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Abstract
Stimulated-emission-depletion (STED) nanoscope achieves super-resolution imaging by using a donut-shaped depletion beam to darken the fluorophores around the excitation spot. As an important factor determining the resolution of imaging, the coaxiality between the excitation and the depletion beam is required to be maintained at the nanoscale, which is often degraded by various interference such as ambient vibration and temperatures etc. Here, we propose a specially designed STED illumination module to guarantee the coaxiality between the two beams while modulating the phase of the depletion beam. This STED illumination module can realize phase modulation, polarization adjustment, pulse delay and two beams coaxial at the same time. With the experiments, the module can guarantee the two beams are stably coaxial for a long time. We imaged fluorescence particles with diameter 40 nm and got images of 40 nm full width at half maximum. Adjacent microfilaments at 80 nm being clearly distinguished with our STED nonoscope demonstrates that it could be well applied to biological samples.
© 2022 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement
1. Introduction
As one of the widely used methods in the life science, fluorescence microscopes can observe the detailed structure of cells and proteins. However, limited by the diffraction, it can only observe structures that are half the wavelength of light (∼200 nm). Several microscopic systems circumventing this diffraction limit have been developed and verified in experiments, such as Structured Illumination Microscopy(SIM) [1], Stochastic Optical Reconstruction Microscopy(STORM) [2], Photo-Activated Localization Microscopy(PALM) [3], Stimulated-emission-depletion(STED)microscopy [4–8] and Reversible Saturable Optical Linear Fluorescence Transition(RESOLFT)microscopy [9]. The STED nanoscope was proposed by Stefan W. Hell in 1994 [4,10], as one of the earliest far-field super-resolution technologies, which achieved a resolution of 25 nm or even smaller. The super-resolution makes it possible to observe organelles at the nanoscale [11–13]
The concept of the STED nanoscope and its related methods are to use a ‘donut’ depletion light to suppress the fluorescence emission from the fluorophore located outside the excitation spot, so that the fluorescence can only be emitted from the center of the focused donut. Theoretically, the central dark region of the depletion light can be infinitely small, enabling the STED nanoscope to distinguish fluorophore smaller than the diffraction limit of the excited beam. The resolution of STED nanoscope is usually given by Eq. (1) [14,15]
Since the inception of the STED nanoscope, several simplified STED nanoscopic methods have been proposed in order to further optimize the STED nanoscope system [16,17]. In these schemes, a single supercontinuum laser source is used to replace the previous high-cost laser system and related supporting devices, and can obtain the similar imaging resolution. In this way, the excitation light and depletion light come from the same pulse, and there is no synchronization imbalance. Meanwhile, the wavelength for excitation and STED can be selected conveniently. However, these methods did not optimize the stability of the STED system. The biggest difficulty in building a STED system is to align the excitation beam and the depletion beam at the nanoscale, the deviation between the center of the depletion beam and excitation beam should be less than 50 nm [18]. The reduction of coaxial precision will degrade the super-resolution imaging quality and even loss the super-resolution. The common solution to this issue is manual calibration, or algorithm automatic calibration, which means high requirements on the operator or additional devices. Several improved methods are proposed. Nàndor Bokor divided the phase plate into different regions [19], and these regions can selectively pass the depletion light or excitation light, so the modulation of the two beams do not interfere with each other. However, this method will inevitably result in a waste of light power. Stefan W. Hell had designed a four-region phase plate [18] to introduce phase change to the depletion light without affecting the phase of the excitation beam, ensure the co-axial stability of the two beams. But this method cannot generate optimal donut. They also designed a phase plate that used the dispersion to discriminatively modulate excitation light and depletion light. However, these phase plates were designed for a single wavelength only, and for alterative wavelength, this phase plate will fail to work [20].
