Cell imaging using GaInAsP semiconductor photoluminescence

We demonstrate label-free imaging of living cells using a GaInAsP semiconductor imaging plate. The photoluminescence (PL) intensity is changed by immersing the semiconductor wafer in different pH solutions and by depositing charged polyelectrolytes on the wafer. Various observations indicate that this phenomenon arises from the radiative and surface recombination rates modified by the Schottky barrier at the charged semiconductor surface. HeLa cancer cells were cultured on the semiconductor, and PL was observed using a near-infrared camera. The semiconductor areas with the cells attached exhibited characteristic PL profiles, which might reflect the attachment and surface condition of the cells, cellular matrix, and other substances. ©2016 Optical Society of America OCIS codes: (170.3880) Medical and biological imaging; (280.1415) Biological sensing and sensors. References and links 1. J. H. Warner, A. Hoshino, K. Yamamoto, and R. D. Tilley, “Water-soluble photoluminescent silicon quantum dots,” Angew. Chem. Int. Ed. Engl. 44(29), 4550–4554 (2005). 2. M. Bruchez, Jr., M. Moronne, P. Gin, S. Weiss, and A. P. Alivisatos, “Semiconductor nanocrystals as fluorescent biological labels,” Science 281(5385), 2013–2016 (1998). 3. P. D. Simonson, E. Rothenberg, and P. R. Selvin, “Single-molecule-based super-resolution images in the presence of multiple fluorophores,” Nano Lett. 11(11), 5090–5096 (2011). 4. L. K. Gifford, I. E. Sendroiu, R. M. Corn, and A. Lutak, “Attomole detection of mesophilic DNA polymerase products by nanoparticle-enhances SPR imaging on glassified gold surfaces,” J. Am. Chem. 132(27), 9265–9267 (2011). 5. S. Kita, S. Hachuda, S. Otsuka, T. Endo, Y. Imai, Y. Nishijima, H. Misawa, and T. Baba, “Super-sensitivity in label-free protein sensing using a nanoslot nanolaser,” Opt. Express 19(18), 17683–17690 (2011). 6. S. Hachuda, S. Otsuka, S. Kita, T. Isono, M. Narimatsu, K. Watanabe, Y. Goshima, and T. Baba, “Selective detection of sub-atto-molar Streptavidin in 10-fold impure sample using photonic crystal nanolaser sensors,” Opt. Express 21(10), 12815–12821 (2013). 7. H. Abe, M. Narimatsu, T. Watanabe, T. Furumoto, Y. Yokouchi, Y. Nishijima, S. Kita, A. Tomitaka, S. Ota, Y. Takemura, and T. Baba, “Living-cell imaging using a photonic crystal nanolaser array,” Opt. Express 23(13), 17056–17066 (2015). 8. K. Watanabe, Y. Kishi, S. Hachuda, T. Watanabe, M. Sakemoto, Y. Nishijima, and T. Baba, “Simultaneous detection of refractive index and surface charges in nanolaser biosensors,” Appl. Phys. Lett. 106(2), 021106 (2015). 9. L. Tang, I. S. Chun, Z. Wang, J. Li, X. Li, and Y. Lu, “DNA detection using plasmonic enhanced near-infrared photoluminescence of gallium arsenide,” Anal. Chem. 85(20), 9522–9527 (2013). 10. H. A. Budz, M. M. Ali, Y. Li, and R. R. LaPierre, “Photoluminescence model for a hybrid aptamer-GaAs optical biosensor,” J. Appl. Phys. 107(10), 104702 (2010). 11. Y. S. Liu, Y. Sun, P. T. Vernier, C. H. Liang, S. Y. C. Chong, and M. A. Gundersen, “pH-sensitive photoluminescence of CdSe/ZnSe/ZnS quantum dots in human ovarian cancer cells,” J Phys Chem C Nanomater Interfaces 111(7), 2872–2878 (2007). 12. P. W. Barone, S. Baik, D. A. Heller, and M. S. Strano, “Near-infrared optical sensors based on single-walled carbon nanotubes,” Nat. Mater. 4(1), 86–92 (2004). 13. E. Nazemi, S. Aithal, W. M. Hassen, E. H. Frost, and J. J. Dubowski, “GaAs/AlGaAs heterostructure based photonic biosensor for rapid detection of Escherichia coli in phosphate bufferd saline solution,” Sens. Actuators B Chem. 207, 556–562 (2015). #262863 Received 12 Apr 2016; revised 11 May 2016; accepted 11 May 2016; published 13 May 2016 © 2016 OSA 16 May 2016 | Vol. 24, No. 10 | DOI:10.1364/OE.24.011232 | OPTICS EXPRESS 11232 14. T. Baba, K. Inoshita, H. Tanaka, J. Yonekura, M. Ariga, A. Matsutani, T. Miyamoto, F. Koyama, and K. Iga, “Strong enhancement of light extraction efficiency in GaInAsP 2-D photonic crystals of columns,” J. Lightwave Technol. 17(11), 2113–2120 (1999). 15. J. Nozik and R. Memming, “Physical chemistry of semiconductor liquid interfaces,” J. Phys. Chem. 100(31), 13061–13078 (1996). 16. J. Peng, X. He, K. Wang, W. Tan, Y. Wang, and Y. Liu, “Noninvasive monitoring of intracellular pH change induced by drug stimulation using silica nanoparticle sensors,” Anal. Bioanal. Chem. 388(3), 645–654 (2007).


