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Abstract
We demonstrate a novel electrowetting liquid combination using a room temperature ionic liquid (RTIL) and a nonpolar liquid, 1-phenyl-1-cyclohexene (PCH) suitable for focus-tunable 3-photon microscopy. We show that both liquids have over 90% transmission at 1300 nm over a 1.1 mm pathlength and an index of refraction contrast of 0.123. A lens using these liquids can be tuned from a contact angle of 133 to 48° with applied voltages of 0 and 60 V, respectively. Finally, a three-photon imaging system including an RTIL electrowetting lens was used to image a mouse brain slice. Axial scans taken with an electrowetting lens show excellent agreement with images acquired using a mechanically scanned objective.
© 2024 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement
1. Introduction
Multiphoton fluorescence microscopy is a mature technique for biomedical imaging at millimeter depths in scattering tissue [1–5]. In two-photon excitation (2PE) microscopy, two near-infrared photons are absorbed by a molecule which fluoresces at visible wavelengths [6–8]. The two-photon absorption rate depends on peak power [7,9], leading to intrinsic optical sectioning. Additionally, near-infrared wavelengths experience less scattering and can thus penetrate deeper in biological tissue. Even with these improvements, the maximum achievable imaging depth in scattering tissue when using 2-photon excitation wavelengths (800 - 1000 nm) is fundamentally limited to less than one mm [10,11]. The use of longer excitation wavelengths (>1000 nm) in 2-photon microscopy allows for deeper imaging depths. However, most common 2PE fluorescent molecules are not sensitive to these wavelengths.
Three photon excitation (3PE) microscopy is an alternative multiphoton microscopy technique that uses the absorption of three longer wavelength near-infrared photons (often 1300 nm or 1700 nm) by a fluorescent molecule to increase the imaging depth in biomedical tissue [12–14]. Using an excitation wavelength of 1700 nm, Horton et al. were able to perform 3-photon imaging at depths up to 1.3 mm in tissue [13]. Three-photon microscopy has been used to image neurons in a mouse brain through an intact skull [12,13,15], opening the door for groundbreaking research in memory formation and disease progression. Recently, a 3PE system was integrated into a head mounted miniature microscope for live neuronal imaging of a freely moving mouse [16,17]. By imaging multiple cortical layers at different depths inside the brain, Klioutchnikov et al. [16] were able compare neural activity from several populations simultaneously while a mouse performed a single task. However, many microscopy systems use a mechanically scanned objective [18,19] or other mechanical methods [20,21] to remotely change the focal plane while imaging. Mechanical scanning techniques are capable of a large range of imaging depths and excellent imaging quality, but typically require expensive and bulky stages that can be prone to misalignment and failure.
An attractive alternative are adaptive optical devices based on the electrowetting-on-dielectric (EWOD) effect. Electrowetting describes the change in contact angle of a liquid droplet on a conductive substrate as a potential is applied between the droplet and the substrate [22–24]. The conductive substrate is commonly coated in a dielectric layer (called electrowetting-on-dielectric) to enhance this effect [25,26]. Electrowetting-based optical devices use an applied electric field to change the curvature of a liquid-liquid interface, resulting in a change in effective focal length. Optical lenses and prisms based on the EWOD effect have recently been demonstrated for aberration compensation [27–29], beam steering [30–32], and multiphoton microscopy [33–36]. These devices are particularly attractive due to their compact nature, large range of tuning, and low power consumption [37–40]. We focus on electrowetting optical devices that consist of a cylindrical glass container filled with two immiscible liquids (polar and nonpolar) of differing refractive index [37,38,41–43]. The inner sidewall of the cylindrical container is coated with an electrode, dielectric, and hydrophobic layer. When a potential is applied between the polar liquid and the sidewall electrode, an electric field forms across the dielectric layer which changes the interfacial surface tension between the polar liquid and the container sidewalls. This change in surface tension results in a decrease of the contact angle of the polar liquid and a corresponding increase in the radius of curvature of the liquid-liquid meniscus. The change in contact angle as a function of applied voltage is given by the Lippmann-Young equation (Eq. (1)):
where $\theta _{0}$ is the initial contact angle, $\epsilon _0$ is the permittivity of free space, $\epsilon _D$ is the dielectric permittivity, $\gamma$ is the surface tension between the liquid and surrounding medium, $d$ is the dielectric thickness, and $V$ is the applied voltage. In the case of a single sidewall electrode, the contact angle of the liquid-liquid meniscus changes symmetrically and the device behaves as a tunable lens. If there are multiple isolated sidewall electrodes, the contact angle of various points of the liquid-liquid interface can be controlled independently and the device functions as a tunable optical prism [44–46].Deionized (DI) water is often used as the polar liquid in electrowetting devices because it is conductive, readily available, has a high surface tension and is immiscible in a wide range of liquids. However, DI water has multiple strong absorption bands in the near-infrared (IR) region, presenting a significant challenge for these applications.The absorption coefficient of DI water exceeds $\alpha =$ 1 cm$^{-1}$ starting at 1300 nm and increases to $\alpha =$ 114 cm$^{-1}$ at 2500 nm [47,48]. For a 2.5 mm long water-based electrowetting device, this results in approximately 77% transmission of 1300 nm light (as predicted by Beer’s law [49]). Additionally, commercial electrowetting lenses use a combination of a nonpolar hydrophobic oil and a polar water solution and have transmission at 1300 nm of below 80% [50]. Such significant loss is detrimental for a wide range of applications. For example, if water-based electrowetting lenses were used with MW peak power lasers necessary for 3PE microscopy, strong absorption would cause the water to heat and the device to ultimately fail.
