Two-photon STED nanoscopy realizing 100-nm spatial resolution utilizing high-peak-power sub-nanosecond 655-nm pulses

: We developed two-photon excitation stimulated emission depletion (STED) nanoscopy using high-peak-power sub-nanosecond 655-nm pulses. The STED pulse exhibited ideal optical properties and sufficient pulse energy to realize a 70-nm spatial resolution in the compact setup with electrically controllable components. For biological applications, we screened suitable fluorescent dyes or proteins and realized the sub-100 nm spatial resolution imaging of presynaptic protein clusters in fixed primary cultured neurons without severe photobleaching. We expect this method to enable visualization of ultrastructures and the cluster dynamics of biomolecules representing physiological functions in living cells and tissue.


Introduction
Two-photon excitation fluorescence microscopy has been widely used for bioimaging at subcellular to tissue levels [1] because it demonstrates superior penetration depth and reduced invasiveness for biological specimens by utilizing a near-infrared pulsed beam for two-photon excitation. Two-photon excitation fluorescence microscopy has realized in vivo volumetric imaging at depths of ~1.6 mm below the surface of a mouse brain, allowing us to visualize the morphology, network, and activity of numerous neurons in a living brain [2][3][4]. Recently, super-resolution technologies [5,6] to improve the spatial resolution of two-photon microscopy have been investigated in various biological fields [7][8][9][10][11][12][13][14][15].
Stimulated emission depletion (STED) microscopy [5] is one such technology that utilizes a doughnut-shaped beam to induce stimulated emission and restrict the emission area below the diffraction limit. Irradiation of a high-average power STED beam often causes severe photodamage to fluorescent probes and biological specimens. Photodamage can be reduced by utilizing pulsed beams to decrease the average power [8][9][10][11]15]. Most two-photon excitation STED microscopy systems utilize a mode-locked femtosecond laser light source similar to that in conventional two-photon excitation fluorescence microscopy [7][8][9][10][11]13,14]. To superimpose the excitation and STED light pulse temporally, the system requires a STED light source composed by another pulsed laser light source, an optical parametric oscillator, continuous-wave (CW) STED light source with time-gated detecting methodology [14], and using two types of compact laser diode (LD)-based light pulse sources [15].
Recently, we have developed compact two-photon excitation pulsed STED microscopy with electrically controllable components, including LD-based light sources for both excitation and STED [15]. Here, a key component is a set of transmissive liquid crystal devices (tLCDs) that are a type of the spatial light modulator (SLM) used to shape the STED beam and compensate for chromatic aberration. Unlike reflective SLMs, tLCDs can be directly inserted in front of the objective lens without adding a new optical path for SLMs, resulting in the compact setup. In contrast, a mass-produced LD-based pulsed STED light source does not have sufficient pulse energy to achieve spatial resolution below 100 nm.
Herein, to solve this low-power problem of the STED light source, we utilized our recently developed light source for STED, which can generate high-peak-power subnanosecond 655-nm optical pulses based on LD-controlling technologies [16]. We demonstrated that the 655-nm optical pulse had ideal optical properties and sufficient pulse energy to realize that the spatial resolution reached 70 nm without using supportive techniques such as a confocal aperture or image processing using deconvolution algorithms. The compact setup was maintaining using LD-based light sources and tLCDs [15], using which presynaptic protein clusters in a fixed primary neuron were successfully visualized at sub-100-nm spatial resolution without severe photobleaching.

Optical setup
The optical setup was based on our previous two-photon excitation STED microscope [15]. As Fig. 1 shows, we employed three different types of pulsed LD light sources driven by a homemade electric pulse generator at a repetition rate of 5 MHz.
For two-photon excitation, a 7.5-ps optical pulse source comprising an in-house 1064-nm GS-LD and multistage optical fiber amplifiers were used [17]. The average power was approximately 1 W, sufficient for two-photon excitation of general fluorescent probes in biological specimens [3,4,15,18]. Using this laser as the two-photon excitation light source at a repetition rate of 5-10 MHz, we successfully realized the non-invasive in vivo observation of the hippocampal dentate gyrus granule cells at a depth of over 1.5 mm from the brain surface in mice [4,18].
For STED, we employed our recently developed 650-nm-band optical pulse source [16]. This optical pulse source featured a 1.3-μm gain-switched semiconductor-laser optical amplifier under CW laser light injection to generate smooth-shaped, sub-nanosecond seed optical pulses. Thereafter, the seed pulses were amplified using a Praseodymium-doped fiber amplifier and converted to second harmonic (SH) pulses. The SH pulse had a pulse width of 260 ps ( Fig. 2(a)) and a peak wavelength of 655 nm ( Fig. 2(b)). Immediately after the output, average power and pulse energy reached 7 mW and 1.4 nJ, respectively. The output beam was already linear polarized and the polarization extinction ratio reached 25 dB, which was sufficient to be converted by liquid crystal (LC) molecules oriented toward its polarization direction [15]. We refer to this SH pulse as 655-nm STED pulse. Note that, for comparison, an optical pulse (peak wavelength = 638 nm, pulse duration = approximately 3 ns) generated from a mass-produced LD light source was also used ( Fig. 2(c, d)) [15]. Here, the average power and pulse energy was 1.5 mW and 0.3 nJ, respectively. We refer to this optical pulse as 638-nm-LD STED pulse.
The two-photon excitation and the STED pulses were tightly synchronized using an electrical timing controller and introduced into a galvano-mirror scanner (C2; Nikon) equipped with an upright microscope (ECLIPSE FN1; Nikon). The tLCDs [15] were placed between the microscope revolver and a water immersion objective lens with a numerical aperture of 1.27 (CFI Plan Apo IR 60XWI; Nikon). A detailed description of the tLCDs can be found in the literature [15]. Briefly, the tLCDs enable the modification of optical properties ove voltages appli of tLCDs. Th convergence objective lens tLCDs do not orientation of as an applied circularly pola beam, making

