2.1.

Chemical Synthesis

Methods for chemical synthesis and characterization of DTE-Ph can be found in the previous work.²⁰ The synthesis of the DTE-Ph nanoparticles (DTE-Ph @ NPs) is stated below.

2 mL tetrahydrofuran (THF) solution mixture with a DTE concentration of 0.1 g/L and a poly(styrene-comaleic anhydride) (PSMA, cumene-terminated, average $\mathrm{MW}\approx 1700$, styrene content 68%, purchased from Sigma-Aldrich) concentration of 0.01 g/L was quickly added into 10 mL of water in a bath sonicator for 3 min. The solution was bubbled by nitrogen and heated to 90°C to remove THF. Then the suspension solution was filtered by a $0.2\mu \mathrm{m}$ filter.

2.2.

SR-SRS Nanoscopy Setup

Our imaging setup was based on a conventional SRS microscope coupled with an additional visible and UV laser source and is illustrated in Fig. 1. For the SRS microscope, pulsed femtosecond laser beams from a commercial optical parametric oscillator (OPO) laser (Insight DS+, Newport, California, United States) were used as the laser source. The fixed fundamental 1040 nm laser was used as the Stokes beam, while the tunable OPO output (680 to 1300 nm) served as the pump beam. Chirped by SF57 glass rods, pulse durations of the pump and Stokes beams were stretched to $\sim 2.3$ and $\sim 1.2\mathrm{ps}$, respectively. The intensity of the 1040 nm Stokes beam was modulated at 1/4 of the laser pulse repetition rate ($f_0=80\mathrm{MHz}$) using an electro-optical modulator (EOM). The two pulse trains were spatially and temporally overlapped through a dichroic mirror (${\mathrm{DM}}_3$) and delay stage (DS), delivered into a laser scanning microscope (FV1200, Olympus), and focused onto the sample with an objective lens (Olympus, UPLSAPO60XW, NA = 1.2). The forward-going pump and Stokes beams, after passing through the samples, were collected in transmission with a high-NA condenser lens (oil immersion, 1.4 NA, Nikon). The stimulated Raman loss signal was optically filtered (CARS ET890/220, Chroma), detected by a homemade reverse-biased photodiode (PD), and demodulated with a lock-in amplifier (LIA) (HF2LI, Zurich Instruments) to feed the analog input of the microscope to form images. In order to induce the conversion of the DTE molecules, an additional HeNe laser served as the visible beam and was engineered into a donut shape with a vortex retarder (VR) (VR1-633, Lbtek), and a solid-state diode laser (UV-FN-360, Changchun New Industry Optoelectronic Technology Co., China) provided the activation UV beam. Visible and UV beams were coupled into the path of pump and Stokes beams through dichroic mirrors (${\mathrm{DM}}_1$ and ${\mathrm{DM}}_2$).

Fig. 1

Design of SR-SRS microscopy. (a) Optical layout of SR-SRS; (b) spatial profiles of the visible beam before and after VR; (c) PSF of donut-shaped visible beam and UV beam; and (d) schematic diagram of the depleted and activated SRS at the detection frequency (${\mathrm{\Omega }}_0$) of the closed-ring isomer. Scale bar: 2 mm in (b) and $1\mu \mathrm{m}$ in (c). VR, vortex retarder; EOM, electro-optical modulator; LIA, lock-in amplifier; OB, objective; Con, condenser; DM, dichroic mirror; SM, single-mode fiber; PD, photodiode; F, filter; PC, personal computer; DS, delay stage; SU, scanning unit; SL, scan lens; and TL, tube lens.

All laser powers were measured after the objective lens. For experiments in Figs. 2 and 3, the Stokes laser power was 9 mW and the pump laser power was 6 mW, while the visible light is $200\mu \mathrm{W}$ and UV power was kept at $20\mu \mathrm{W}$ for SR-SRS. For the cell imaging shown in Fig. 4, the laser powers were used as $P_{\mathrm{Stokes}}=40\mathrm{mW}$ and $P_{\mathrm{pump}}=20\mathrm{mW}$, $P_{\mathrm{VIS}}=300\mu \mathrm{W}$, and $P_{\mathrm{UV}}=50\mu \mathrm{W}$. Pixel dwell times were set at $40\mu \mathrm{s}$. To meet the Nyquist sampling theorem, an imaging area of $20.8\mu \mathrm{m}\times 20.8\mu \mathrm{m}$ with $800\mathrm{pixels}\times 800\mathrm{pixels}$ was set (pixel sampling size of 26 nm).

