Development of pH‐Sensitive Film for Detection of Implant Infection via Ultrasound Luminescent Chemical Imaging

A new hybrid ultrasound luminescent chemical imaging technique is described along with a pH sensor to image chemical concentrations at the surface of implanted medical devices. The purpose is to detect and study local biochemistry during infection. The sensor comprises a mechanoluminescent film (SrAl2O4:Eu, Dy microphosphors embedded in a biocompatible polymer film) and a pH indicator dye. A focused ultrasound beam generates green luminescence at the ultrasound focal point. By pulsing the ultrasound ON and OFF, the modulated luminescence can be distinguished from persistent luminescence, for high spatial resolution imaging. A red fluorescent dye and the pH indicator dye bromothymol blue are added to the coating to modulate the red‐light transmittance via pH dependent absorbance. Acidosis is observed as an increase in red luminescence intensity in spectroscopy and imaging. The films are sensitive to biologically relevant changes in pH (6.0–8.0) and can be imaged through optically scattering media to mimic tissue. The images have a knife edge spatial resolution of ≈3 mm through optically scattering phantoms, limited by the focused ultrasound spot size. This novel technique may permit the elucidation of implant infection at the implant surface and can be further developed for the measurement of other relevant chemical species in the future.


Introduction
Implanted medical devices, which include heart valves, pacemakers, stents, catheters, orthopedic rods, screws, plates, hernia meshes, and prosthetic joints, are used to treat many medical conditions that improve patient quality of life and extend life expectancy.However, implant-associated infection continues to be a concern.For example, ≈5-10% of the 2 million fracture fixation surgeries performed each year result in implantassociated infection. [1]In war traumarelated fractures and treatment scenarios, infection is reported to be as high as 40%. [2]Infection risk factors include smoking, diabetes, immunosuppressed states, fracture energy, and location, open wounds, and revision surgeries for previously infected implants. [3,4]Implants increase not only the risk of infection but also infection severity because the surface forms a haven for the growth of bacterial biofilms.These infections are highly resistant to antibiotics due to factors involving the penetration of antibiotic throughout the biofilm, and the formation of varied niche microenvironments with biochemistry that can affect antibiotic action, especially pH, and oxygen.Moreover, persistent dormant regions with low nutrients and oxygen that grow slowly are formed that are not susceptible to antibiotic mechanisms based on interference with cell growth and division.Early detection of implant-associated infection is crucial for maximizing treatment efficacy.For example, if a prosthetic joint infection is diagnosed within four weeks of implantation, surgical irrigation and debridement of the implant surface with the administration of antibiotics is often sufficient to eradicate the infection. [5,6]However, early detection is often difficult, especially when the bacteria are localized near the implant surface.[9] Device removal requires additional surgery and hospitalization, is expensive, and there is an increased risk of infection for the new device.Hence, early detection of implant infection may reduce the need for invasive treatment.In addition, elucidating the biochemical environment at the implant surface, where persistent biofilms grow, can guide efforts to develop therapeutics capable of targeting biofilms regions with reduced antibiotic susceptibility.
Currently, infection-specific molecular imaging is performed using radiolabeled probes, imaged with either positron emission tomography or single-photon emission computed tomography.However, these techniques are expensive and require proximity to radiochemical synthesis facilities with a cyclotron. [10]Alternative imaging modalities that do not use radiopharmaceutical contrast agents, such as computed tomography, magnetic resonance imaging, ultrasound, and X-ray projection imaging, can provide excellent spatial resolution but lack the chemical sensitivity required for early diagnosis of infection or quantification of the local microenvironment. [11]H at the surface of an implant is important to measure both to elucidate the local biochemistry in order to develop therapeutics that work in antibiotic resistant biofilms, and because it might be an early/local indicator of infection.Acidosis is a product of bacterial metabolic fermentation and respiration (i.e., lactic and carbonic acid produced in glycolysis), and bacterial colonies have been shown to produce a local acidic environment in vitro. [12,13]nflammatory reactions by the host can also contribute to acidosis.This acidic environment plays a role in reduced antibiotic susceptibility of biofilms, along with other factors such as nutrient limitation and insufficient antibiotic penetration. [7]Thus, measuring local pH could be useful for early detection and imaging of local biofilms.It would also be relevant for techniques that aim to target low pH environments, [14] select antibiotics that work in that environment, [15] or increase local pH to potentiate antibiotic treatments. [16,17]reviously, our group has reported implantable pH sensors developed to measure local pH at the surface of orthopedic implants to detect and study implant-associated infection that were read using radiography using two different methods.In the first method, the sensor comprised a radiopaque tungsten indicator pin embedded within a chemically responsive hydrogel that exhibits a pH-dependent swelling.This sensor was calibrated in a series of standard pH buffers and tested it during bacterial growth in culture.In addition, radiographic measurements were also performed in cadaveric tissue with this sensor attached to an orthopedic plate fixed to a tibia. [18]In the second method, the sensor comprised a Gd 2 O 2 S: Eu scintillator layer that emitted 620 and 700 nm light upon X-ray irradiation and a pH indicator layer containing bromocresol green dye that absorbs 620 nm luminescence in neutral/basic pH and passes 700 nm light at all pH.Using a focused X-ray beam that irradiated one spot on the sensor, and the 620 to 700 nm peak ratio was measured to determine the local pH.This sensor was further attached to an orthopedic plate affixed to a human cadaveric tibia and imaged through tissue. [19]n addition, our group also demonstrated the first implantable peritoneal fluid sensor for measuring peritoneal fluid pH using plain radiography. [20]Furthermore, the first in vivo use of an implantable tri-anchored methylene blue-based electrochemical pH sensor was demonstrated by another group that allowed accurate tracking of externally induced pH changes within a naturally occurring ovine lung cancer model, which was further validated with laboratory measurements made on extracted arterial blood. [21]erein we introduce Ultrasound Luminescent Chemical Imaging (ULCI), a technique to monitor local pH at the sur-face of a modified implant during infection (Figure 1). [22]Specifically, to image or map a target area via a point-by-point raster scanning with the ULCI system, we modified the X-ray Excited Luminescent Chemical Imaging (XELCI) [12,19,23,24] system that has been successfully used to image pH in human cadaveric tibia [19] and live rabbits. [25]The XELCI system produces high resolution images, but uses a relatively expensive focused X-ray source, and the associated radiation dose that limits repeated measurements. [10,24]ULCI scanning and image generation is similar to XELCI but uses a pulsed ultrasound source to excite a mechanoluminescent pH sensor instead of a focused Xray beam.Traditionally, ultrasound imaging is used as a complementary diagnostic tool to capture structural images through biological tissues.Ultrasound (US) is an attractive optical excitation source because it is non-ionizing and scatters less than light in biological tissues. [26]However, ultrasound alone does not provide the biochemical information necessary to measure pH and monitor local infection.There have been reports on ultrasound modulated fluorescence [27] contrast agents based on ultrasound induced heating and ultrasound enhanced chemiluminescence contast based on reactive oxygen generation. [28]dditionally, mechanoluminescent materials have been used to image ultrasound fields, [29] and to excite optogenetics from nanoparticles, [30,31] and excite photodynamic therapy from implanted microLEDs [32] but this is the first report on mechnoluminescence through tissue mimicks and ultrasound modulated luminescence to image changes in local biochemistry related to implant infection.To impart biochemical sensitivity, an ultrasound luminescent film is paired with a pH-sensitive dye.Specifically, SrAl 2 O 4 : Eu, Dy is a commercially available mechanoluminescent material that emits a maximum luminescence intensity at the wavelength of ≈522 nm, in response to mechanical stimulation such as ultrasound.This emission is attributed to the 4f 6 5d − 4f 7 electronic transition of Eu 2+ ions in SrAl 2 O 4 : Eu. [33] Emission is red-shifted using two different fluorescent dyes, a) a Krylon fluorescent spray paint coating over the sensor, and b) Nile red fluorescent dye entrapped in the HydroMed D3 coating along with the scintillator and pH indicator.
After shifting the emission to >605 nm, the mechanoluminescence overlaps with the absorption of pH-sensitive bromothymol blue at pH 8 (≈627 nm).As pH becomes more acidic, the bromothymol blue film transmits more luminescence, manifesting as an increase in luminescence with decreasing pH.To use a single dye in this sensor design, there are a few important considerations.First, the pH must be sensitive in the physiologically relevant range (i.e., pH ≈6.0-8.0).Second, the dye must be able to modulate the luminescence signal according to pH, (i.e., the excitation wavelength of the dye must overlap with the emission of our mechanoluminescent film, which is 522 nm for SrAl 2 O 4 : Eu, Dy).Third, the dye should emit red or near infrared light (>600 nm) to penetrate biological tissues for pH measurement.Fortunately, many pH indicators are available including both absorption based dyes used here and fluorescent pH probe, e.g., SNARF that could be used in the future. [34]n this study, we show the proof-of-principle for imaging changes in pH using a biocompatible absorption indicator-based sensor using ultrasound luminescence, under different pH conditions through a light scattering media.The ULCI materials were charged prior to the experiment with UV light.In future, the Sensor is attached to or coated on an implant (e.g., fracture fixation plate on a fractured bone).Focused ultrasound excites mechanoluminescence from the film; to form an image, light is collected by a liquid light guide as the ultrasound scans over the sample point-by-point.Sensor Design: Green luminescence of SrAl 2 O 4 :Eu,Dy is shifted using a red dye to overlap absorption of pH-sensitive dye.As a result, phosphorescence is modulated by pH-sensitive dye.Growth of bacterial biofilm generates an acidic environment, which causes the pH-sensitive film to turn yellow.Yellow film transmits more luminescence, resulting in higher transmittance of luminescence emission.Blue/green color film, associated with physiological and basic pH, transmits less luminescence emission.pH is monitored as a function of luminescence emission intensity.
approach may also be extended to materials such as Lu 3 Al 5 O 12 : Ce 3+ -based elastomers that do not require pre-irradiation, [35] or the UV excitation could be replaced by infrared excitation coupled with infrared-to-visible upconversion phosphors, [36] or excitation by a local beta radiation emitting source, [37] or using a minimally invasive process of charging using fiber optic cables.

