, K.Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, S.W. Hell, Sharper low-power STED nanoscopy by time gating, Nat. Methods 8 (2011) 571–573. [18] J.A. Fitzpatrick, Q. Yan, J.J. Sieber, M. Dyba, U. Schwarz, C. Szent-Gyorgyi, C.A. Woolford, P.B. Berget, A.S. Waggoner, M.P. Bruchez, STED nanoscopy in living cells using Fluorogen Activating Proteins, Bioconjug. Chem. 20 (2009) 1843–1847.
Contents lists available at ScienceDirect
Optik journal homepage: www.elsevier.de/ijleo
Original research article
Time alignment optimization of pulsed STED microscopy Fei Gao, Yunhai Zhang ∗ , Haomin Yang, Yun Xiao, Tongda Wei, Jian Chang Jiangsu Key Laboratory of Medical Optics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, PR China
a r t i c l e
i n f o
Article history: Received 22 February 2016 Accepted 15 April 2016 Keywords: Time alignment STED Superresolution
a b s t r a c t Stimulated emission depletion (STED) microscopy implemented with both pulsed excitation and STED beams (pulsed STED) is especially suitable in biological superresolution imaging as it reduces photobleaching rates and provides better resolution. Proper time alignment between the excitation and STED pulse can improve resolution. To get the optimal time alignment the fluorescence depletion effect was analysed under different time alignments in theory first. Then, the relationship between the time alignment and the position of two moveable mirrors used for changing the excitation optical path length was established. Finally, the depletion experiments were performed under different time alignments. By both simulation and experimentation, the optimal time alignment was got which was about 210 ps when the widths of excitation and STED pulses were both 120 ps. STED image with 60 nm resolution was achieved based on the optimal time alignment. The method provides a reliable way to optimize the time alignment of pulsed STED microscopy. © 2016 Elsevier GmbH. All rights reserved.
1. Introduction In 1994, Hell and Wichmann [1] first proposed and demonstrated STimulated Emission Depletion (STED) microscopy which got superresolution in far-field fluorescence microscopy using point spread function engineering. Traditional farfield optical microscopy has a limited resolution which is about 250 nm in lateral and about 500 nm in axial when using visible light [2]. However, STED microscopy can achieve sub–100 nm resolution [3–8] by depopulating the excited states of the fluorophores within the doughnut beam profile. Therefore the effective point spread function of the fluorescence is smaller than the diffraction limit. A theoretically unlimited resolution can be obtained which is given by Eq. (1) [9–11]: d ≈ /(2NA
1 + ISTED /IS ),
(1)
where denotes the wavelength of light, NA the numerical aperture of the objective lens, ISTED the maximum intensity of the STED doughnut, and IS a saturation intensity which is characteristic of the dye. However, due to such factors as photobleaching, the intensity of the STED light has a limit. Thus the resolution cannot be improved unlimitedly. Lateral resolution of /25 and axial resolution of /23 has been achieved in STED microscopy by Westphal et al. [12]. So far, combination of pulsed excitation beam and pulsed STED beam (known as pulsed STED), pulsed excitation beam and continuous STED beam, or continuous excitation beam and continuous STED beam can be applied in STED microscopy [6,13]. Compared with the other two, pulsed STED microscopy reduces fluorescence photobleaching rates as pulsed laser
∗ Corresponding author at: Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, PR China. E-mail address: [email protected] (Y. Zhang). http://dx.doi.org/10.1016/j.ijleo.2016.04.094 0030-4026/© 2016 Elsevier GmbH. All rights reserved.
F. Gao et al. / Optik 127 (2016) 6610–6617
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Fig. 1. The energy levels of a typical fluorophore.
has much higher peak intensity than continuous wave laser with the same average power. The time alignment between the excitation pulse and the STED pulse seems to be less than molecular vibrational relaxed time (generally less than 1 ps) to de-excite excited molecules by stimulated emission effectively. However, because the STED pulse has a Gaussian shape in time domain and high intensity, its rising edge has depletion effect. As a result, a proper time alignment can improve the resolution of pulsed STED microscopy [14–16]. In this paper, we simulated the fluorescence depletion effect versus the time alignment, and then presented an experimental method to obtain the optimal time alignment.