In this work, we present a near-infrared electrowetting lens using room temperature ionic liquids (RTILs). These liquids are molten salts at room temperature and have several key benefits including a negligible vapor pressure [51–54], high thermal stability, and crucially, a low absorbance in the near-infrared region. Previous demonstrations of RTIL-based electrowetting lenses [55,56] relied on liquid combinations that were hazardous and density mismatched ($\Delta \rho$ > 0.5 g/cm$^3$), leading to undesirable gravitational effects. In contrast, we have chosen a non-toxic polar and nonpolar liquid combination of N-Propyl-N-methylpyrrolidinium bis(fluorosulfonyl)imide (N-Pr-Me-Pyrr (SO$_{2}$F)$_{2}$) and 1-phenyl-1-cyclohexene (PCH) [41]. We show that this liquid combination has a high transmittance in the near-IR region, a large focal length tuning range, and is better density matched than previous demonstrations ($\Delta \rho$ $\approx$ 0.35 g/cm$^3$ vs $\Delta \rho$ > 0.5 g/cm$^3$). Finally, we integrate an RTIL electrowetting lens into a conventional 3PE microscope to take images as a function of depth that are in excellent agreement with those acquired from a mechanical scan.
2. Liquid characterization
Room temperature ionic liquids (RTILs) were first synthesized in 1914 and have gained interest since the mid 1980s. These liquids are commonly used in areas such as chemical synthesis due to a unique combination of physico-chemical properties [57–59]. By varying the anion and cation structure of RTILs, one can finely tune these properties to a specific application. For example, the miscibility of an RTIL in water can be completely changed by simply changing the anion in the chemical structure [57].
When choosing RTIL liquid combinations suitable for electrowetting, we require the following properties: a small density mismatch between the RTIL and nonpolar liquid (ideally $\Delta \rho$ < 0.1 g/cm$^3$), a low liquid dynamic viscosity (< 50 cP), high transmission in the near-infrared region (>= 90%), and liquids that are non-toxic. The use of well density-matched liquids in electrowetting reduces the impact of gravitational forces on the meniscus shape [24]. The effect of gravity on the shape and movement of a liquid is expressed by the Bond number, a unitless ratio of gravitational to capillary forces [60]. This ratio is directly proportional to the density mismatch of the two liquids and is given by $B_{0} = \Delta \rho g R^{2}/\gamma$. Where $\Delta \rho$ is the density mismatch of the two liquid system, $g$ is the gravitational acceleration, $R$ is the radius of our device, and $\gamma$ is the interfacial surface tension between the two liquids. A Bond number much less than one for our RTIL and nonpolar liquid combination results in a liquid meniscus resistant to the impacts of gravity-caused deformation. The Bond number of a liquid system also places a constraint on the size of the electrowetting device. For the combination of DI water and PCH ($\Delta \rho = 0.004 g/cm^{3}$) in a 4 mm diameter device, the Bond number is 0.00645 « 1. The Bond number of this liquid combination exceeds unity for device diameters greater than 5 cm.