Results and Discussion
Our ultrasound luminescent (UL) pH sensor and imaging technique is designed to map pH on the surface of orthopedic implants.Herein, we first demonstrate the phenomenon of ultra-sound luminescence (UL) using SrAl 2 O 4 :Eu, Dy polymer films (Section 2.1); we image UL targets to evaluate the spatial resolution (Section 2.2); and we describe UL pH sensors imaged using the ULCI setup (Section 2.3).

Optical Characterization of Ultrasound Modulated Mechanoluminescent Film
Figure 2a shows a bright spot produced by a focused ultrasound beam on a previously UV-irradiated polydimethyl siloxane (PDMS) encapsulated SrAl 2 O 4 :Eu, Dy microphosphor film.The 1 Hz (i.e., the ultrasound was turned ON 5 s and OFF 5 s, and 10 s/cycle).c) Luminescence intensity versus time for one US pulse, depicting the phosphorescence increases rapidly with ultrasound excitation and decays exponentially once ultrasound is turned OFF. [18]d) Spectra from SrAl 2 O 4 :Eu, Dy film as ultrasound is pulsed ON and OFF.After initial excitation with 395 nm light, ultrasound excitation produces a ≈23x increase in luminescence intensity compared to when the ultrasound is switched OFF. [22]chanoluminescence can be turned ON and OFF by pulsing the ultrasound beam as a function of time, as shown in Video S1 (Supporting Information).When the ultrasound is turned ON, a visible bright spot is observed on the film indicating a higher emission intensity, and when it is switched OFF, the emission decreases, and the spot is no longer visible to the naked eye.Moreover, the luminescence intensity increases by a factor of ≈23 when the ultrasound is pulsed ON compared to the spectra taken while the ultrasound is pulsed OFF (see Figure 2b-d; Figure S1, Supporting Information).This factor depends on the duration of UV excitation, saturation of the CCD camera at high intensities, and the background luminescence of the SrAl 2 O 3 : Eu, Dy afterglow that decays over time.For example, Figure S1 (Supporting Information) shows four different trials on linear and log y-axis scales, showing similar modulation patterns, although with different ON and OFF values, as they were excited at different times.
Interestingly, we observed a ≈10 nm blueshift in the luminescence spectrum of SrAl 2 O 4 :Eu, Dy under ultrasound excitation compared to the afterglow emission seen after UV excitation.Typical afterglow emission spectra of SrAl 2 O 4 :Eu, Dy show an emission maximum at 520 nm, [38,39] while our spectrum under ultrasound excitation is 512 nm.A blueshift suggests that emission is occurring from a higher energy state than with UV excitation alone.The mechanism for phosphorescence involves absorption of excitation energy, followed by intersystem crossing to an excited triplet state, from which long-lived phosphorescence is emitted as electrons transition back to the singlet ground state. [40,41][44] The ultrasound luminescence decay behavior in Figure 2c could be fitted to a double exponential: I (t) = I 01 e −t∕ 1 + I 02 e −t∕ 2 , where,  1 ≈ 0.6 s,  2 ≈ 5 s, indicating a slow and fast exponential decay (see Figure S2a, Supporting Information) arising from different detrapping probabilities.Interestingly the UV excitation of our PDMS coated SrAl 2 O 4 : Eu, Dy could not be fitted using a double exponential, rather it followed a power law behavior (I∝t k ), as shown in Figure S2b,c (Supporting Information), where k ≈1, which indicates that tunneling of trapped electrons to the recombination centers is a dominant phenomenon. [45]Regardless of the precise mechanism, Figure 2 shows a large increase in luminescence intensity of the film in the region of the focused ultrasound beam.