2. Theory The energy levels in Fig. 1 shows the excitation and the subsequent emission process of a typical fluorophore. S0 and S1 are the ground and the first excited electronic state, respectively. L0 is a low vibrational level of S0 , and L3 is a higher level of S0 . Similarly, L2 is the relaxed vibrational level of S1 , and L1 is the directly excited level of S1 . The excitation light generates the transition from L0 to L1 , and after vibrational relaxation (from L1 to L2 ), the STED light induces the transition from L2 to L3 by stimulated emission and depletes the excited state before fluorescence takes place. The temporal behaviours of the population probabilities ni (t) of the levels Li (i = 0, 1, 2, 3) of dyes are described by a set of coupled differential equations (Eq. (2)) with initial and normalization condition (Eq. (3)) under the room temperature and isolated system. IEXC is the intensity of excitation beam, and ISTED is the intensity of STED beam. In an excitation and depletion period, IEXC and ISTED are Gaussian distribution in time domain. EXC and STED are the wave frequency of excitation and STED pulses, respectively. 01 and 23 are the cross sections for the absorptions from L0 to L1 and from L3 to L2 , respectively. Typical values for 01 and 23 are 10−16 cm2 . FLUO is the average fluorescence lifetime and VIBR is the average vibrational relaxation time for L1 to L2 and L3 to L0 . FLUO is of the order of 2 ns, VIBR is of the order of 0.1 ps, and the quenching rate Q is typically 108 s−1 . The value of the Planck Constant h is 6.62606957 × 10−34 Js. To simulate the optimal time alignment of the excitation pulse and the STED pulse, it assumes that the widths of the Gaussian shaped excitation and STED pulses are both 120 ps; the peak intensity are both 30MW/cm2 ; and the wavelengths are 488 nm and 592 nm, respectively. So IEXC , ISTED , EXC and STED are given by Eq. (4). IEXC 01 dn0 1 = (n1 − n0 ) + n3 , VIBR dt hEXC IEXC 01 1 dn1 = (n0 − n1 ) − n1 , VIBR dt hEXC dn2 ISTED 23 1 1 = (n3 − n2 ) + n1 − ( + Q )n2 , VIBR FLUO dt hSTED
(2)
ISTED 23 1 1 dn3 = (n2 − n3 ) − n3 + ( + Q )n2 VIBR FLUO dt hSTED
n0 |t=0 = 1, ni |t=0 = 0(i = 1, 2, 3),
3 i=0
ni = 1
(3)
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Fig. 2. (a) (t = −50 ps, 200 ps, 450 ps) and (c) (t = 160 ps, 220 ps, 280 ps) show the relationship between the population probability of L2 (n2 ) and the time (t) under different time alignments (t). (b) (the time alignment range from −100 ps to 450 ps with an interval of 50 ps) and (d) (the time alignment range from −160 ps to 290 ps with a finer interval of 10 ps) show the relationship between the average value of n2 and t.