Here, we will report a specially designed STED illumination module to guarantee the coaxial precision and stability between the two beams while modulate the phase of the depletion beam. The module skillfully uses the light polarization, to stabilize the coaxiality of the two beams to reduce the maintenance burden. The STED nanoscope construction is shown in Fig. 1, the supercontinuum wideband pulse laser is filtered by a double-bandpass filter to obtain both the excitation and depletion beam. Since the excitation beam and depletion beam come from the same laser pulse, there is no synchronization mismatch between the two laser pulse sequences [20]. After coupling, the two laser beams enter the single-mode polarization-maintaining fiber, and then enter the STED illumination module after collimating the outgoing beams, which is modulated by the STED illumination module and then directed to the objective lens with the dichroic mirror.
Fig. 1. STED nanoscopic imaging system with the illumination module. The excitation light (647 nm, yellow) and depletion light (750 nm, red) were filtered out by a double bandpass filter from the supercontinuum wideband pulse laser. Then, the beams were coupled into the single-mode polarization-maintaining fiber (PMF), and enters the illumination module after collimating the outgoing beams. A quarter wave plate (1/4 plate) modulated the linearly polarized light emitted from the illumination module into circularly polarized light and shot into the objective lens. The fluorescence signal (orange) is filtered out by the dichromatic mirror. After purified by a bandpass filter, the fluorescence signal is detected by the APD.
As an integrated part, this illumination module is less disturbed by the surrounding environment, such as ambient vibration and temperatures etc., and guarantees the two beams stably coaxial for a long time. The experiments show this illumination module can generate uniform distribution donut shape and the STED nonoscope (Hereinafter referred to as Module STED) got 40 nm super-resolution.
This illumination module does not improve the coaxial stability of the STED nanoscope only, but simplifies the optical structure and reduces the volume of the STED nanoscope also, which is 2.54 cm × 2.54 cm × 5.21 cm in size (about the size of a coin as shown in Fig. 2). Such an integrated design is very conducive to the mutual use of the STED nanoscope and other instruments.
2. Illumination module structure
Successively, this module consists of a polarization beam splitter (PBS), a quarter-wave plate, a dichromatic mirror (DM), a delay cuboid and a reflection-type phase plate. The structure and function diagram of the illumination module is shown in Fig. 3. For clarity, each part of the module is separated in this figure. In practice, they were bound into a stable one with refractive index matching optical glue (norland61, Refractive index of polymer after curing: 1.56). The optical glue fills the gaps between the components and provides narrow spaces between them. These narrow spaces improve tolerance and ensure that the tilt of the component surface would not affect the coaxiality of two beams. We cured the glue and detected the point-spread functions of the excitation beam and depletion beam at the same time, which allowed us to correct the drift caused by glue solidification shrinkage in time. Finally, a fully cured optical glue keeps the components at the right posture.
Fig. 3. Schematic setup of the illumination module. Red and yellow stripe line: two beams modulated by the module, excitation light (yellow) and depletion light (red). Module composition from left to right: polarization beam splitter (PBS), quarter-wave plate (λ/4 Plate), Dichromatic Mirror (DM), Delay cuboid and Reflection-type Phase Plate. Above and below of the red and yellow stripe line: the polarization states of two beams after passing through different components (yellow arrow: excited light; Red arrow: depletion light, straight arrow: linear polarized light; circular arrow: circularly polarized light). The phase states of the incident/reflected light are shown on the upper and lower sides of the reflection-type phase plate. The backward depletion beam merges with the excitation beam at the dichromatic mirror, and the two beams return to the coaxial. Two beams then exit the module reflected by the PBS splitter.
The module is designed for excitation wavelength below 700 nm and depletion wavelength of 750 nm from the same laser pulse sequence. The beams pass through the single-mode polarization-maintaining fiber to obtain a pure single-mode P-polarized Gaussian beam, which is collimated and then shot into the module for modulation. Outgoing from the optical fiber, the P-polarized beam passes through the polarization beam splitter (PBS) and converted into circularly polarized light by the quarter wave plate. The excitation beam is reflected in front of a customized dichromatic mirror (DM) at an incident angle of 0°, and the depletion beam passes through this dichromatic mirror and enters the delay cuboid.