Target Imaging Using ULCI Scanner
To measure the knife-edge spatial resolution of the ULCI scanning system, we imaged black triangular and rectangular electrical tape targets on the SrAl 2 O 4 :Eu, Dy film (see Figure 3a) using ultrasound excitation and a point-by-point raster scanning through a scattering media.Figure 3b shows a schematic of the UL scanner.SrAl 2 O 4 :Eu, Dy is first "charged" by briefly irradiating with ultraviolet (395 nm) light.The film then emits longlasting afterglow luminescence, which decays following a power law behavior.By pulsing the ultrasound ON and OFF, we observe a modulated bright spot at the ultrasound focus, along with a continuous background at other regions.Separating the signal blinking at the ultrasound cycle frequency allows us to distinguish between the ultrasound modulated signal, and the signal attributed to background afterglow luminescence as depicted in Figure 3.If our ultrasound source were to be continuously ON, we would be unable to distinguish between background afterglow luminescence and ultrasound modulated luminescence, as shown in Figure S3 (Supporting Information), where the target is imaged with ultrasound continuously ON.This is especially true for targets larger than the ultrasound focal point/spot size (≈3 mm), where the combined background luminescence can be higher than the luminescence from the ultrasound excited region.
Figure 3c shows the luminescence intensity of the mapped target area (30 x 30 mm) obtained from a PMT, when the ultrasound signal was modulated at a frequency of 2 Hz (i.e., the US was turned ON and OFF every 0.5 s, 2 cycles/s).We raster scanned the 30 x 30 mm area at a scan speed of 1 mm s −1 with 15 horizontal scans as can be seen in Figure 3c,f,i.A Video S2 (Supporting Information) shows an example of UL during raster scanning.Mapping the UL source could clearly identify the rectangular and triangular areas (covered by black tape) through the creamer solution, with observed spikes that depict ultrasound modulation (Figure 3c,d).Figure 3d shows the luminescence intensity along selected raster scan lines corresponding to the dashed lines (L3, L9, and L15) through the image in Figure 3c.As expected, the intensity of the bright areas of L9 line matches that of the bright area of L3.Despite a significant background level (as indicated by L15), the luminescence intensity from ultrasound pulsing is observed as well as the absence of signal in areas covered by black tape that absorbs luminescence.The spikes observed in each of these linear scans correspond to the modulation of the luminescence intensity with respect to the US modulation using a frequency of 2 Hz.Although measured as a function of position during each raster scan, the amplitude of these oscillations could be computed as a function of time (in second) using the raster scan speed (1 mm s −1 ), and the distance traveled (30 mm along the x-direction).
The ultrasound modulation is also evident from the fast Fourier transform (FFT) showing a blinking signal at 2 Hz (Figure 3d), except where there is no mechanoluminescence film (line L15).Furthermore, the 0 and 2 Hz components of the sample area are mapped using Matlab's spectrogram function (a short-time Fourier Transform with a 2 s, or 2 mm, window, stepped every 1 s or 1 mm), as shown in Figure 3f,i, respectively.These show that the 2 Hz blinking signal generates a higher contrast and spatial resolution image of the target than the 0 Hz component.Specifically, in Figure 3i,j for horizontal line H9, we observe the signal with the sharp edge of the triangle increases from ≈6000 counts to 45 000 counts in 3 mm, and at the edge of the mechanoluminescent film falls from 50 000 counts to 5000 counts over 3 mm, showing a 3 mm knife edge resolution.This signal is clearly absent at position H15, where there is no meachnoluminescent film or target (there is a little residual signal, which is likely from the edge of the ultrasound beam hitting the edge of the mechanoluminescent film).A similar knife-edge transition is also evident in the vertical direction (Figure 3i,k).
By contrast, the 0 Hz component of the image (Figure 3f,g,h) has much poorer spatial resolution.Some of the blinking signal shows up in the 0 Hz component image in part because as we turn on the ultrasound we increase the average luminescence, and in part because the collection optic does provide some position dependence albeit with a large point spread function.If we examine the falling edge of the film (H9), we see a continuous change from ≈12 000 000 counts at x-position 14 mm to 5 000 000 counts at 28 mm, with no obvious edge, and only a small difference at H15 below the film indicating a resolution >13 mm and possibly significantly more.Similarly, it is difficult to discern the target in the vertical direction (Figure 3f,h).This low resolution luminescence imaging through the scattering media is expected because our collection optic is ≈2 cm above the target and is able to collect light that has diffused laterally from its starting position and angle.The ultrasound modulation allows us to define a specific region in the ultrasound focus.
The spatial resolution of ULCI technique is essentially limited by the size of the ultrasound beam, which only generates light in the excited spot on the UL film.Here, we calculate the theoretical Abbe diffraction-limited spot size as follows: where, BD is the minimum resolvable beam diameter according to the Rayleigh criterion,  is ultrasound wavelength (≈0.9 mm), calculated using the speed of sound in water (1530 m s −1 ) and the ultrasound transducer frequency ( = 1.7 MHz), and  is the halfangle. [46]Based on an approximate half angle of 10°(0.17rad) and the wavelength of our ultrasound, we calculate a diffractionlimited spot size of ≈3.2 mm.Our current focused ultrasound source generates a spot (Figure 2a) with a width of ∼3 mm, similar to the Abbe diffraction-limit, however, when the beam is angled with respect to the mechanoluminescence film, it forms an elliptical spot, and the larger dimension in Figure 2 is 5 mm.We note that the 1.7 MHz source frequency is low compared to most diagnostic ultrasound (typically 2-20 MHz) and this limits the focal spot size.In the future, we plan to use a higher frequency ultrasound source with a smaller spot size (e.g., a 10 MHz ultrasound source with the same numerical aperture would be expected to generate a spot diameter of 0.2 mm while still allowing reasonable transmittance through soft tissue). [47]