Fig. 2

Properties of photoswitchable Raman probe and SR-SRS imaging of native probe molecules. (a) SRS spectra of DTE-Ph in the open- and closed-ring isomers upon visible and UV light; (b) SRS signal of DTE-Ph acquired at the UV-induced Raman frequency ($2194{\mathrm{cm}}^{-1}$) shows on/off switching behavior under UV/visible pulsed irradiations; (c) relationship of on-switching transition time under different powers of the UV beam; (d) SRS signal depletion under different visible laser powers and fixed $20\mu \mathrm{W}$ UV power; (e) SRS and (f) SR-SRS images of the probe powder; and (g), (h) normalized intensity profiles along the line cuts in (e) and (f). Scale bar: $1\mu \mathrm{m}$.

Fig. 3

SR-SRS imaging of synthesized DTE-Ph NPs. (a) Schematics of NP synthesis; (b) size distribution of NPs determined by DLS; (c) SRS and SR-SRS images of the NPs spread on glass slide; (d) zoom-in images of the labeled rectangle regions in (c) and data fitted by Gaussian function of the single dots pointed with green arrowheads; (e) SRS and SR-SRS images of the NPs; (f) intensity profile along the lines marked in (e); and (g) statistical analysis of multiple particles ($n=10$). Scale bar: $2\mu \mathrm{m}$ in (c), (e) and 500 nm in (d).

Fig. 4

SR-SRS imaging of live cells. (a), (c) SRS images of NPs treated HeLa cells at different Raman frequencies: ${\mathrm{CH}}_2$ symmetric stretch ($2850{\mathrm{cm}}^{-1}$), ${\mathrm{CH}}_3$ symmetric stretch ($2930{\mathrm{cm}}^{-1}$), alkyne stretches of the closed-ring ($2194{\mathrm{cm}}^{-1}$) isomers, and corresponding SR-SRS at the activate frequency ($2194{\mathrm{cm}}^{-1}$). (b), (d) Data fitted by Gaussian function of the dots pointed with green arrowheads in (a) and (b). Scale bar: $20\mu \mathrm{m}$ in zoom-out images and $2\mu \mathrm{m}$ in zoom-in images.

2.3.

SRS Imaging of DTE-Ph @ NPs

The DTE-Ph @ NPs suspended solution (370 ppm; parts per million) was resuspended in deionized water by a 1:200 dilution. The solution is sonicated for 10 min at room temperature before being dropped onto a glass slide. When the solution is naturally dried, the slide is sandwiched by a cover glass for SRS imaging.

2.4.

Cell Culturing and Imaging

Before labeling experiments, HeLa cells were cultured with a DMEM medium (Invitrogen, 11965092) supplemented with 10% FBS (Invitrogen, 16000) and 1% penicillin–streptomycin (Invitrogen, 15140) at a humidified environment at 37°C and 5% ${\mathrm{CO}}_2$. All samples were assembled into a chamber using a homemade punched slide filled with PBS solution for imaging. In the experiment of NP treatment, cells were seeded and cultured on a glass coverslip in a 6-well plate for 24 h and then incubated with 2 ppm DTE-Ph @ NPs for 4 h. Before imaging, cells were washed with PBS 3 times.

2.5.

Fourier Transform Infrared Spectroscopy

The IR spectra presented in Fig. S9 in the Supplementary Material were measured using a Bruker Fourier transform infrared (FTIR) spectrometer (Vertex 70v) equipped with a Hyperion 2000 microscope at room temperature. Before the test, DTE powder ($\sim 1\mathrm{mg}$) was dissolved ahead of time in 0.5 mL poly(methyl methacrylate), and the mixture was spin-coated onto a quartz substrate. The incident light was focused on the coated DTE sample using a 15× IR objective, and IR radiation was collected by a liquid nitrogen cooled mercury cadmium telluride detector.

3.1.