Introduction
Fluorescence labels are widely used for biomolecular and cellular imaging [1], including semiconductor quantum dots [2] and super-high-resolution microscopy [3].However, fluorescence imaging is costly and time consuming due to label modifications, invasiveness and denaturing of the sample, and noise from sample fluorescence under excitation.In particular, invasiveness has recently become a major problem in studying unspecialized cells including induced pluripotent stem cells, and thus less invasive and label-free imaging is becoming increasingly important.So far, surface plasmon resonance has been employed to enable label-free bioimaging in which only the environmental refractive index is visualized, and the resolution and image contrast are tradeoffs [4].
We developed GaInAsP semiconductor photonic crystal nanolasers for label-free protein sensing, which detects a change in the environmental index from a shift in lasing wavelength [5,6].We also demonstrated living cell imaging using a nanolaser array, where the spatial resolution is limited to several microns by the pitch between nanolasers [7].Recently, we found further evidence that the nanolaser detects the surrounding pH and surface charges as modified on the laser emission intensity [8].Simultaneous detection of the refractive index and surface charge was demonstrated for the adsorption of charged polyelectrolytes and hybridization of deoxyribonucleic acids.Here, the mechanism for detecting surface charges is mainly due to surface recombination at the sidewalls of the air holes in the GaInAsP quantum-well photonic crystal, which is modified by the Schottky barrier at the charged semiconductor surface.This suggests that this technique can be applied in a simpler bulk wafer with no photonic crystal patterns for photoluminescence (PL) bioimaging.This semiconductor wafer is referred to as a semiconductor imaging plate.
PL from some semiconductors and nanomaterials used for labeling and/or protein sensing exhibits a dependence on pH [9][10][11][12][13] but has never been employed as such an imaging plate.This is likely due to a decrease in sensitivity in semiconductors such as GaAs because the surface recombination rate is so high that the Fermi level is pinned by many surface states.In contrast, the moderate surface recombination rate of GaInAsP maintains the emission intensity and sensitivity.An obvious disadvantage of GaInAsP is that emission wavelengths longer than 1.2 μm from the alloy are undetectable by Si-based photodiodes or highresolution imagers.However, in recent years, near-infrared cameras using GaInAs semiconductors have been greatly improved: 640 × 512 pixels and 16-bit gradation are now available with a dark current suppressed by cooling.Besides, longer wavelengths are especially advantageous for suppressing pump absorption, light emitted by biomaterials, and PL noise from both.In this paper, we discuss the sensitivity of the semiconductor imaging plate based on various PL characteristics of the unpatterned GaInAsP bulk epiwafer and demonstrate its ability to image cancerous cells on the plate.It is expected to yield a very simple technique to observe some cell activities which cannot be observed usually by optical microscope without fluorescence labels.