Lowering the viscosity of the polar liquid in electrowetting results in a faster electrowetting actuation speed [61,62]. For example, Zhao et al. found that increasing the kinematic liquid viscosity of their polar liquid from 1.9 to 665 cP ($\rho = 1.9 g/cm^{3}$) increased the settling time of a 5 mm diameter device from 0.232 s to 1.243 s [61]. This imposes a limit on the density matching between our RTIL and our nonpolar liquid. Room temperature ionic liquids with densities close to common electrowetting nonpolar liquids ($\rho \approx$ 1 g/cm$^3$ for PCH) typically have a large dynamic viscosity [56,63]. For example, one common RTIL, tetradecyl(trihexyl)phosphonium bis(trifluoromethylsulfonyl)imide, has a density of 1.07 g/cm$^3$ and a dynamic viscosity of 304 cP at 30 °C [63]. This is over 300 times greater than the dynamic viscosity of water (0.890 cP) and would result in a slower actuation speed compared to water/PCH based devices [61].
We chose to focus on the liquid combination of 99.9% purity N-Propyl-N-methylpyrrolidinium bis(fluorosulfonyl)imide (N-Pr-Me-Pyrr (SO$_{2}$F)$_{2}$) and 1-phenyl-1-cyclohexene (PCH). N-Pr-Me-Pyrr (SO$_{2}$F)$_{2}$ and PCH have densities of 1.343 g/cm$^3$ and 0.994 g/cm$^3$, respectively, resulting in a density mismatch of $\Delta \rho$ $\approx$ 0.349 g/cm$^3$. We chose an RTIL with a lower dynamic viscosity of 52.7 cP at 25 °C compared to less-dense RTILs (dynamic viscosity > 300 cP [56,63]). The choice of N-Pr-Me-Pyrr (SO$_{2}$F)$_{2}$ as our polar liquid results in as close density matching as possible without the reduced liquid actuation speed found in higher viscosity RTILs. For 3PE microscopy, we require that our chosen RTIL has greater than 90% transmittance in the near-infrared (IR) region. Fortunately, high purity RTILs have weak absorption ($\alpha <$ 1 cm$^{-1}$) in the 800 - 1600 nm region, owing to an absence of molecular overtones and fundamental combination vibrations [64–67]. Finally, for safety considerations we avoided RTILs based on the [PF$_6$] anion as these have been shown to decompose into dangerous compounds such as HF vapor [57,68,69].
2.1 Transmission
The transmission of N-Pr-Me-Pyrr (SO$_{2}$F)$_{2}$ from 450 to 1500 nm is measured and plotted in Fig. 1 (red dotted line). These measurements were taken through a 1.1 mm pathlength corrected cuvette (International Crystal 0004H-1323W) using a UV-VIS-NIR spectrophotometer (Agilent Carry 5000). The transmission of PCH through the same 1.1 mm cuvette is shown by the green solid line in Fig. 1. A baseline measurement was also taken across the measurement range using an empty cuvette. Both liquids have over 90% transmission through the visible region and up to 1500 nm, after which they exhibit strong absorption bands. We measure the transmission of PCH and N-Pr-Me-Pyrr (SO$_{2}$F)$_{2}$ at 1300 nm as 90.35% and 93.06%, respectively, through a 1.1 mm pathlength corrected cuvette. As a comparison, we also plot the transmission of DI water in the same cuvette (blue dash-dotted line). The transmission of DI water at 1300 nm is measured to be 77.84% and significantly decreases past this point.
Fig. 1. The percent transmission of the RTIL (red dotted line), and nonpolar liquid PCH (solid green line) used. The transmission of DI water (blue dash-dotted line) is included for comparison. The transmission of all three liquids was measured through a 1.1 mm pathlength corrected Infrasil quartz cuvette (International Crystal 0004H-1323W) using a UV-VIS-NIR spectrophotometer (Agilent Carry 5000). A baseline measurement was taken by measuring the transmittance of an empty cuvette. At 1300 nm, we find the transmittance of N-Pr-Me-Pyrr (SO$_{2}$F)$_{2}$) to be 93.06% and of PCH to be 90.35%.
2.2 Index of refraction
A large index of refraction contrast between our polar and nonpolar liquid is essential to maximize the range of focal length tuning. We measure the refractive index of N-Pr-Me-Pyrr (SO$_{2}$F)$_{2}$ using a Metricon prism coupler (Metricon Model 2010/M) at wavelengths ranging between 400-1600 nm (Fig. 2, blue points). The index of refraction at each wavelength was measured three times, resulting in a variance too small to distinguish. We fit to these measured points using the Sellmeier equation (Eq. (2)) and find the Sellmeier coefficients for N-Pr-Me-Pyrr (SO$_{2}$F)$_{2}$ within 95% confidence (shown in Table 1).