RESOLFT-Based SR-SRS Microscopy

Typical far-field SR nanoscopy breaks the diffraction barrier by manipulating molecules into different states in the time (localization-based) or spatial (PSF engineering-based) domain.^17,21–24 Localization-based methods differentiate identical single molecules on the basis of individual stochastic behavior in time with either photobleaching or reversible blinking under wide-field illumination.^23,24 Such wide-field imaging modality is incompatible with the point-scanning-based SRS; hence we chose to realize SR-SRS by PSF engineering combined with photoswitching properties.

The optical setup of our SR-SRS was constructed by integrating a basic SRS signal detection/imaging framework with a RESOLFT focal engineering part [Fig. 1(a)]. For the conventional SRS, a fixed fundamental laser beam (1040 nm) stretched to $\sim 1.2\mathrm{ps}$ was used as the Stokes beam, and the tunable OPO output (680 to 1300 nm, stretched to $\sim 2.3\mathrm{ps}$) served as the pump beam, both of which kept Gaussian-shaped intensity profiles with diffraction-limited overlapping focal spots (see Fig. S1 in the Supplementary Material). Since the photoswitchable Raman probes follow UV activation and visible quenching when imaged at the alkyne Raman band of the closed-ring isomer ($\sim 2194{\mathrm{cm}}^{-1}$ for DTE-Ph),²⁰ a Gaussian-shaped UV beam (CW solid-state diode laser, 360 nm) was used to turn on the molecules, whereas a visible donut-shaped beam (CW HeNe laser, 633 nm) was used to switch off the SRS signal to sharpen the PSF. Figure 1(b) indicates that the donut beam was converted from a Gaussian beam by a VR as verified by CCD imaging before and after the VR. In the end, the four beams were combined and overlapped through DMs to interact with samples under the laser scanning microscope. The PSF profiles of the visible and UV beams after the objective lens were determined by imaging the light scattering of single Au NPs [Fig. 1(c)]. Thus the working principle of SR-SRS could be clearly pictured: at the particular SRS detection frequency, only molecules at the very center of the focal spot remain in the “on-state” by continuous UV activation, whereas the peripheral molecules in the donut area are erased as they frequency-shifted to the “off-state” by visible-light-induced isomerization [Fig. 1(d)]. The competition of UV and visible spots effectively compresses the PSF of the final SRS signal, improving the spatial resolution beyond the diffraction limit.

3.2.

Reversible Photoswitching Properties of Raman Probes

Fluorescence-based SR microscopy mostly relies on the sensitive photochemical or photophysical properties of electronic transitions in fluorescence probes, which are usually lacking in vibrational transitions, hindering Raman probes from possessing photoactive properties for SR imaging. In our previous work, we screened out two photochromic vibrational probes—DTE-TMS and DTE-Ph (see Fig. S2 in the Supplementary Material) and demonstrated reversible photoswitchable SRS imaging.²⁰ Here we chose to use a DTE-Ph molecule to investigate SR-SRS because of its enhanced SRS signal intensity and superior on–off ratio ($\sim 50$) in the background-free spectral region, which are the keys to optimizing the spatial resolution of RESOLFT-based SR microscopy.

We first characterized the critical photoswitching properties of DTE-Ph to obtain the optimum activation and depletion parameters. The photoisomerization of DTE-Ph renders a large SRS frequency shift ($\sim 20{\mathrm{cm}}^{-1}$) of the alkyne vibration, switching between the closed-ring ($\sim 2194{\mathrm{cm}}^{-1}$) and the open-ring ($\sim 2214{\mathrm{cm}}^{-1}$) isomers under UV/visible light irradiations [Fig. 2(a)]. Unless specifically stated, SRS detection frequency was kept at $2194{\mathrm{cm}}^{-1}$ throughout the work; hence the on/off states were defined as the closed/open-ring isomers. Figure 2(b) shows robust reversible on/off switching behavior and a high on/off ratio of the probe molecule under periodic and out-of-phase UV/visible irradiations ($n=3$), with clear transition times during the activation and depletion processes. We measured the onset transition time under various UV doses to determine the optimum activation parameters for the laser point-scanning imaging mode. As the UV dose increased, the transition time continuously shortened till it reached a constant level ($\sim 40\mu \mathrm{s}$) at the UV power of $\sim 20\mu \mathrm{W}$ [Fig. 2(c), $n=3$]. A prolonged UV irradiate dose is likely to cause irreversible photoreactions to kill the switchable capability;^25,26 thus the preferred UV activation power was set to 20 to $30\mu \mathrm{W}$ with a pixel dwell time of $40\mu \mathrm{s}$. The depletion property of visible irradiation was characterized by measuring the SRS intensity of the probe molecules with varying visible power under a fixed UV dose ($20\mu \mathrm{W}$). It can be seen that visible irradiation could effectively suppress the SRS signal, and almost completely depleted it when the visible power reached above $100\mu \mathrm{W}$ [Fig. 2(d), $n=9$].