−
(t − 258 × 10−12 )
IEXC = 30 × e −
ISTED = 30 × e
vEXC = vSTED =
c EXC
=
(120 × 10−12 )
2
2
MW/cm2 ,
(t − 258 × 10−12 − t) (120 × 10−12 ) 3 × 108 m/s 488 × 10−9 m
2
2
MW/cm2 ,
(4)
= 6.147541 × 1014 s−1 ,
c 3 × 108 m/s = 5.067568 × 1014 s−1 = STED 592 × 10−9 m
The population probability of L2 reveals the intensity of spontaneous fluorescence and the depletion efficiency. In order to obtain the optimal time alignment, the rate equations of fluorophore (Eq. (2) and Eq. (3)) were solved by 4-order Runge-Kutta algorithm, and the population probability of L2 (n2 ) versus the time (t) under different time alignments (t) were simulated. Firstly, the time alignment was varied from −100 ps to 450 ps with an interval of 50 ps, and under each time alignment, the population probability of L2 versus the time was simulated. Fig. 2(a) shows some of the curves that n2 as function of t. To directly show the depletion effect, an average of n2 was calculated by Eq. (5): n2 = (n2 (t1 ) + n2 (t2 ) + ... + n2 (tn ))/n, n = 5000, t1 = 0ps, t2 = 1ps, ......, t5000 = 4999ps
,
(5)
where n2 (ti ) is the value of n2 at time ti , and n is the algorithm steps. Fig. 2(b) indicates that less population probability of L2 , i.e., higher depletion efficiency is under the time alignment range from 150 ps to 300 ps. To view the detail of the depletion effect, we varied the time alignment from −160 ps to 290 ps with a finer step of 10 ps, and under each time alignment, the simulation mentioned above was performed again. The results were illuminated in Fig. 2(c) and (d). Fig. 2(d) shows that the least population probability of L2 , i.e., the highest depletion efficiency is under the time alignment around 210 ps. Therefore, the optimal time alignment about 210 ps was achieved by simulation when the width of excitation and STED pulses were both 120 ps.
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Fig. 3. (a) shows the scheme and (b) is a photograph of the experimental setup. The supercontinuum laser is split into two beams using a dichroic beam splitter (D1) from which the excitation and STED wavelengths are extracted by means of two band pass filters (BP1 and BP2). Both beams are expanded and collimated by beam expanders (L1, L2 and L3, L4), and coupled into the main light path using two dichroic beam splitters (D2 and D3). D2 is installed in a kinematic mirror mount with two piezo adjusters to align the excitation and STED beams accurately. Two moveable mirrors (M2 and M3) are installed on an optical delay line to adjust the time alignment between the excitation pulse and the STED pulse. The doughnut shape is produced by using a vortex-phase plate (VPP). Both excitation light path and depletion light path have a half-wave plate (AHWP1 and AHWP2) for adjusting the polarization, and a quarterwave plate (AQWP) generating circular polarization. P1, P2: polarizer; BP3: band pass detection filter; L5: pinhole lens; SM: scan mirrors of galvanometer type; SL: scan lens; TL: tube lens; OL: 100× objective lens.
3. Experimental method After simulation, we presented an experimental method to obtain the optimal time alignment of a customized pulsed STED microscopy system. Fig. 3(a) shows the scheme, and Fig. 3(b) is a photograph of the experimental setup. A commercial
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Fig. 4. The excitation pulse and the STED pulse displayed on the oscilloscope. (a) x1 = 0 mm, t1 = −716.80 ps; (b) x2 = 220 mm, t2 = 750.40 ps.