pH Sensor Development
Local acidosis is an indicator of bacterial growth on biomedical implant surfaces. [12]Accordingly, we leveraged our ultrasound luminescent imaging methodology to detect local chemical changes such as pH.Emission wavelength is one of the first considerations in designing a luminescent sensor to be read through tissue.Longer wavelengths of light are absorbed and scattered less than shorter wavelengths by biological tissues. [48]ence, red emission is ideal for designing an optical sensor to be read through tissue.To maximize light collection efficiency through tissue, we added a fluorescent dye to redshift the green luminescence of SrAl 2 O 4 :Eu, Dy) (Figure 2).While these experiments were done in vitro, in vivo we would expect some signals that could interfere with the modulated ultrasound signal including breathing, muscle motion, and pulsatile blood flow.In previous studies with X-ray excited luminescence chemical imaging through >1 cm of tissue, we saw no signals that would significantly interfere with our measurements. [25,49]However, if it is a problem for the ultrasound because we are extracting a weaker signal at 2 Hz, one could either try to measure variation in transmittance with external pulse oximeters, or vary the signal to avoid common backgrounds, although the ≈1 s luminescence response time means that increasing the blinking rate would attenuate the modulated signal.
For human and animal studies, light passes through both tissue and skin.Skin absorbs and scatters (attenuates) light, with optical transmittance depending upon wavelength, patient skin pigmentation, and anatomical region.Pigmentation from melanin absorbs more light in the blue than in the red and especially near infrared spectral regions.At 620 nm, diffuse reflectance of dark pigmented skin (Fitzpatrick scale V-VI) measures ≈20% versus 60% for fair pigment skin (Fitzpatrick scale I-II) [50] and dermal reduced scattering coefficients are 2,3 mm. [51]ased on these values, red light is able to penetrate skin and scatter from underlying tissue for fair and dark skin, albeit with some attenuation.Variations in transmittance through skin could be considered part of the variations in optical collection efficiency, which also depend on position and angle of collection optics.Importantly, the spatially separated reference region on the implant accounts for most of these optical transmission issues as long as Vertical lines evident in these images are due to ultrasound modulation at 2 Hz (1 mm s −1 scan rate). [22]e tissue is relatively uniform between the reference and sensor regions.Our previous studies also demonstrate that optical absorption at 620 and 700 nm could be detected through 1 cm of skin and tissue in both live rabbits and a human cadaveric leg.[19,52] If needed, scattering can be reduced by applying glycerol to make refractive index more uniform, [53] and fluorescence can be used to shift light to the near infrared where melanin and tissue absorption are much lower.[54] We targeted a mechanoluminescent film emission wavelength of ≈600-650 nm, which would penetrate through reasonably thick tissue and overlap well with bromothymol blue indicator dye.At basic pH (≈8), bromothymol blue appears blue and absorbs red light (≈627 nm), while at acidic pH (≈4), bromothymol blue appears yellow and absorbs ≈474 nm light.As a result, bromothymol blue can be used to modulate the luminescence intensity of red-emitting phosphors as a function of pH.[55] SrAl 2 O 4 :Eu, Dy exhibits a luminescence maximum at ≈522 nm, and only slightly overlaps with the bromothymol blue absorption maxima at 627 nm.Thus, the emission of SrAl 2 O 4 :Eu, Dy must first be shifted to ≈627 nm to overlap with the absorption of bromothymol blue (pH 8) to maximize the luminescence modulation as a function of pH (Figure S4, Supporting Information).We demonstrate two methods to shif SrAl 2 O 4 :Eu, Dy emission: 1) Krylon fluorescent spray paint and 2) Nile red fluorescent dye, using two pH sensor designs.The first design is a 2-layer proofof-concept design using Krylon cerise fluorescent spray paint to shift SrAl 2 O 4 :Eu, Dy emission, and a polyacrylamide hydrogel impregnated with bromothymol blue to modulate luminescence emission as a function of pH.The second sensor design also contains bromothymol blue but incorporates all components into a single hydrophilic polyurethane (HydroMed D3) film.Nile red is used in this design to shift SrAl 2 O 4 :Eu, Dy emission rather than Krylon spray paint.

Two-Layer pH Sensor: Krylon Spray Paint Modified UL Film and pH-Sensitive Hydrogel
As proof of principle, cerise (reddish pink) colored Krylon spray paint was spray-coated on top of the SrAl 2 O 4 :Eu, Dy, and PDMS film to shift the luminescence emission to overlap with the absorption of a pH-sensitive dye, bromothymol blue (Figure 4).When coated with Krylon spray paint, the luminescence emission is absorbed, and fluorescence is emitted by the paint at ≈607 nm.This emission wavelength overlaps well with the absorption maximum of bromothymol blue at pH 8 (Figure 4).When the surrounding medium is at a higher pH, more of the luminescence emission is absorbed by the bromothymol blue dye.As a result, when a hydrogel impregnated with bromothymol blue is placed on top of the Krylon™ coated SrAl 2 O 4 :Eu, Dy film, the luminescence intensity decreases as pH increases.This is because the bromothymol blue film under acidic conditions does not absorb as much of the luminescence emission as compared to more basic conditions.
The pH-modulated luminescence intensity at a given wavelength can be estimated according to the following relationship between the percent transmittance of bromothymol blue at a given pH based upon the Beer-Lambert law of protonated and deprotonated forms of the dye and the Henderson-Hasselbalch equation to determine the fraction of protonated and deprotonated forms (Equation 2).The derivation of Equation 2 is provided in the Supporting Information.Generally, the light intensity at a given pH, L PH , depends on the light intensity with no pH indicator film, L 0, (which can be measured in an uncoated reference region), the effective optical path length that relates to the film thickness, b, formal dye concentration, F, the molar absorption coefficient of deprotonated and protonated forms of the dye  In-, and  HIn , respectively, and the pK a of the dye.
When the pH is high compared to pK a , the larger fraction of deprotonated bromothymol blue, 10 (pH − pKa) /(1 + 10 (pH − pKa) ), results in a higher red light absorbance.In other words, as pH increases, the SrAl 2 O 4 :Eu, and Dy luminescence transmission decreases.This is seen clearly in Figure 4a, where the luminescence spectra of the Krylon™ coated SrAl 2 O 4 :Eu, Dy film is collected as it passes through the pH-sensitive bromothymol blue hydrogel at pHs between 4 and 9.The luminescence emission intensity, under consistent UV excitation, is highest at low pH (pH 4), and lowest at high pH (pH 9).
The pH sensor film, which also contains a reference region of hydrogel without bromothymol blue, was imaged in a solution of coffee creamer (≈1.0 g L −1 ) to simulate light scattering in biological tissue (a simple but commonly used method to make tissue phantoms). [56]Figure 4d,e shows images at pH 4 and pH 9, using an ultrasound modulated frequency of 2 Hz, with a scan speed of 1 mm s −1 .The pH-sensitive target is clearly distinguishable from background, in comparison to the control target.Furthermore, the target at pH 9 appears darker, according to the luminescence color map, when compared to the target in the image at pH 4. The decrease in luminescence with an increase in pH is consistent with the spectroscopic data shown in Figure 4a.
The use of Krylon ™ spray paint to redshift SrAl 2 O 4 :Eu, Dy film was successful but impractical for application in vivo.The fluorescent dye used in the Krylon ™ spray paint is considered proprietary, however, it is not intended for in vivo use and spray paint usually contains toxic hydrocarbons such as toluene, benzene, and xylene, which are associated with an increased risk of renal tubular acidosis, hypokalemic paralysis, and various hematological disorders. [57,58]While it may be possible to mitigate potential leaching and toxicity by encapsulating the fluorescent dye within the mechanolumionescent SrAl 2 O 4 :Eu, Dy/PDMS film, and the proprietary formulation also may affect the ability to reproduce data in the future if the formula changes.Consequently, we inves-tigated alternative dyes in the next section(s) that similarly shift the SrAl 2 O 4 :Eu, Dy luminescence emission spectrum.
Another challenge with this approach is the attachment of the hydrogel pH-sensitive target to the paint-coated SrAl 2 O 4 :Eu, Dy film.The hydrogel is somewhat brittle and can fracture during attachment to the mechanoluminescent film.In the images in Figure 5, the hydrogel is attached to the luminescent film using cyanoacrylate.Occasionally this adhesive interacts with the pH-sensitive target, preventing reliable pH response, and color change.Ideally, the goal is to make a pH-sensitive, ultrasound luminescent film that contains all the necessary components in a single layer.The next section discusses preliminary work and imaging of a single-layer composite film.