PL characteristic and mechanism
The wafer used in this experiment was a 300-nm undoped GaInAsP layer epitaxially grown on an InP substrate by metal-organic chemical vapor deposition (Fig. 1(a)).The layer exhibits weak n-type conductivity and a PL peak wavelength at 1.36 μm at room temprature.To measure various PL characteristics shown below, we first coated the wafer with a 3-nm ZrO 2 film using atomic layer deposition for chemical protection (we observed the surface etching for strong acid and alkali without this coating).Then we hydrophilized it with oxygen plasma, set in a polydimethylsiloxane micro-fluidic channel, and successively injected different solutions, measuring the PL; since we measured a 13% nonuniformity in the PL intensity over the 140 × 140 μm 2 wafer area, we eliminated its influence from the following results by measuring the PL at the same wafer position in this procedure.We prepared different pH solutions, which included HCl or KOH as well as 10 mM KCl, and measured the PL intensity I and lifetime τ in a way similar to those described in Ref. [8].As shown in Figs.1(b)-1(e), I decreased to less than 30% and τ decreased from 0.84 to 0.60 ns as the pH increased from 2 to 11.In general, I is proportional to the quantum yield of light emission η, which is expressed as , where τ r and τ nr are radiative and nonradiative lifetimes, respectively.The latter is also given as , where τ d and τ s are defect and surface recombination lifetimes, respectively.The former is a constant value for the applied wafer, whereas the latter is given by (v s /t) −1 for the surface recombination velocity v s , which has been experimentally evaluated as 1.3 × 10 4 cm/s in similarly grown GaInAsP [14] with thickness t = 300 nm.We estimated τ r and τ s from I and τ at different pH, assuming τ d = 2 ns to obtain the reasonable behaviors of the PL intensity and lifetime with the change of τ r and τ s .As shown in Fig. 1(e), τ r increased (the radiative recombination rate decreased) gradually while τ s decreased (the surface recombination rate increased) rapidly as the pH increased from 2 to 11.Based on these lifetimes, the Schottky barrier effects can be discussed in more detail.The Schottky barrier is formed near the semiconductor surface by acid dissociation equilibria, as illustrated in Fig. 2. At low acidic pH, the n-type semiconductor is positively charged and the band in the semiconductor becomes flat near the surface.The holes (minority carriers) are then equally distributed, which does not lead to any radiative or surface recombination effects (Fig. 2(a)).At high alkaline pH, the n-type semiconductor is negatively charged and an upward Schottky barrier is formed [15].In this case, the holes are concentrated near the surface, and the surface recombination is accelerated.The spatial segregation of electrons and holes at the barrier also decreases the radiative recombination.Thus these two effects result in low-intensity PL (Fig. 2(b)).

Various observations
To verify this mechanism, we measured the PL intensity of n-doped (N D = 3 × 10 18 cm −3 ) and p-doped (N A = 4 × 10 18 cm −3 ) wafers, as shown in Fig. 3(a).The n-doped wafer exhibited a sensitivity curve similar to that shown in Fig. 1(d), whereas the p-doped wafer exhibited an opposite curve.These behaviors can be explained by the downward Schottky barrier in the ptype semiconductor (Figs.2(c) and 2(d)).The PL characteristics of the former wafer were also measured with different thicknesses of ZrO 2 , as shown in Fig. 3(b).Thinner ZrO 2 exhibited greater dependence on pH and led to more intense PL compared with thicker ZrO 2 .Electrons in thin ZrO 2 can tunnel and interact with ions in the solution.Thicker ZrO 2 suppresses tunneling and the band is fixed by pinning.Figure 3(c) shows the dependency of PL intensity on pH at different KCl concentrations (100, 10, and 1 mM).PL was only sensitive to pH when the pH was less than 2 at 1 mM, whereas the sensitivity is extended over the full range of pH at 100 mM.One candidate explanation for this is the Debye length λ D , which is inversely proportional to the square root of the ion concentration; at a high salt concentration, λ D decreases and ions accumulate near the surface, which might increase the surface concentration of protons, resulting in the sensitivity being maintained even close to the neutral pH.We also conducted a test experiment to confirm that the PL intensity responds to the surface charge, as shown in Fig. 3(d).Here, we first formed the self-assembled monolayer of 3-aminopropyl triethoxysilane (APTES) on the ZrO 2 -coated wafer.Then we set the wafer in the micro-fluidic channel and alternately deposited negatively charged polyelectrolyte (polystyrene sulfonate: PSS, 100 kDa, 0.1 wt%) and positively charged one (polyallylamine hydrochloride: PAH, 90 kDa, 0.1 wt%) by injecting solutions including them in the channel, measuring the PL intensity.We observed reproducibly that the PL intensity decreased for PSS and increased for PAH.This result is consistent with the above mechanism and indicates that the PL intensity reflects the charge of attached medium.