3.3.

SR-SRS Imaging of Native Probe Molecules

With the determined activation and depletion parameters, we then performed SR-SRS on native probe molecules in powder form. The applied laser powers of the four beams were set as $30\mu \mathrm{W}$ for the UV (360 nm), $200\mu \mathrm{W}$ for the visible (633 nm), 6 mW for the pump (845 nm), and 9 mW for the Stokes (1040 nm). A pixel dwell time of $40\mu \mathrm{s}$ was chosen to match the switching time. In the presence of the visible beam, the SR-SRS image showed overall reduced signal intensity compared with conventional SRS due to the compressed effective PSF, as shown in Figs. S3(a) and S3(b) in the Supplementary Material. Meanwhile, more details of the morphological features could be visualized [see arrowhead in Fig. S3(a) in the Supplementary Material]. To make a more direct comparison, the line-cut profiles were taken across the imaged region with normalized intensities [Fig. S3(c) in the Supplementary Material]. As illustrated in the shadow-colored regions of the line profiles, the conventional SRS image only depicted three broad lumps, whereas the SR-SRS image resolved a number of much sharper peaks, indicating improved spatial resolution. For quantitative evaluation, the intensity distributions across the edges of the imaged powder were extracted and shown in Figs. 2(e) and 2(f). The data were fitted with error function [$\alpha (x)=A_1{\int }_{-\infty }^x\exp (-t^2/x_0^2)\mathrm{d}t+A_2$] to determine the resolution quantitatively, where $A_1$, $A_2$, and $x_0$ are the fitting parameters.^11,15 The lateral resolution could be calculated as the full width at half maximum (FWHM) of the Gaussian function in the fitting function $\mathrm{\Delta }=2\sqrt{\ln 2}{x}_0$ [Figs. 2(g) and 2(h)]. Although conventional SRS provided a resolution of $\sim 380\mathrm{nm}$, SR-SRS presented a value of $\sim 150\mathrm{nm}$, offering an improvement factor of $\sim 2.5$. However, this approach only offers a quick and rough estimation; it might underestimate the resolution because the physical interface of the sample might not be a true sharp step.

3.4.

SR-SRS Nanoscopy of Optimized DTE-Ph NPs

To more precisely characterize the resolution of SR-SRS, the probes were synthesized into DTE-Ph @ NPs as detailed in Sec. 2. Briefly, DTE-Ph molecules were swelled by PSMA in a concentration of around 90% and formed nanoscale spherical particles [Fig. 3(a)]. The size of the DTE-Ph @ NPs in water suspension was determined by dynamic light scattering (DLS), showing an average diameter of $\sim 18\mathrm{nm}$ [Fig. 3(b)]. The compact particle size is suited for the demonstration and characterization of SR imaging capability.

DTE-Ph @ NPs were spread onto a glass slide by evaporating a drop of NP solution at room temperature and were imaged without further processing. SRS spectroscopy verifies the same photoswitching properties of DTE-Ph @ NPs as the native probe (see Fig. S4 in the Supplementary Material). Meanwhile, we noticed a slightly increased transient absorption background in the SRS spectrum under UV irradiation. Nonetheless, such a background was not seen in the open-ring isomer upon visible-light illumination; thus the on/off ratio remained sufficient to ensure a high improvement factor of the spatial resolution.