Single-Layer: pH-Sensitive HydroMed Film with Nile Red Modified UL
To incorporate and immobilize all pH sensor components into a single layer, commercial HydroMed D3 was selected.HydroMed is a biocompatible, hydrophilic, and ether-based polyurethane intended for coating biomedical implants.The hydrophilic properties permit diffusion of hydronium ions while keeping pH sensor film components (SrAl 2 O 4 :Eu, Dy, and dyes) immobilized.Another advantage is the structural pliability, making it ideal for conformal coating of implant surfaces.To replace Krylon fluorescent spray paint, we employ Nile red dye (9-(Diethylamino)−5Hbenzo[a]phenoxazin-5-one) to redshift the green emission of SrAl 2 O 4 :Eu, Dy.Nile Red is a lipophilic biological reagent suitable for fluorescence and has applications as a membrane dye in cell biology. [59]Unlike the spray paint that was coated on top of the luminescent component of the film in our first pH sensor, the Hy-droMed film encapsulates the Nile Red in our second sensor.Our leaching studies aimed to understand the stability of our sensor suggested that the Nile Red did not leach from the PDMS coating (cf. Figure S5a, Supporting Information).Thus, compared to spray paint, our pH-sensitive HydroMed D3 film containing Nile red, is certainly more biocompatible.To confirm there is no diffusion of pH sensor components, we performed leaching study by incubating the sensors in the pH buffers (pH 6-8) and recorded the absorbance and emission spectra of the buffer solutions.Our results indicated that, bromothymol blue leached but no leaching of strontium aluminate (Sr 2 Al 2 O 4 :Eu,Dy) and Nile red.
When a polar solvent (i.e., ethanol) is used to deposit a film of HydroMed D3 (hydrophilic polyurethane), SrAl 2 O 4 :Eu, Dy, and Nile red, the absorption of Nile red overlaps sufficiently with the emission of SrAl 2 O 4 :Eu, Dy.As a result, the fluorescence maximum of Nile red in the UL film is observed at ≈624 nm (dry film), compared to ≈522 nm luminescence with SrAl 2 O 4 :Eu, and Dy alone (Figure S4, Supporting Information).The fluorescence of Nile red overlaps with the absorption of pH-sensitive bromothymol blue at pH 8 (≈627 nm), which makes Nile red a suitable replacement for the Krylon cerise spray paint.
Nile red is a solvatochromic dye, which means the shape and spectral position of absorption and emission bands vary (up to 100 nm) with solvent polarity.Although Nile red is solvatochromic, the ionic strength of the physiological salts, e.g., sodium, a major cation of extracellular fluid (normal physiological concentration ≈135-145 mmol/L) is well regulated within 5%. [60]Hence, we expect our sensor to still work in the presence of physiological ions.[63] This phenomenon is seen in fluorescent compounds that have aromatic rings with polar substituents, details of which have been explored elsewhere. [64]Due to its solvatochromic properties, Nile red response to pH change is characterized separately using a HydroMed D3 film containing only Nile red and SrAl 2 O 4 :Eu, Dy (Figure S6, Supporting Information).The film exhibits emission maxima at 503 and 624 nm before submersion in buffer (dry).Immediately after submersion in pH 5 buffer, the film changes from a deep pink color to a more pastel purple color.After equilibration in buffer for 30 min, the color does not change dramatically from pH 5 to 9. The excitation peak at 503 nm shifts to 510 nm at pH 5, 508 nm at pH 7, and 506 nm at pH 9. The maximum emission peak at 624 nm, which is the region of luminescence modulated by bromothymol blue, remains at 638 nm at pH 5, 7, and 9. (cf. Figure S6, Sup-porting Information).The absorption maximum for bromothymol blue at pH 8 is ≈627 nm, so the redshift in emission maximum of Nile red means there is less overlap between the fluorescence and absorption.However, the shift is not dramatic enough to prevent this film design from working, and the advantages of a single-layer film outweigh the potential drawbacks.Additionally, once the film is equilibrated in an aqueous solution (of any pH studied), the emission remains consistent at 638 nm.Nile red, is within our target pH range, and remains an appropriate choice to shift the emission spectrum of SrAl 2 O 4 :Eu, Dy.
Bromothymol blue was added to the HydroMed D3 film along with SrAl 2 O 4 :Eu, Dy, and Nile red to impart pH sensitivity.As expected, this film is much more flexible and easier to manipulate without tearing or fracturing.In Figure 5a, the single-layer film response to pH 6.0-9.0 is demonstrated by taking afterglow luminescence spectra following UV excitation, after equilibration in various buffer solutions.Spectra acquired with UV ON can be found in Figure S7 (Supporting Information).Spectra acquired with UV OFF have lower emission intensity overall, which is because the afterglow of SrAl 2 O 4 :Eu, Dy decays exponentially with time.Because the pH response of this film is based on changes in luminescence intensity, the time between switching the UV flashlight OFF and collecting spectrum needs to be consistent for each pH spectrum collected.For this reason, the characterization is compared with spectra taken while the UV light remains ON.For both UV ON and UV OFF, the emission intensity at 638 nm decreases as pH increases.The wavelength 638 nm is chosen because this is the fluorescence maximum determined for Nile red within this pH range (Figure S6, Supporting Information).As expected, the bromothymol blue dye absorbs more of the luminescence emission as it changes from yellow to green (i.e., as pH increases).Despite the 14 nm redshift in Nile red fluorescence (Figure S6, Supporting Information), the single-layer film behaves in a similar manner to the two-part film and is most sensitive to changes between pH 6.0 and 8.0.Normal muscle tissues have relatively neutral pH (between 7.0 and 7.4), and a study of implant surface pH during infection (using microelectrodes) showed that pH dropped to ≈6.0. [65]Therefore, this film is sensitive within the correct range to measure biologically relevant pH for localized acidosis.In Figure 5b, a calibration curve is developed by plotting the ratio of the measured luminescence intensities at 638 to 520 nm (from panel a), as a function of pH in the range 6 to 9, which is modeled using Equation 2, derived from the Henderson-Hasselbalch equation for pH-dependent fraction of protonated indicator dye and the absorption spectrum of protonated and deprotonated species.The uncertainty was determined from the noise level within the single spectra.
The film was imaged at pH 6.0 and 9.0 (cf. Figure 5c) using the ULCI scanner as shown in Figure 3b.According to the color map generated in MATLAB, the luminescence intensity is ≈5x brighter at pH 6.0 when compared to pH 9.0.The luminescence in the control target remains the same at each pH value.SrAl 2 O 4 :Eu, Dy in HydroMed D3 was used as a control (C) to demonstrate that the imaging system is unaffected by changing the pH (cf. Figure 5d).The film behaves as expected, and the ULCI images reflect a change in luminescence intensity consistent with an increase in surrounding pH.