supercontinuum fiber laser (SC-450-PP-HE, Fianium, UK) was used to provide both excitation and STED pulses by using a dichroic beam splitter. The excitation wavelength of 488 nm and the STED wavelength of 592 nm were extracted by two band pass filters, respectively. Since originating from the same light source, the excitation and STED pulses are inherently synchronized. Thus, the time alignment can be adjusted by changing the excitation optical path lengths with a proper difference compared with the depletion optical path. The supercontinuum laser has the total power of 2W and the width of the pulses is 120 ps, which are appropriate for STED. A 1 MHz repetition rate was used to limit photobleaching of the fluorophore. The key points of the time alignment optimization method were as follows: (1) Two moveable mirrors installed on an optical delay line (ODL220-FS/M, Thorlabs, USA) were used to change the excitation optical path length. The relationship between the time alignment and the position of the moveable mirrors was determined quantificationally. In Fig. 3(a), the difference of the excitation path length and the STED path length is S = 2 × (S1 -S2 ). S was increased while S2 was decreased when the moveable mirrors were moved from the position of 0 mm to 220 mm. Next, the relationship between the time alignment and the position of the moveable mirrors was determined quantificationally. The excitation and STED beams were collected into a same fast photodiode (G4176-03, 30 ps rise time, Hamamatsu, Japan). The photodiode’s output was connected to a bias tee (5541A, Pulse Labs, USA) to remove the DC component, and then fed into a fast oscilloscope. The two pulse trains can be observed approaching together when the moveable mirrors along the optical delay line are properly moved. Then the time alignment can be read out from the display of the oscilloscope. When the mirrors were moved to the position of x1 = 0 mm and x2 = 220 mm, the time alignment read from the display were t1 = −716.80 ps and t2 = 750.40 ps respectively, as shown in Fig. 4. It is noted that the oscilloscope cannot resolve the peaks of the excitation and STED pulses if they are too close. The relationship between the time alignment (t) and the position of the moveable mirrors (x) is linear, so it could be described by Eq. (6): t =
t1 − t2 t2 × x1 − t1 × x2 ×x+ = 6.67x − 716.80 x1 − x2 x1 − x2
(6)
(2) The depletion experiments at different positions of the moveable mirrors were performed and the depletion efficiency was calculated. The optimal time alignment was corresponding to the position of maximum depletion efficiency. The depletion experiments needed to be performed using Gaussian shaped STED beam. Therefore the blank area of the vortex-phase plate was placed to the STED path instead of taking it away, so that the STED path length was almost unchanged. The mirrors were moved to different positions, and at each position, 40 nm fluorescent beads were imaged in excitation mode (excitation beam on, STED beam off), depletion mode (excitation beam on, STED beam on) and excitation mode (excitation beam on, STED beam off) in sequence. The imaging was performed at an excitation/STED wavelength pair of 488 nm/592 nm. The laser power at the back aperture of the objective lens was ∼1 W for excitation and ∼100 W for STED. I1 was used to denote the total summed pixel value of the first excitation mode image, and I2 and I3 were used to denote that of the depletion mode and the second excitation mode images. The depletion efficiency could be described as 1-I2 /I1 , and the higher depletion efficiency meant the better time alignment. The experimental data was presented in Table 1.
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Table 1 The data of the depletion experiments. x (mm)
t (ps)
Vx, Vy (volts)
1-I2 /I1
I3 /I1
120 125 130 135 140 145 150 155 160 165
83.6 116.95 150.3 183.65 217 250.35 283.7 317.05 350.4 383.75
90,80 95,80 100,80 100,85 100,80 100,85 95,85 90,85 90,80 90,85
0.3106 0.392 0.4803 0.4842 0.511 0.494 0.491 0.4631 0.4676 0.4504
0.9936 0.9986 0.9962 0.9953 1.0058 0.9955 0.9926 1.0049 1.0018 0.9977
Fig. 5. The depletion efficiency as function of the position of the moveable mirrors and the time alignment between the excitation pulse and the STED pulse.
In STED microscopy, the excitation beam and the STED beam should be combined together, so that their focus point by the objective could be precisely overlapped. The measurement above might be influenced by the alignment precision of the excitation and STED beams and the fluorescence photobleaching. In order to exclude the possible influence by the fluorescence photobleaching, a lower intensity of the excitation and STED beams should be used. The I3 to I1 ratios (as I3 /I1 listed in Table 1) were very closed to 1 and this indicated that there was almost no photobleaching during the experiments. Some ratios value were larger than 1 because of noise during the image process. Since the movement of the moveable mirrors might cause drifts of the excitation beam, we must align the excitation beam and the STED beam accurately at each position of the moveable mirrors. The alignment experiments were performed through imaging the excitation and STED point spread functions by measuring the scatter light from 60 nm gold nanoparticles. To avoid frequently switching between the depletion experiments and the alignment experiments, we adopted a dichroic mirror which was installed in a kinematic mirror mount with two piezo adjusters. We combined the excitation beam and the STED beam precisely at each position of the moveable mirrors and recorded the corresponding piezo voltages (as Vx, Vy listed in Table 1). Since the piezo adjusters have extremely accurate repeatability, when the moveable mirrors position was changed, the excitation beam and the STED beam can be rapidly combined together with high precision by reloading the piezo voltages recorded before. Therefore, we could perform the above mentioned depletion experiments uninterruptedly. Fig. 5 shows that there is maximum depletion efficiency when the moveable mirrors are at the position of 140 mm, and the corresponding time alignment is 217 ps. So the optimal time alignment of the developed pulsed STED microscopy system is 217 ps that accords with the simulation result. Fig. 6 shows the excitation image before depletion, the depletion image and the excitation image after depletion under the optimal time alignment.