Fig. 2. The index of refraction as a function of wavelength of N-Pr-Me-Pyrr (SO$_{2}$F)$_{2}$ measured using a Metricon prism coupler (Metricon Model 2010/M), shown by the blue plotted points. The Sellmeier equation was fitted to these points using the Sellmeier coefficients as our fitting parameters. We predict the index of refraction of N-Pr-Me-Pyrr (SO$_{2}$F)$_{2}$ at 1300 nm to be 1.43367 at 20°C.
The fitted Sellmeier equation is shown in Fig. 2 (red curve). Using our fitted coefficients, we determine the refractive index of N-Pr-Me-Pyrr (SO$_{2}$F)$_{2}$ at 1300 nm to be $\sim$ 1.433 at room temperature (20°C). We used a similar approach to measure the refractive index of PCH at various wavelengths (see Supplement 1). From this, we predict the refractive index of PCH at 1300 nm to be $\sim$1.556. Therefore, the liquid combination of PCH and N-Pr-Me-Pyrr (SO$_{2}$F)$_{2}$ provides an index of refraction contrast of $\Delta$n $\approx$ 0.123.
Table 1. Sellmeier coefficients of N-Pr-Me-Pyrr (SO$_{2}$F)$_{2}$
3. Lens characterization
3.1 Device fabrication
Our electrowetting lenses consist of a borosillicate glass cylindrical container attached to a glass ground electrode window and connected to a custom printed circuit board (PCB) [38,41]. Equal volumes ($\approx$ 35 $\mathrm{\mu}$L) of our polar and nonpolar liquid are sealed in the cylindrical container and a potential is applied between the polar liquid and the sidewall electrode. Note that we overfill our devices with our nonpolar liquid to avoid the introduction of air bubbles when sealing the device. A cross-sectional view of the fabrication design of our 4 mm diameter electrowetting lens design is shown in Fig. 3(a). The ground electrode window and the cylindrical glass container are fabricated separately and bonded together to create the final device. For our cylindrical glass container, we use a borosilicate glass tube with an inner diameter of 4 mm and a height of 5 mm. The entire tube is uniformly coated with 250 nm of indium tin oxide (ITO) via sputter deposition. The conductivity of the deposited ITO is increased by annealing at 275°C for 2 hours. We uniformly coat the inner and outer walls of the glass tube to allow electrical connection to be made through the electrode on the exterior of the tube. Each tube is next coated in 3 $\mathrm{\mu}$m of Parylene HT (Specialty Coating Systems), serving as the dielectric layer. Finally, the entire tube is dip-coated for 60 s in a 10% weight hydrophobic Cytop solution and baked at 185°C for one hour. This results in a 600 nm thick layer of Cytop used to increase the initial contact angle of the polar liquid.
Fig. 3. (a) A cross-section view of the fabrication design of a 4 mm diameter electrowetting lens. A 4 mm inner diameter, 5 mm tall cylindrical glass tube is uniformly coated with a 250 nm electrode layer (ITO), a 3 $\mathrm{\mu}$m thick dielectric layer (Parylene HT), and a 600 nm thick hydrophobic layer (Cytop). The glass tube is then bonded to a ground electrode window containing an annular ring electrode (gold) and an SU-8 insulation layer. The cavity is filled with equal volumes of a polar and nonpolar liquid (35 $\mathrm{\mu}$L) and is actuated by applying a potential between the electrode on the walls of the tube and electrode on the ground window. (b) Shows an image of a fully assembled and fabricated electrowetting lens prior to filling with liquid. The final device is attached to a custom PCB and connection is made through a pin connector. The overall device dimensions including the custom PCB are $11\times 15\times 6$ mm.
For the ground electrode window, we use a 1 $\times$ 1 cm, 500 $\mathrm{\mu}$m thick borosilicate glass chip. This allows us to use standard 2D microfabrication processing and maintain optical transparency. The chip is first uniformly coated with NR-7 photoresist which is lithographically patterned with a mask corresponding to a single annular ring electrode serving as our ground connection. The chip is then coated, via sputter deposition, with 10 nm of a titanium adhesion layer and 250 nm of gold. The excess metal in undesired regions is removed from the chip with a lift-off process in an acetone bath. We then deposit and pattern an approximately 50 $\mathrm{\mu}$m thick layer of SU-8 3050 photoresist onto the chip, serving as an additional separation layer between the ground and side electrodes.