The length of the optical delay cuboid was designed to produce fixed pulse time delay (120 ps for our nanoscope) between the excitation beam and depletion beam (Eq. (2)).
Where, c is the velocity of light in vacuum, d is the delay cuboid length, n is the refractive index of the delay cuboid and t is the pulse delay time. For optical glass N-BK7, the optimal length of the pulse delay cuboid is 1.2 cm. At the other end of the optical delay cuboid, a reflection-type phase plate combining a half-wave phase plate and a reflective mirror is responsible for the 2π phase delay of depletion beam. The final phase delay is: where, d is the length of the geometric path, Δn is the refractive index difference between the phase plate medium and air.Passing through the half-wave phase plate twice, the opposite depletion beams obtains π phase difference. This reflection-type phase plate allows us to reduce module dimension and improve illumination module integration. The backward depletion beam merges with the excitation beam at the dichromatic mirror. Then two circularly polarized beams pass through the achromatic quarter-wave plate again, convert into S-polarized beams, and exit the module by PBS. These two S-polarized beams are converted into circularly polarized beams again before shooting into the objective lens.
3. Results
We built a super-resolution STED nanoscope to validate the performance of our module (Fig. 1). Firstly, the excitation beam(647 nm) and depletion beam(750 nm) are separated from the laser beam emitted by the supercontinuum pulse laser using a double band pass filter, and then they are coupled together into the single-mode polarization-maintaining fiber (pm-630-hp, Thorlabs GmbH, Dachau, Germany). The two outgoing beams from the fiber are collimated by the lens and then enter into the illumination module. The two outgoing beams from the illumination module were converted into circularly polarized light with the quarter-wave plate, and shot into the objective lens.
To avoid chromatic aberration, the achromatic collimating lens(AC127-050-A-ML, THORLABS)and quarter wave plate(AQWP10M-580, THORLABS) were selected. The fluorescence signal is filtered out by the band pass dichromatic mirror and the fluorescence filter, and then was detected by the APD detector (Excelitas, SPCM-AQRH-14-FC).
To characterize the co-axial state of excitation and depletion beam, a PMT(PMM02, THORLABS) was used to detect the scattering light of 80 nm-diameter gold nano-spheres. By superimposing both the excitation and depletion beam scattered by the gold particle, Fig. 4(a) shows the point spread functions (PSF) of excitation (yellow) and depletion beam(red) respectively in the objective focal plane. As shown in the Fig. 4(a), the depletion beam turns into the donut shape as expected. The light intensity distribution on the donut ridge is uniform (As shown in Fig. 4(b)), and the ratio of maximum intensity to minimum intensity is 1.13. The excitation beam is wrapped in the center of the donut and its peak coincides perfectly with the center of the donut. To characterize the alignment of two beams, we fitted the center area of the point spread function with 2D Gaussian function, and found that the deviation between the excitation peak and donut valley is less than 20 nm(Fig. 4(c)).
Fig. 4. (a) The point spread function of the module STED nanoscope. The intensity distribution of the depletion beam (red) and excitation beam (yellow) in the focal plane. scale bar:200 nm. (b) The corresponding intensity profile marked by orange dotted line. (c) 2D Gaussian fit of the point spread function of module STED. Red: depletion beam; Yellow: excitation beam. The depletion beam turns into the donut shape as expected, and the excitation beam is wrapped in the center of the donut and its peak coincides perfectly with the center of the donut.
Then we tested the coaxial stability between the excitation PSF and depletion PSF(as shown in Fig. 5), and compared it with that of our self-build common STED nanoscope (Hereinafter referred to as Common STED) [21,22].
Fig. 5. The deviation between the excitation PSF and STED PSF. (a) The test of the module coaxial stability in four weeks. Top: The depletion beam (red) and the excitation beam (yellow); Bottom: The line profile of the PSF marked by gray dotted line. (b) The test of coaxial stability on the self-build common STED system in four weeks. Top: The depletion beam (red) and the excitation beam (yellow); Bottom: The profile of the PSF marked by gray dotted line. During testing, the module is at the same environment temperature as the common STED nanoscope and both the nanoscopes were not calibrated manually, scale bar:200 nm. (c) The deviation between the excitation PSF and STED PSF versus time. Red broken line for the Self-build Common STED and blue broken line for the Module STED.