Limitations
The ULCI technique has some limitations that are important to describe: 1) It requires coating the surface of an implant with a mechanoluminescent and chemical sensing agent.2) It requires an ultrasound transducer that usually needs to be in contact with the sample.3) Ultrasound has some inherent limitations with tradeoffs between frequency (resolution), and depth, especially for maintaining intensity or focus through interfaces such as bone.4) To avoid heating effects in tissue, diagnostic ultrasound is usually limited to ≈700 mW cm −2 , [66] this is sufficient for most cases but may be a concern especially for imaging through deep tissue.5) The current approach used a UV light to excite the SrAl 2 O 4 :Eu, Dy traps prior to mechanoluminescence generation.However, there may be other approaches that could be used to recharge the sensor including the use of X-ray excited luminescence, [67][68][69] or similar beta luminescence, [37] or near infrared upconversion luminescence with an upconversion phosphor [70] or chemiluminescence.It may also be possible to use other mechanisms to convert ultrasound to light (e.g., piezoelectric transducer with an electroluminescent signal). [71]Finally, the current embodiment is a first proof of principle using an inexpensive (≈$15) ultrasound transducer (AW 1605) from a mist maker that had relatively low resolution.Future improvements would involve higher frequency transducers for higher resolution images, and eventually phased array to rapidly scan the ultrasound focal spot and superimpose ULCI with ultrasound images.