The cylindrical glass tube and bottom electrode window are epoxy bonded with a UV curable epoxy (Norland UVS-91) and attached to a custom printed circuit board (PCB) allowing electrical connection to be made to our device with a pin connector. A silver conductive epoxy (MG Chemicals 8331-14G) is used to make connection between the PCB and the electrodes on both the ground window and exterior of the glass tube. An image of a fabricated device before filling with liquid is shown in Fig. 3(b). The overall dimensions of the device attached to the custom PCB is approximately $11\times 15\times 6$ mm. The final assembled device is filled with equal volumes ($\approx$ 35 $\mathrm{\mu}$L) of N-Pr-Me-Pyrr N(SO$_2$F)$_2$ and PCH using a micro-pipette.
3.2 Contact angle tunability
To characterize the contact angle tuning of our device, we position a CMOS camera parallel to the side of the cylindrical glass tube. Because the device is coated in a transparent electrode, ITO, we can take images of the liquid meniscus at various actuation voltages. Figure 4(a) shows images of an RTIL electrowetting lens when actuated with 0 V (top) and with 32 V (bottom). These images allow us to derive a relationship between contact angle and actuation voltage [shown in Fig. 4(b)]. By recording the change in the height of the center of the liquid meniscus over a range of actuation voltages, we can calculate the change in contact angle assuming a constant liquid volume. We observe that the contact angle of the liquid meniscus for our combination of N-Pr-Me-Pyrr (SO$_{2}$F)$_{2}$ and PCH is tunable from 133 to 48° with an actuation voltage of 0 and 60 V, respectively. We model our lens in Zemax Opticstudio and find that this contact angle range corresponds to a focal length tuning range of −22.95 to $-\infty$ mm in the diverging regime and $+\infty$ to + 23.39 mm in the converging regime. Our lens behaves as a diverging lens at contact angles $>90^\circ$ and a converging lens at contact angles $<90^\circ$. At 90° contact angle, the liquid meniscus is flat and the optical power of our device becomes zero. Actuation repeatability was verified for three different electrowetting lenses, shown by the three different colored symbols in Fig. 4(b).
Fig. 4. (a) Images of the liquid-liquid meniscus of an electrowetting lens filled with N-Pr-Me-Pyrr (SO$_{2}$F)$_{2}$ and PCH when actuated to 0 V (top) and 32 V (bottom) corresponding to a contact angle of 133° and 64°, respectively. (b) A plot of contact angle as a function of applied voltage for three different electrowetting lenses (plotted as the multi-colored symbols). These results were used to fit the Lippmann-Young equation (Eq. (1)) using the interfacial surface tension between N-Pr-Me-Pyrr (SO$_{2}$F)$_{2}$ and PCH as a fitting parameter. We find excellent agreement between our measured device contact angle and that predicted by the Lippmann-Young equation up until approximately 32 V, after which contact angle saturation causes deviation from the Lippmann-Young equation.
To verify the tuning range of our devices, we compare our measured contact angle as a function of actuation voltage to those predicted by the Lippmann-Young equation (Eq. (1)). We use 133° as our initial contact angle and a dielectric thickness of 3 $\mathrm{\mu}$m (Specialty Coating Systems) to fit Eq. (1) to our measured points using the interfacial surface tension, $\gamma$, as our fitting coefficient. Note that we exclude points measured above 32.5 V as our device shows contact angle saturation [70,71] beyond this point, causing the response to deviate from the Lippmann-Young equation. Our fitted Lippmann-Young curve is shown by the black dashed line in Fig. 4. Our fit shows excellent agreement with our measured data (R-squared = 0.95) using an interfacial surface tension of 3.193 mN/m.
3.3 Liquid switching time
The response time of our device determines the speed of switching to different imaging focal planes. To measure this, we pass a 2 mm diameter 632.8 nm incident laser beam (HeNe) through the center of our electrowetting lens and onto a photodetector (Thorlabs DET36A) with a 50 $\mathrm{\mu}$m pinhole on the front. As we actuate our device, a capillary wave is generated along the liquid-liquid meniscus [72–74]. These oscillations are recorded as a change in intensity over time on our photodetector. The blue plot in Fig. 5 shows the output of a photodetector in time as we actuate one of our devices with a 30 V step input function. The liquid meniscus takes approximately 11.64 s to reach within 2.5% of its steady state value when applying a 30 V step input function. The high viscosity of our RTIL and the low interfacial surface tension of our liquid combination results in an overdamped response of the liquid meniscus upon actuation [61,75]. N-Pr-Me-Pyrr (SO$_{2}$F)$_{2}$ has a reported dynamic viscosity of 52.7 cP at 25 °C. For reference, the dynamic viscosity of DI water is only 0.890 cP at 25 °C. Additionally, the interfacial surface tension of N-Pr-Me-Pyrr (SO$_{2}$F)$_{2}$ and PCH is 3.193 mN/m (calculated by fitting the Lippmann-Young equation to our contact angle curve). In contrast, the interfacial surface tension of deionized water and PCH is 24.83 mN/m, resulting in the liquid meniscus reaching 2.5% of it’s steady-state value within 100 ms [41].