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Fig. 6. The excitation image before depletion (a), the depletion image (b), and the excitation image after depletion (c) under the optimal time alignment. The scale bar represents 1 m.
Fig. 7. Comparison between confocal (a) and STED (b) images of fluorescent beads. The confocal cross section along the traces indicated by the arrows fits to Gaussion and The STED cross section along the traces indicated by the arrows fits to Lorentzian (c). The scale bar represents 1 m.
4. Results and discussion To test the optimal time alignment, a sample of 40 nm fluorescent beads (Yellow-Green FluoSpheres, 40 nm, Molecular Probes, USA) immobilized on a coverslip was first imaged in confocal mode and then in STED mode. When imaged in STED mode, the effective region of the vortex phase plate was inserted into the STED light path. The imaging was performed at an excitation/STED wavelength pair of 488 nm/592 nm. The laser power at the back aperture of the objective lens was ∼1 W for excitation and ∼1 mW for STED. Images were generated by raster scanning the sample over the beams, using a piezoelectric stage (P733.3DD, Physik Instrumente (PI) GmbH & Co. KG, Germany) which has sub-nanometer accuracy. Imaging area was 250 × 250 pixels with 20 nm pixel size and 250 s pixel dwell time. The fluorescence photons were detected by an avalanche photodiode module (SPCM-AQRH-16-FC, Perkin Elmer, Canada) which was connected to a DAQ card (PCIe-6361, National Instruments, USA). Fig. 7 presents images recorded in the confocal mode and the STED mode. Closely spaced nanospheres cannot be resolved in the confocal image, but are clearly discernible in the STED image. The STED cross section indicated by the arrows was fit to a Lorentzian, for which there is precedent in the literature [17,18], and it yielded a FWHM of 70 nm. Taking into account the actual bead diameter the STED image has about 60 nm resolution. In Fig. 8, a microtubule network was imaged both in confocal and STED mode. Images were generated by raster scanning the beams over the sample, using a pair of x-y galvanometers (6215H, Cambridge Technology, USA) with 28 nm pixel size and 500 × 500 pixels. It can be seen that detailed structures and closely spaced fibers are revealed in the STED image while blurred in the confocal image. The significant improvement of resolution in the STED mode verifies the effectiveness of above time alignment optimization method. 5. Conclusions As the STED pulse has the Gaussian shape in time domain and high intensity, its rising edge has depletion effect. As a result, a proper time alignment between the excitation and STED pulse can improve the resolution of pulsed STED microscopy. The optimal time alignment about 210 ps was obtained by simulation when the pulse width of the excitation and STED pulses were both 120 ps. An experimental method was presented to obtain the optimal time alignment of pulsed STED microscopy after simulation. Through the depletion experimental method, the 217 ps optimal time alignment was got for a customized STED microscopy system which was similar to the simulation result 210 ps. Superresolution image with resolution of 60 nm was achieved in the STED microscopy using the optimal time alignment. The simulation and experimental methods in this paper provide a reliable way to optimize the time alignment of pulsed STED microscopy.
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Fig. 8. The confocal (a) and the STED (b) images of the microtubule network. All images are raw data. The scale bar represents 1 m.
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