Conclusion
We designed a novel ultrasound luminescent chemical imaging technique, along with two novel UL pH-sensitive films.Twolayer films, with a primary layer containing mechanoluminescent SrAl 2 O 4 :Eu, Dy, and Krylon fluorescent spray paint and a secondary layer containing pH-sensitive bromothymol blue, show sensitive pH response between 6.0 and 8.0.To improve upon the initial design, we developed a single-layer pH sensor film.This film contains HydroMed™ D3, SrAl 2 O 4 :Eu, Dy, Nile red, and bromothymol blue.After characterization of solvatochromic Nile red at pHs 5, 7, and 9, we found that the shifted signal at 638 nm is modulated effectively by bromothymol blue.This film is sensitive to changes in pH between 6.0 and 8.0 and is easy to fabricate.
The technique provides high spatial resolution images (limited by the ultrasound focus spot size) with low background (as tissue is not mechanoluminescent) and good chemical specificity from the indicator dyes.By changing indicator dyes, we expect multiple different analytes can be imaged, or an array of sensors could be used for the analysis of multiple analytes in well-mixed fluids.Unlike X-ray excited chemical luminescence imaging (XELCI), ULCI is free from ionizing radiation, which allows for repeated measurements (although in principle, both could be used synergistically as SrAl 2 O 4 can be excited by X-ray).
Last, we plan to optimize the optical setup and collection efficiency, as well as determine the best ultrasound pulse frequency for imaging.For example, in addition to the 625 nm filter that collects the pH modulated signal, a 475 or 750 nm filter could potentially serve as a reference.The ratio of reference wavelength emission to pH modulated wavelength emission can be used to account for scattering and absorption due to the presence of biological tissue (or mimics).In addition, the positioning of the liquid light guide and ultrasound source can be optimized with respect to excitation and collection efficiency.A custom holder can then be fabricated to maintain optimized positioning and aid reproducibility.Eventually, a phased ultrasound array could be used to rapidly scan the ultrasound beam without moving the actual sample.
This series of studies demonstrates proof-of-concept for an ultrasound luminescence-based chemical imaging methodology applied to imaging changes in pH.Localized acidosis is associated with implant-associated infection, and an ultrasound-based method to probe the chemical environment of the implant surface would be useful to detect infection at an early stage.With improvements to sensor design, light collection, and imaging setup, we hope to investigate this sensor in an animal model of infection in the future.fluorescent dye (N0659, TCI America, Portland, OR, USA).All recipe calculations were based on the total volume of solvent (95% ethanol) chosen and did not account for volume changes due to the addition of individual components.First HydroMed D3 (120 mg mL −1 ) was added to ethanol.The mixture was placed on a 360°sample rotator for 24 h to completely dissolve polymer.The following components were added once HydroMed was dissolved: SrAl 2 O 4 :Eu, Dy (300 mg mL −1 ), and Nile Red (0.08 mg mL −1 ).An example recipe is 0.75 g SrAl 2 O 4 :Eu, Dy, and 0.3 g HydroMed D3, 2.5 mL ethanol (95%), and 200 μL Nile Red (1 mg mL −1 in acetone).The mixture was then placed on the 360°sample rotator for 3 h to ensure a homogeneous combination of all components.The mixture was then carefully poured onto a glass microscope slide and placed into a vacuum chamber for 12 h to evacuate air bubbles and evaporate solvent.
The film was then placed face down on the microscope stage (DMI 5000, Leica Microsystems, Germany), centered over the objective lens.The film was irradiated from the top with a UV flashlight (395 nm, EscoLite, La Palma, CA, USA), held using a ring stand and clamp, ≈10 cm from the sample in a perpendicular orientation.A 5X objective lens directs light to a spectrometer (DNS 300, DeltaNu, Laramie, WY, USA) to collect spectral data.An exposure time of 0.01 s was used, and the spectra were collected immediately after UV flashlight was switched off (to collect fluorescence only from Nile red excited by SrAl 2 O 4 :Eu, Dy, and eliminate any direct excitation of the Nile red dye by UV).A background spectrum was collected in the same way, and subtracted from the fluorescence spectra, manually, in Microsoft Excel.Each spectrum was plotted as an average of three spectra acquired back-to-back.To obtain spectra of the film at pH 5, 7, and 9, the film was submerged in the corresponding phosphate-buffered saline (Standard Buffer, BDH LLC, Minneapolis, MN, USA) solution for 30 min prior to acquiring spectra.Measurement of pH was done with a pH electrode (Mettler Toledo InLab Routine Pro, Columbus, OH) and pH meter (6230 N JENCO, San Diego, CA), calibrated at pH 4.0, 7.0, and 10.0.
Preparation of Single-Layer pH-sensitive ULCI Film: All recipe calculations were based on the total volume of solvent (ethanol) chosen and did not account for volume changes due to adding individual components.To fabricate the pH-sensitive all-in-one target, HydroMed D3 (120 mg mL −1 ) was first added to ethanol.The mixture was placed on a 360°sample rotator for 24 h to completely dissolve polymer.The following components were added once the HydroMed dissolved: SrAl 2 O 4 :Eu, Dy (300 mg mL −1 ), Nile Red (0.04 mg mL −1 ), and bromothymol blue (0.6 mg mL −1 ).An example recipe was 3 g SrAl 2 O 4 :Eu, Dy, 1.2 g HydroMed D3, 10 mL ethanol, 400 μL Nile Red (1 mg mL −1 in acetone), and 1 mL bromothymol blue in ethanol (6 mg mL −1 ).The mixture was then placed on the 360°sample rotator for 3 h to ensure homogeneous combination of all components.The mixture was then carefully poured onto a glass microscope slide and placed into a vacuum chamber for 12 h to evacuate air bubbles and evaporate solvent.To remove the film from the glass slide, the film and slide were placed into a petri dish with pH 7 phosphate buffered saline for 3 h.The film automatically detached from the glass slide as it absorbed water.
To fabricate the control film, the same recipe was used, without the addition of Nile red and bromothymol blue dyes.An example recipe is 3 g SrAl 2 O 4 : Eu, Dy, 1.2 g HydroMed D3, and 10 mL ethanol.The mixture was then placed on the 360°sample rotator for 3 h, to ensure homogeneous combination of all components.Next, the mixture was carefully poured onto a glass microscope slide and placed into a vacuum chamber for 12 h to evacuate air bubbles and evaporate solvent.To remove the film from the glass slide, the film, and slide were placed into a petri dish with pH 7 phosphate buffered saline for 3 h.The film automatically detached from the glass slide as it absorbed water.
The target holder was fabricated using a self-sealing laminating pouch (LS851-10G.Scotch Self-Sealing Laminating Pouches, Business Card Size, 2 × 3.5-inches, 3 M, St. Paul, MN, USA).The target windows were created before placing the films into the laminating pouch.First, black duct tape was applied to the front side of the laminating pouch in a single layer (Duck 1265013 Black Color Duct Tape, Duck Brand, Avon, OH, USA).This serves to block luminescence from any film in the laminating pouch that was not in the defined target window.To generate the target windows, a single hole punch was used to create two holes (8 mm in diameter, each).The holes were punched through the front and back layers of the laminating pouch.The pH-sensitive film, and control film, were roughly cut into ≈10 mm diameter circles and placed inside the laminating pouch, centered over the target window (on the back side of the laminating pouch).The protective sheet on the inside of the top layer was removed and carefully applied to the bottom layer, over the two films still centered over the target window.This target holder design serves to hold the UL films in place at the edges (without the use of cyanoacrylate glue), hold the targets stationary while they were submerged in pH buffer for ULCI scanning, and expose films to changing pH in the surrounding fluid (that permits color change in pHsensitive film).
Target Imaging with ULCI Scanner: For imaging the SrAl 2 O 4 :Eu, Dy-based mechanoluminescent target using a modified XELCI scanner, [12,55,19] the SrAl 2 O 4 :Eu, Dy film was secured to the bottom of a glass dish containing water and placed on an X-Y moveable stage (cf. Figure 3b).The immersed ultrasound probe and liquid light guide were held stationary while the sample was raster scanned in X-Y directions relative to these components.The ultrasound probe was controlled by a function generator, which pulses the ultrasound at a chosen frequency (2 Hz).The liquid light guide collects light emitted from the SrAl 2 O 4 :Eu, Dy film and delivers it to a 50/50 beam splitter, which splits the signal between two photomultiplier tubes (PMTs).Apart from local heating effects, the strength or the duration of the ultrasound signal does not affect the pH sensor, it just modulates the light at 2 Hz.This work shows that the modulated signal produced a higher contrast compared to the non-blinking signal.No bleaching effect was observed either.
pH Sensor Response Characterization: ULCI film response to pH was characterized using the same Leica microscope and DeltaNu spectrometer previously described.Figure S8 (Supporting Information) shows a schematic of the setup, with the separate UL film, and pH-sensitive PEG hydrogel target as an example.The target was placed face down on the microscope stage, so the UV flashlight irradiates the UL film from underneath, and the light collected by the objective was only the light that has passed through the pH-sensitive target.A black piece of cardboard, with a hole the same size and shape as the target, was used to block UV and fluorescent light from the UL film that had not passed through the pHsensitive target.
For the UL film coated with cerise Krylon spray paint, and the pHsensitive PEG hydrogel, spectra were acquired at the pH values 4.0, 6.0, 6.5, 7.0, 7.5, 8.0, and 9.0.The film was submerged in the corresponding buffer (Standard Buffer, BDH) for 30 min prior to spectra acquisition to ensure a complete color change of pH-sensitive target.An exposure time of 0.005 s was used.Spectra were normalized to 700 nm from pH 4. For the HydroMed™ D3 2-part film, containing Nile red and bromothymol blue layers, spectra were acquired at the pH values 5.0, 6.0, 7.0, 7.5, 8.0, 8.5, and 9.0.The film was submerged in the corresponding buffer (Standard Buffer, BDH) for 30 min prior to spectra acquisition to ensure a complete color change of pH-sensitive target.The exposure time was set to 0.00003 s.Spectra were normalized to 750 nm from pH 7.5.
For the 1-layer film, spectra were acquired at the pH values 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0.The film was submerged in the corresponding buffer (Standard Buffer, BDH) for 30 min prior to spectra acquisition to ensure a complete color change of pH-sensitive target.An exposure time of 0.05 s was used.Measurement of pH for each experiment was done using a pH electrode and meter.After three iterations of Gaussian smoothing were performed (averaged over a window size of 3), the spectra were normalized to the intensity of pH 6.5 at 475 nm.With changing pH conditions, the pH sensor starts to respond within 2 min, and undergoes a complete change in color in ≈10 min from orange to blue under acidic to alkaline conditions.ULCI Scanning and pH Mapping: ULCI film was first attached to the bottom of an 8″ × 8″ glass dish (Easy Grab Baking Dish, Pyrex®, Corning, NY, USA) using Scotch tape, then filled with 1.5 L PBS (P4417-100TAB, Sigma-Aldrich, St. Louis, MO, USA).The pH of this solution was adjusted using 0.1 M hydrochloric acid (HCl) and 0.1 M sodium hydroxide (NaOH).Measurement of pH for each experiment was done using the pH electrode and meter.To mimic scattering effects of biological tissue, powdered coffee creamer was added to the solution (1 g L −1 ) (Coffee-Mate Original Powder Coffee Creamer, Nestle, Arlington, VA, USA).
The scanning system itself was a modified version of the setup used for XELCI.The glass dish was positioned on an x-y motorized stage (Models: LTS300 and LTS150, Thorlabs Inc., Newton, NJ, USA) such that the sample was aligned with ultrasound focus and light collection optics.Below the transparent platform of the stage, UV LED strip lights (385-400 nm) were taped to charge the UL film prior to scanning (Ontesik 20 ft LED Black Light Strip Kit, Amazon, Seattle, WA, USA) (Figure S9, Supporting Information).The collimated ultrasound beam was generated by a mist-maker ultrasound transducer with ≈3 mm diameter spot size.Initial testing was done using the transducer (disc, AW 1605) removed from a basic humidifier (unknown make and model), and later iterations of testing used a mist/fog maker (Aluminum Mist Maker, AGPTEK, Brooklyn, NY, USA) purchased individually.Light was collected using a liquid light-guide (Model 77638, Newport Corporation, Irvine, CA, USA) attached to a 50/50 beam splitter containing photomultiplier tubes (PMTs) (Model P25PC-16, Sen-sTech, Surrey, UK) that was an artifact of the XELCI setup.A 625 nm bandpass filter (Figure S6, Supporting Information), was connected to one of the beamsplitters, and the readout from 625 nm filter was used to generate the pH images in this study (Figure 5).No filters were attached to the PMT for generating the images in Figure 3-5.PMT pulses were counted with a data acquisition board (DAQ) (NI cDAQ™−9171, National Instruments, Austin, TX, USA).The stage was controlled with a custom program written in LabVIEW (National Instruments, Austin, TX, USA).This program also records PMT output and position of stage versus time, displaying the image as it is being acquired.A more nuanced description of the scanning system can be found in previously published work. [55]or image acquisition and scanning, the film was first charged with the UV strip lights for 1 min, and imaging was initiated 30 s after switching the UV lights off.The mist-maker/ultrasound and the liquid light guide were held stationary while the sample inside the glass dish was rasterscanned using the stage beneath (Figure 3).A scan speed of 1 mm s −1 was used.All imaging components were housed inside a light-tight box, with controls outside the box.Images were exported and analyzed using custom MATLAB scripts.
Ultrasound Modulation: Ultrasound output was pulsed by switching the direct current to the transducer power supply on and off using a fieldeffect transistor (FET) (IRFP260N, Infineon Technologies, Neubiberg, Germany).The FET was coupled in series on the ground leg of the 24 V power supply, which allows the FET to have a ground reference, thereby simplifying connection to the external function generator (DS345, Stanford Research Systems, Sunnyvale, CA, USA).The function generator was programmed to output a 5 V square wave with a frequency of 2 Hz. [22]re-Processing of Data: Data included videos, spectra, and ULCI images.The videos and photos used the standard camera settings; the spectra were normalized as follows: In Figure 4a, spectra were normalized to 700 nm from pH 4. In Figure 5a, after three iterations of Gaussian smoothing were performed (averaged over a window size of 3), and the spectra were normalized to the intensity of pH 6.5 at 475 nm, and the raw data is shown in Figure S10 (Supporting Information).Raw data from images was luminescence and motor position in time, which were converted into luminescence versus position via a MATLAB interpolation script.
Data Presentation: The calibration curve shows mean and error bars for stdev.The uncertainty in Figure 5b was determined from the noise level within the single spectra.
Sample Size (n) for Each Statistical Analysis: Several pulsing trials were performed and 4 pulsing trials are shown in Figure S1 (Supporting Information).The leaching study was performed twice, and the second one was shown in Figure S5 (Supporting Information), and the blank reading was taken at least 3 times, for the pH buffers 7, 8, and 9.
Statistical Methods: For the leaching study, the LOD depended on pH.It was estimated from 3s y /m, where s y was the standard deviation of the area under the absorbance curve (from 520 to 700 nm) for blank samples, and m was the slope of the calibration curve, for each tested pH.The LODs were as follows: 0.14, 0.09, and 0.25 μg mL −1 for pH 7, 8, and 9, respectively.
Software Used for Statistical Analysis: Microsoft Excel was used to perform each statistical analysis.