Fig. 5. A plot of the measured intensity from a photodetector of a beam passing through the center of our lens as we actuate from 0 to 30 V. The blue plot shows the output of our photodetector when we actuate our lens with a 30 V step input function. For this case, we find the liquid meniscus takes 11.64 seconds to relax to within 2.5% of its final steady-state value. The orange plot shows the output of the photodetector when the device is actuated with a 1.75x overdrive input function for 50 ms. We find that overdriving our lens significantly decreases the liquid-liquid interface response time, relaxing to within 2.5% of its steady-state value within 5.34 s.
The use of a 1.75x overdrive input step with a length of 50 ms significantly decreased the settling time of the lens [61,76]. Figure 5 shows the response of our device when applying an over-driven input with a final voltage of 30 V (orange curve). The device is actuated at $t$ = 0 and reaches 2.5% of its steady-state value within 5.34 seconds, a significant improvement over the device’s step response. This can be further improved through the use of a shaped input voltage function [61,74] or by increasing the interfacial surface tension by using an alternative nonpolar liquid.
4. Three-photon imaging
Finally, we incorporated our electrowetting lens into a benchtop 3-photon microscope system (Fig. 6 [77]) to demonstrate its ability to perform focus-tunable 3PE imaging. This system uses a 1030 nm center wavelength, 70 W average power regenerative amplifier (Spirit-1030-70, Spectra Physics) wavelength converted by a non-collinear optical parametric amplifier (NOPA-VIS-IR, Spectra Physics) to generate a pulsed 1.1 W, 1300 nm excitation source at a one MHz repetition rate. The pulse width is measured to be $\approx$ 60 fs (measured using frequency-resolved optical gating method) at the sample plane prior to inserting the electrowetting lens. A two-axis galvo scanning mirror system is used to raster scan the excitation beam at the sample plane. After passing through the galvo mirrors, the excitation source passes through a scan lens, tube lens, and a 25X water-immersion objective (XLPLN25XWMP2, 25X/1.05 NA, Olympus) which focuses the light onto the sample.
Fig. 6. Schematic of a 3PE microscopy system. A regenerative amplifier (Spirit-1030-70, Spectra Physics), a non-colinear optical parametric amplifier (NOPA-VIS-IR, Spectra Physics), and a dual prism compressor produces a 60 fs, up to 1.1 W average power 1300 nm pulsed excitation source with a one MHz repetition rate. The power of our source is remotely tuned by rotating $\lambda$/2 waveplate and polarizing beamsplitter (PBS). This source is directed through our electrowetting lens, galvo mirrors, a scan lens and tube lens, and a 25X water-immersion objective lens. The fluorescence and harmonic emission are separated from the incident light using a dichroic mirror, spectrally separated and collected on three separate photomultiplier tubes (PMTs).
Fluorescence and harmonic emission are collected back through the objective and reflected using a dichroic mirror from the excitation path, spectrally filtered, and detected by separate photomultiplier tubes (H10770PA-40, Hamamatsu). Data was acquired using Slidebook 2021 (Intelligent Imaging Innovations). Second and third harmonic generation arise from blood vessels and the myelin fibers in the corpus collosum, respectively [78,79]. We inserted our electrowetting lens in the conjugate plane to the back aperture of the objective and include a low magnification (1.33X) telescope to relay the beam exiting our device as close to the galvo mirror plane as possible. This prevents our diverging excitation beam from clipping on the imaging optics. We measure the maximum power of our excitation beam at our electrowetting lens to be approximately 85 mW. The size of our electrowetting device (including the custom PCB) is 11 $\times$ 15 $\times$ 6 mm. In comparison, the mechanical stage used to axially translate the imaging objective (Sutter Instruments, MOM) is significantly larger at 12 x 5 x 4 cm. To perform depth scanning, we actuate our device throughout its actuation range (0 to 60 V) which changes the divergence of the beam exiting the device and incident on the back aperture of our objective. As a result, the focal plane imaged by our objective shifts axially and we take an image at each of these planes. At lower voltages (high contact angles), the large divergence angle of the incident light after passing through the electrowetting lens results in a higher laser power necessary for 3-photon imaging. To reduce the risk of damaging the sample, we chose to collect fluorescence images when our device is actuated at 20 V (100° contact angle) and higher.