Fluoresc
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COS-7 cells with fluorescent dyes or proteins
COS-7 cells (DS Pharma Biomedical Co., Ltd.) were cultured on the coverslips with Dulbecco's modified Eagle's medium (DMEM; FUJIFILM Wako Pure Chemical Co.) supplemented with 10% fetal bovine serum in a CO 2 incubator at 37 °C.
To express fluorescent proteins, plasmid DNAs encoding EYFP, mCitrine, mOrange2, DsRed2, tdTomato, mRuby2 or mCherry were transiently expressed in COS-7 cells using Lipofectamine2000 transfection reagent (Invitrogen) according to the manufacturer's instructions. The transfected cells were grown in the DMEM in a CO 2 incubator at 37 °C for 48 hours, and then were fixed with 4% formaldehyde in PBS. The fixed cells were mounted with ProLong diamond reagent.
The fluorescent images of the COS-7 cell stained with AlexaFluor546, which were constructed by averaging four acquired images, had a pixel size and dwell time of 28 nm and 44.2 µs, respectively.

Primary neuronal culture
Cortical neurons from the brains of P0 ICR mice were cultured on the coverslips coated with poly-D-lysine and collagen at a density of 6,000 cells per 35-mm-diameter dish in Neurobasal-A medium containing 2% B27 supplement. The neurons were cultured in a CO 2 incubator at 37 °C for 21 days in vitro, and then were fixed with 4% formaldehyde in PBS. The fixed neurons were immunostained with an anti-Bassoon antibody (1:1,000 dilution, ab82958, Abcam), followed by ATTO532-conjugated goat anti-mouse IgG antibody (1:400 dilution, 610-153-121S, Rockland Immunochemicals, Inc.). SlowFade diamond reagent (Invitrogen) was used to mount the specimens.
The fluorescent images of the cultured neuron stained with ATTO532, which were constructed by averaging 16 acquired images, had a pixel size of 28 nm (or 207 nm in the low magnification image) and a pixel dwell time of 44.2 µs.

Results and discussion
First, we assessed the fluorescence depletion efficiency of 655-nm STED pulses using a 1-µm Nile red bead excited with the two-photon absorption of 1064 nm pulses. For reference, we compared the efficiency of a 638-nm-LD STED pulse used in our previous STED study [15]. To induce fluorescence depletion of the whole bead area at the focal plane, these pulsed STED beams were used as a Gaussian pattern rather than being converted to a doughnut shape. The average power of the 655-nm STED, 638-nm-LD STED, and two-photon excitation beams at the focal plane was ~3.0 mW, ~0.7 mW, and 1.5 mW, respectively. Figures 3(a) and (b) show fluorescent images and intensity profiles of the 1-µm Nile red bead irradiated with 655-nm and 638-nm-LD STED beams, respectively. The depletion efficiency induced by the 638-nm-LD STED beam was approximately 80%, consistent with our previous results [15]. The 655-nm STED beam demonstrated much higher depletion efficiency, reaching approximately 95%. Figure 3(c) shows the depletion efficiencies relative to the STED beam power (P STED ) at the focal plane. These results indicate that the 655-nm STED beam beams always beam even tho indicated that red beads.   To estima beads by ove shaped 655-n red bead exci the axial foca photon excita power of the 6 and 2.0 mW, nm STED be values. As Fig  shaped 655 [24][25][26]. A lon hotobleaching. for the incomp that a properly ciency without e super-resolut ED beam irrad high laser pow ecimens. The p two-photon ex photobleaching is a componen nsmitters are r e we confirme g. 4). The ave beams at the f lowest pixel dw high-power be Fig. 7(a)) was t fluorescence lly visualized puncta patterns Bassoon protei ful realization o mages of culture 2-conjugated secon the area marked nes.
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Conclusion
The 655-nm STED pulse utilized in two-photon tLC-STED nanoscopy has ideal optical properties and sufficient pulse energy to realize 70-nm spatial resolution. Furthermore, by utilizing pulsed light sources, we realized sub-100-nm spatial resolution bioimaging that does not suffer from severe photobleaching. The proposed nanoscopy is expected to be a powerful tool for super-resolution imaging for live-cell and tissue under optimal sample conditions.