Cell imaging
In the following, we present images of HeLa cancer cells cultured on the wafer.In general, pH inside and even on the periphery of cancer cells can be lower than 7.4 in normal cells and culture fluid, although it depends on the cultural environment [16].The surface of cancer cells is also charged more positively due to abnormal carbohydrate chain of the cell surface.Therefore, the PL intensity at the semiconductor surface with HeLa attached is expected to increase.In this experiment, we employed pulsed photopumping using InGaAs/AlGaAs semiconductor laser (pulse width = 1 μs, repetition frequency = 10 kHz, wavelength = 976 nm) to suppress speckle noise and excess heating of the semiconductor.This wavelength is not absorbed by the cells, but the power density of the pulse peak was set as low as 60 W/cm 2 .Reflected pump light was filtered out by a combination of notch filter and long pass filter in front of the near-infrared camera.The camera used for the observation was a Princeton Instruments NIRvana 640 (512 × 640 pixels, 16-bit dynamic range, cooled at −60°C).Fluctuation between neighboring pixels ΔPL min in a uniform PL was measured as 455 counts on average for a maximum number of counts of 65536.The pH sensitivity of PL, S ≡ (ΔPL/PL)/ΔpH, as shown in Fig. 4, was evaluated from the fitting curve in Fig. 3(c) with a salt concentration of 100 mM.The sensitivity at around neutral pH is relatively low: S = 0.12 pH −1 at pH = 7.4.The pH resolution R can be obtained by R ≡ (ΔPL min /PL)/S, as also shown in Fig. 4. We evaluated R = 0.057 at pH = 7.4.Thus, if a difference in pH between the periphery of HeLa cells and culture fluid is larger than this value, it is detectable.
The wafer was first hydrophilized by the oxygen plasma, sterilized by ethanol, and rinsed with a phosphate buffer solution.The wafer was then soaked in culture fluid (Dulbecco's Modified Eagle's Medium, Sigma-Aldrich, pH = 7.4), which includes a HeLa cell line, and cultured with 5% CO 2 in a 37°C incubator for 24 h.Since HeLa cells were not cultured on the ZrO 2 -coated wafer and the culture fluid is close to neutral, which limits etching of the wafer, we refrained from depositing ZrO 2 onto the wafer.Considering the dependency on film thickness as shown in Fig. 3(b), we expect a higher sensitivity and finer pH resolution without ZrO 2 .Figure 5 shows example images acquired after the culture.In the optical micrograph shown in Fig. 5(a), approximately 10 cells are attached, each of which is 10−30 μm, although the image quality might be affected by the microscope aberration at near-infrared wavelengths.We also confirmed that a scattered light image observed under illumination of pump light without filtering resembles the optical micrograph closely.The near-field pattern of PL (Fig. 5(b)) after controlling the image gradation exhibits a characteristic pattern.It appears similar to the optical micrograph but its details are very different.To make more easily understandable, these two pictures are superimposed with the green colored PL image, as shown in Fig. 5(c), which looks like an image with fluorescence labels.The colored areas (e.g., as indicated by symbol A) overlap with many cells.Moreover, some exceptional cases are shown: the area in the absence of cells exhibits the color represented by symbol B, and the areas below some cells do not exhibit any color as represented by symbol C.They can reflect any factors that change the pH and charge at the semiconductor surface, e.g. the surface and attachment condition of the cells, cellular matrix, proteins and ions, which are of great interest for investigation.In other words, one may be able to monitor the reaction without labels if one injects some regent which stimulates a specific factor.

Conclusion
In conclusion, using a simple unprocessed bulk wafer, this imaging technique shows potential for visualizing cell behaviors undetectable by conventional optical microscopy without fluorescence labels.The simplicity and spatial resolution, which are comparable with those of the optical microscope, are superior to those of other label-free imaging methods such as surface plasmon resonance.By applying this technique to various cells and systematically analyzing the images, it is expected to reveal unknown cell behavior and reactions with reagents.

Fig. 1 .
Fig. 1.(a) Schematic of the GaInAsP bulk wafer coated with ZrO 2 .(b) PL intensity.(c) Temporal decay of PL.(d) pH dependency of PL intensity and lifetime.(e) Measured lifetime τ, estimated radiative lifetime τ r , and surface recombination lifetime τ s .Arrows in (d) and (e) indicate pH for measuring (b) and (c).

Fig. 3 .
Fig. 3. Various PL intensity characteristics.(a) pH dependency for different doping types of semiconductor.(b) pH dependency for different thicknesses of ZrO 2 .(c) pH dependency for different salt concentrations.Dashed line shows the fitting curve of a fourth-order function.(d) Variations when charged polyelectrolytes are alternately deposited.

Fig. 4 .
Fig. 4. Sensitivity and pH sensing resolution estimated from the dashed line in Fig. 3(c).