Images of a 300 $\mathrm{\mu}$m thick brain slice mounted in 1x Phosphate Buffered Saline and 0.02% Sodium Azide (NaN$_{3}$) from a Thy1-GCaMP6f [80] transgenic mouse are shown in Fig. 7. Adhesive imaging spacers (654006, GRACE Bio-Labs) were used to reduce tissue compression. All animal procedures were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Colorado Anschutz Medical Campus. The image in Fig. 7(a) shows a widefield image of the region of interest taken using an Olympus BX41 microscope with a 4x objective. The images in Fig. 7(b) were acquired using the 3-photon microscope shown in Fig. 6. The approximate region imaged is marked by the red box in the widefield image. The 3-photon fluorescence signal shows a subset of labelled cell bodies and dendrites at various focal planes. These images show a composite image of three recorded channels corresponding to second harmonic generation (SHG, red), green fluorescent protein (GFP, green), and third harmonic generation (THG, blue) when actuating our electrowetting device to 24.5 V (top) and 40 V (bottom). The difference in imaging focal plane when our device is actuated to 24.5 and 40 V is found to be approximately 20 $\mathrm{\mu}$m. The amount of focal plane shift when actuating our electrowetting lens was verified by comparing captured images at each focal plane to images of the same field of view taken by performing a mechanical z-scan through 150 $\mathrm{\mu}$m depth in the sample.
Fig. 7. Images taken of a 300 $\mathrm{\mu}$m thick brain slice mounted in 1x Phosphate Buffered Saline (PBS) and 0.02% Sodium Azide (NaN$_{3}$) from a Thy1-GCaMP6f transgenic mouse. The image in (a) is a widefield image taken using an Olympus BX41 microscope with a 4X objective. The approximate region of interest imaged by our benchtop 3PE microscope is marked by a red box. This region is located between the cerebral cortex and the corpus collosum. The images in (b) show a composite of the three collected channels corresponding to second harmonic generation (SHG, red), third harmonic generation (THG, blue) and green fluorescent protein (GFP, green) when our device is actuated at 24.5 V (top) and 40 V (bottom). These two voltages correspond to an imaging plane approximately 20 $\mathrm{\mu}$m apart. Bright GFP cell bodies with connected dendrites (green) are visible in this region as well as the corpus callosum imaged by THG (blue).
To assess the imaging quality of our 3PE microscope with an electrowetting lens, we compare the images taken actuating our device throughout its actuation range with those acquired using a linear motorized stage that shifts our objective axially. The images in Fig. 8(a) were taken through the electrowetting lens when actuating to 30 V (top), 40 V (middle), and 60 V (bottom). The images in Fig. 8(b) were taken by axially scanning our 25X objective. The electrowetting lens was removed before taking the mechanical scan images and the system had to be realigned to find the same region of interest. This resulted in a slight deviation in the lateral position of the images taken when actuating our electrowetting lens compared to those taken using a mechanical scan. However, the size of the imaging field of view (FOV) was the same for both scans. We identify corresponding imaging planes between the electrowetting and mechanical scan by identifying fluorescent cell bodies and dendrites. Additionally, we use a small step size mechanical scan to identify the change in focal plane as we actuate our device through its full tunability range. For this system, we find that actuating our device in the range of 20 V to 60 V (100° to 48°, respectively) results in a total axial scan range of approximately 32 $\mathrm{\mu}$m.
Fig. 8. A comparison between three separate focal planes of the same field of view taken by actuating our electrowetting lens (a) and mechanically shifting the sample stage (b). The three images in (a) were taken when actuating our device at 30 V (top), 40 V (middle), and 60 V (bottom). The three images (b) are the corresponding imaging planes taken using a mechanical scan. We identify corresponding imaging planes between the electrowetting and mechanical scan by identifying fluorescent cell bodies and dendrites.
To verify that the introduction of our electrowetting lens does not affect the resolution of the 3PE system, we plot the intensity profile across a single fluorescent cell body in both our electrowetting scan and mechanical scan images. The intensity profile region (yellow line) for each scan image is shown in Fig. 9(a). The image on the left shows the GFP signal acquired when actuating our electrowetting lens at 40 V. The image on the right shows the corresponding focal plane found using the mechanical scan. The plot in Fig. 9(b) shows an intensity profile across the region outlined in Fig. 9(a) for both our electrowetting scan image (blue plot), and our mechanical scan image (black dashed plot). We fit the structure on the right side of the plot to a Gaussian function and find the full width half maximum (FWHM) for each scan. The FWHM of the fitted Gaussian is 10.2 $\mathrm{\mu}$m for the electrowetting scan and 9.7 $\mathrm{\mu}$m for the mechanical scan. As can be seen, the FWHM of the structure on the right is not significantly changed when introducing our electrowetting lens to the 3PE microscope system.