Obviously, the module does play the role in stabilizing the coaxiality between the excitation and the depletion beam. We used 2D Gaussian function to fit the point spread function of the Module STED and the Common STED, and recorded the deviation between the excitation PSF and donut PSF (Fig. 5(c)). In the second week, the uncalibrated Common STED nanoscope had excitation beam spots about 300 nm away from the center of the donut, while in the fourth week, the excitation beam spots appeared about 1000 nm away from the center of the depletion beam spot. However, the deviation of the Module STED was kept within 30 nm.
With the Module STED nanoscope, we imaged fluorescence particles with diameter 40 nm (Exc/Em: 660/680 nm), and the Fig. 6 shows the imaging results (Excitation beam: 647 nm, depletion beam: 750 nm). The comparison between STED and Confocal imaging showed that the resolution had significantly improved. We selected a same fluorescent particle in the confocal and STED images (indicated by the yellow arrow in the figure) for intensity profile analysis, and its full width at half maximum (FWHM) is 40 nm, while the FWHM of the confocal image is about 280 nm.
Fig. 6. Characterizing the imaging resolution with fluorescence particles. (a) The image of 40 nm fluorescence particles using the confocal microscope. (b) The image of 40 nm fluorescence particles using our module STED nanoscope. (c) Line profile of the fluorescent particles indicated by yellow arrows in the images. scale bar:1µm.
Finally, we used our module STED nanoscope to characterize the biological samples. Figure 7 shows the imaging of Hela cell microfilaments stained with ATTO647. The excitation and depletion wavelengths were 647 nm and 750 nm respectively, with repetition frequency of 1 MHz. The average power of the depletion beam measured at the rear port of the objective lens is 1.3 mW. The imaging results are shown in Fig. 7. Compared with confocal image, the resolution of STED image is significantly improved.
Fig. 7. The imaging of Hela cell microfilaments stained with ATTO647 by using our module STED nanoscope. (a) The imaging of Hela cell microfilaments under confocal microscope. (b) The imaging of Hela cell microfilaments under the Module STED nanoscope. Excitation beam: 647 nm ; Depletion beam: 750 nm ; repetition frequency of the laser pulse: 1 MHz. scale bar:1µm. (c) The intensity profile at gray dotted line in the image.
We selected several microfilaments in the module STED image (indicated by the gray dotted line) to make an intensity line profile, as shown in Fig. 7(c). The line profile shows that the adjacent microfilaments at 80 nm could be clearly distinguished, and the results show that super-resolved STED imaging could be well applied to biological samples.
4. Conclusion
We designed a special STED illumination module, which can realize phase modulation, polarization adjustment, pulse delay and two beams coaxial at the same time. As an integrated part, the illumination module can avoid the inherent vibrational and thermal instability of the mechanical structure, so that the Module STED nanoscope can work in variety of environments reliably for a long time without subsequent complicated maintenance and calibration.
This illumination module can generate uniform intensity distribution of the depletion beam on the focal plane, which is very crucial for super-resolution imaging. In addition, because the cut-off wavelength of the long-pass dichromatic mirror in the illumination module is at 700 nm, any laser wavelength less than 700 nm can be used as excitation light, which means that our illumination module can be adaptive to multicolor super-resolution STED nanoscope as well, which depletion light wavelength at 750 nm. The module was tested on a custom-built piezo-stage scanning STED microscope. A galvo scanning system can be installed between the module and microscope to improve scanning speed of the system.
Due to its small size, the illumination module can also be embedded into other microscopes as illuminator. This illumination module will simplify the operation of the STED nanoscope and extend the STED nanoscope to wider applications.
Funding
National Natural Science Foundation of China (21127901, 22077124); Chinese Academy of Sciences.
Disclosures
The authors declare no conflicts of interest.
Data Availability
No data were generated or analyzed in the presented research.
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