For direct comparison, we imaged the DTE-Ph @ NPs with conventional SRS and SR-SRS, as shown in Fig. 3(c). Individual bright dots of the NPs indicated that the particles were dispersed with small amount of aggregation, well suited for quantifying the resolution. As we switched from conventional SRS to SR-SRS by coupling the UV-activation and visible-depletion beams, the imaged NPs appeared much sharper, with reduced signal intensity and particle sizes, indicating a significant increase of spatial resolution [Fig. 3(c)]. This could also be verified by comparing the imaging results of UV-only, visible-only, and UV + visible (see Fig. S5 in the Supplementary Material), showing the resolution enhancement truly originated from the combination of UV and visible beams. Single-dot analysis showed that measured NP sizes as small as $\sim 82\mathrm{nm}$ [arrowhead in Fig. 3(d)] could be identified by FWHM fitting using Gaussian functions in the magnified areas of the SR-SRS image [red dotted rectangle in Fig. 3(c)]. Compared with the 380 nm size determined by conventional SRS of the same NP, an enhancement of the lateral resolution by a factor of $\sim 4.6$ could be reached.

We also demonstrated SR imaging in regions with more crowded particles, where dots/aggregates were often too close to be resolved in conventional SRS. In contrast, the fine features could be clearly identified in SR-SRS images, including the isolation of two nearby particles and the differentiation of multiple substructures in the aggregates [Fig. 3(e)]. More obvious distinction could be visualized in the merged image, as the dots imaged with SR-SRS (red) appeared more compact than that in conventional SRS (green). The resolution enhancement could also be clearly shown in the intensity profiles along the line cuts across particles and aggregations, as shown in Fig. 3(f) and the lines in Fig. 3(e). Unresolvable peaks in conventional SRS could now be well differentiated as multiple peaks in SR-SRS, and the sharpest peak in each profile was characterized by Gaussian function to verify the sub-100 nm resolution [see lines #1 and #2 in Fig. 3(f)]. More data for intuitive comparison of NP images may be found in Fig. S6 in the Supplementary Material. We have performed a statistical analysis of imaged particles to present the PSF size improvement [see Fig. 3(g)]; these were all the particles selected within a single image to meet the criteria of the weakest measurable signal to avoid aggregates larger than the SR PSF.

3.5.

SR-SRS Imaging of Live Cells

To further demonstrate the potential of SR-SRS nanoscopy for biological imaging, we fed HeLa cells with the DTE-Ph @ NPs as detailed in Sec. 2, on account of the cellular uptake of NPs via endocytosis with low toxicity.^27,28 The treated live HeLa cells were imaged with both conventional and SR-SRS modalities. In Fig. 4(a), we first checked the cellular regions using rapid-scanning mode in the C–H vibrational band for lipid/protein imaging ($2850{\mathrm{cm}}^{-1}$ for ${\mathrm{CH}}_2$ and $2930{\mathrm{cm}}^{-1}$ for ${\mathrm{CH}}_3$) to locate the cells of potential interest under low magnification. Then the field of view was zoomed-in to observe finer intracellular features, including subcellular compartments and lipid droplets, as shown in the C–H channels, as well as the endocytosed NPs imaged in the alkyne channel ($2194{\mathrm{cm}}^{-1}$) without any background inference from the cells. To meet the Nyquist sampling theorem, an imaging area of $20.8\mu \mathrm{m}\times 20.8\mu \mathrm{m}$ with $800\mathrm{pixels}\times 800\mathrm{pixels}$ was set (pixel sampling size of 26 nm). From the SRS images of NPs and cells, we can see the dots tend to stay in cytoplasm with sparse distribution because of the low uptake concentration, offering the basis of resolution quantification for live cell imaging. The sizes of the smallest dots found inside cells matched well with the in vitro results [Figs. 3(d) and 4(b)]. The FWHM of the dot in conventional SRS image was analyzed to be $\sim 338\mathrm{nm}$, in contrast to that of $\sim 84\mathrm{nm}$ in the corresponding SR-SRS image, proving the ∼fourfold enhancement of resolution of our system [Fig. 4(b)]. Similar results could be seen repeatedly in different cells with sub-100 nm resolution [Figs. 4(c) and 4(d)]. Figure S7 in the Supplementary Material shows the statistical results of the resolution measurement.