Fig. 9. (a) The GFP signal acquired when actuating our electrowetting lens to 40 V (left) and the corresponding focal plane found using the mechanical scan (right). (b) The intensity profile of the line cut marked by the yellow line in both images in (a). The blue plot is the line profile in the image taken using our electrowetting lens actuated to 40 V and the black dashed plot is the line profile in the image taken using the mechanical scan. We fit both of these intensity profiles to a Gaussian and find the FWHM of the structure on the right to be 9.7 $\mathrm{\mu}$m for the mechanical scan and 10.2 $\mathrm{\mu}$m for the electrowetting scan.
5. Discussion and conclusions
In summary, we have demonstrated an electrowetting lens liquid combination of N-Propyl-N-methylpyrrolidinium bis(fluorosulfonyl)imide (N-Pr-Me-Pyrr (SO$_{2}$F)$_{2}$) and 1-phenyl-1-cyclohexene (PCH) usable for applications using wavelengths ranging from visible to 1600 nm. The transmittance of N-Pr-Me-Pyrr (SO$_{2}$F)$_{2}$ and PCH through a 1.1 mm pathlength corrected cuvette was found to be over 90% at 1300 nm, justifying their use in 3-photon microscopy systems. We have measured the refractive index contrast between these liquids to be $\Delta$$n$ $\approx$ 0.123, allowing our electrowetting lens to be used to axially shift the focal plane of a 3p imaging system across its actuation range. The contact angle of this liquid combination can be tuned from 133° to 48° with an applied voltage of 0 and 60 V, respectively. Finally, we have demonstrated that these lenses can perform focus-tunable 3-photon imaging with comparable quality to a conventional mechanical z-stage.
Electrowetting lenses show great promise as an alternative to mechanical focal scanning methods in conventional 3-photon imaging systems. The compact size, weight, and minimal power usage of electrowetting tunable optics make them ideal for integration into head-mounted miniature microscopes [16,81]. Electrowetting lenses using RTILs as the polar liquid are able to withstand the large excitation power requirements of 3-photon fluorescence imaging without sacrificing tuning range. However, the relatively slow response time of these lenses as well as the density mismatch between RTILs and common nonpolar electrowetting liquids leaves room for future enhancements. The use of an alternative nonpolar liquid with a smaller density mismatch and larger interfacial surface tension with our chosen RTIL would decrease the Bond number of the liquid system, reducing the effects of gravity on the shape of the liquid-liquid interface and improving the response time. Additionally, further optimization of the input voltage function could allow for a 29% improvement in the response time of our lenses [74]. Though this work focuses on using RTIL electrowetting lenses in 3PE microscopes, the high near-IR transmission of these liquids and excellent tuning range opens the door for the use of these devices in a wide range of near-IR applications such as free-space optical communications and LiDAR.
Funding
National Science Foundation (1919148, 1926668, 1926676); National Institutes of Health (R01NS123665, UF1NS116241); Office of Naval Research (N00014-20-1-2087); University of Colorado (Anschutz-Boulder Nexus Seed Grant).
Acknowledgments
The authors would like to thank Dr. Curtis Beimborn (CU Boulder, JILA) for his valuable assistance and advice in the JILA cleanroom. We would like to thank Colter Oroke (CU Boulder) for technical assistance with the distillation of PCH. We would like to thank Dr. Wei Lim (CU Boulder, Mechanical Engineering) for useful technical discussions and Professor David Jonas (CU Boulder, Chemistry) for the suggestion to investigate RTILs. We would also like to thank Arlo Marquez-Grap and Professor Bob McLeod (CU Boulder, ECEE) for the use of their Metricon prism coupler. Finally, we would like to thank Professor Tom Finger’s lab (CU Anschutz, Biomedical sciences) for the use of a microscope for widefield images. Publication of this article was funded by the University of Colorado Boulder Libraries Open Access Fund.
Disclosures
The authors declare no conflicts of interest.
Data availability
The data that support the findings of this study are available on request from the corresponding author.
Supplemental document
See Supplement 1 for supporting content.
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