Figure 1 .
Figure1.Illustration of ULCI pH sensor design and application.Sensor is attached to or coated on an implant (e.g., fracture fixation plate on a fractured bone).Focused ultrasound excites mechanoluminescence from the film; to form an image, light is collected by a liquid light guide as the ultrasound scans over the sample point-by-point.Sensor Design: Green luminescence of SrAl 2 O 4 :Eu,Dy is shifted using a red dye to overlap absorption of pH-sensitive dye.As a result, phosphorescence is modulated by pH-sensitive dye.Growth of bacterial biofilm generates an acidic environment, which causes the pH-sensitive film to turn yellow.Yellow film transmits more luminescence, resulting in higher transmittance of luminescence emission.Blue/green color film, associated with physiological and basic pH, transmits less luminescence emission.pH is monitored as a function of luminescence emission intensity.

Figure 2 .
Figure 2. Ultrasound modulated luminescence in a PDMS film containing SrAl 2 O 4 :Eu, Dy. a) Frame from a video similar to Video S1 (Supporting Information) showing a bright green luminescence spot where the ultrasound is focused.b) Luminescence intensity of SrAl 2 O 4 :Eu, Dy film due to application of a square wave ultrasound modulation frequency of 0.1 Hz (i.e., the ultrasound was turned ON 5 s and OFF 5 s, and 10 s/cycle).c) Luminescence intensity versus time for one US pulse, depicting the phosphorescence increases rapidly with ultrasound excitation and decays exponentially once ultrasound is turned OFF.[18] d) Spectra from SrAl 2 O 4 :Eu, Dy film as ultrasound is pulsed ON and OFF.After initial excitation with 395 nm light, ultrasound excitation produces a ≈23x increase in luminescence intensity compared to when the ultrasound is switched OFF.[22]

Figure 3 .
Figure 3. ULCI imaging of a target.a) Photograph of the target.b) Schematic of ULCI setup for target scanning.The ultrasound probe and liquid light guide are held stationary over the sample, which is attached to a container on the motorized stage.The liquid light guide is fed to the optical filters and the PMTs attached to the 50/50 beam splitter.Information from PMTs is fed to a data acquisition board, then sent to a computer for image display using LabVIEW and MATLAB.c) ULCI image obtained with light collected from one of the PMTs.d) Plot of intensity versus linear position taken from b) in the regions indicated by the red, blue, and yellow dotted lines (L3, L9, and L15).Ultrasound modulation temporarily increases luminescence while ultrasound is ON.[22] e) FFT of line profiles L3, L9, and L15 showing the dominant frequency component at 2 Hz.f) Image c filtered at 0 Hz; i) image c filtered at 2 Hz.The 2 Hz-filtered image has higher contrast than the 0 Hz image.g,h) Selected horizontal and vertical line scans, respectively of the 0 Hz filtered image.j,k) Selected horizontal and vertical line scans, respectively of the 2 Hz image.The colormaps indicate luminescence intensity (counts).

Figure 4 .
Figure 4. pH sensing with Krylon Spray Paint Modified UL Film a) Plot depicts changes in luminescence intensity of Krylon ™ coated SrAl 2 O 4 :Eu, Dy film modulated by bromothymol blue pH-sensitive dye impregnated PEG hydrogel target at pH 4.0-9.0.b) Schematic of film fabrication: pH sensor comprising the SrAl 2 O 4 :Eu, Dy film coated with Krylon spray paint and the bromothymol blue PEG hydrogel attached on top using cyanoacrylate glue, while the control gel consists of only the hydrogel glued to the film.c) Image of pH sensor.Leftmost hydrogel contains bromothymol blue, while the rightmost gel does not contain bromothymol blue, and hence should not exhibit any pH response (control gel).Hydrogel targets only cover a portion of the Krylon™ coated SrAl 2 O 4 :Eu, Dy film.d) ULCI image at pH 4. e) ULCI image at pH 9.Vertical lines evident in these images are due to ultrasound modulation at 2 Hz (1 mm s −1 scan rate).[22]

Figure 5 .
Figure 5. Ultrasound luminescence pH sensors.a) Luminescence spectra at pH 6.0-9.0 for a single-layer pH-sensitive film containing HydroMed D3, Nile red, SrAl 2 O 4 :Eu,Dy, and bromothymol blue (normalized to luminescence intensity at 475 nm).UV OFF, spectra acquired using afterglow.b) The ratio of luminescence peaks at 638 to 520 nm (from panel a), modeled using the Henderson-Hasselbalch equation versus pH; line shows regression to Equation 2. c) Laminating pouch sample holder with windows punched through the front and back layers.Films held in place around the outer circumference.Leftmost film contains only HydroMed and SrAl 2 O 4 :Eu, Dy (C: Control).Rightmost film contains HydroMed, SrAl 2 O 4 :Eu, Dy, Nile red, and bromothymol blue (S: Sensor).I. Photo of film after equilibration at pH 6. II.Photo of film after equilibration at pH 9. d) I. and II.ULCI images at pH 6 and 9, respectively.The control samples did not show any pH response, as expected.