Graphene quantum dot composite with multiphoton excitation as near infrared-II probe in bioimaging

2.1 Preparation of GQD-based materials

GQDs: Graphene oxide was prepared using a natural graphite powder (Bay Carbon Inc., Bay City, MI, USA) through a modified Hummers’ method (Hummers et al., 1958). Graphite (8.5 M) and NaNO₃ (0.6 M) (Merck & Co., Kenilworth, NJ, USA) were mixed with H₂SO₄ (FUJIFILM Wako Chemicals USA Inc., Richmond, VA, USA). KMnO₄ (2.0 M, FUJIFILM Wako Chemicals USA Inc.) was slowly added with continual stirring at 35 °C (Corning, New York, NY, USA) overnight. Subsequently, deionized water (ddH₂O) was gradually added and continually stirred before H₂O₂ (35%; Sigma Aldrich Co., St Louis, MO, USA) was added to terminate the reaction. The graphene oxide was washed several times with ddH₂O before it was collected and placed in a tube furnace (Tainan, Taiwan) and heated to 400–600 °C in the presence of argon for 4–6 h. Concentrated HNO₃ (16.0 M, Sigma Aldrich Co.) was added and stirred for 2 d. The mixture was placed in a water bath sonicator (Rocker Scientific Co. Ltd., New Taipei City, Taiwan) for 2 d at 45°, then placed in an oven at 160 °C (Yotec Instruments Co. Ltd., Hsinchu City, Taiwan) for 1 d to vaporize all the liquid. Washing and centrifugation (approximately 847,200 RCF, > 10 min; Optima TLX Ultracentrifuge, Beckman Coulter Inc., Brea, CA, USA) with ddH₂O were conducted several times before the supernatant was filtered through a 0.22 μm microporous membrane (Nylon filter membrane, Merck & Co.). The pH (7.4; Hanna Co. Ltd., Thane, Maharashtra, India) of the resulting black suspension was adjusted with NaOH (Sigma Aldrich Co.) to obtain the GQDs specimens.

GQDs doped with nitrogen (N-GQDs): Graphene oxide was prepared from a natural graphite powder using a modified Hummers’ method. Graphite (8.5 M) and NaNO₃ (0.6 M) were mixed with H₂SO₄ and KMnO₄ was slowly added with continual stirring at 35 °C overnight. Subsequently, ddH₂O was gradually added and continually stirred before the reaction was terminated with H₂O_2. Several washing and centrifugation steps with ddH₂O were performed and the graphene oxide was collected. The as-prepared graphene oxide was placed in a tube furnace and heated to 400–600 °C in the presence of ammonia (Sigma Aldrich Co.) for 4–6 h before the addition of concentrated HNO₃ (16.0 M) and stirred for 2 d. The mixture was placed in a water bath sonicator for 2 d at 45 °C, then placed in an oven at 160 °C for 1 d to vaporize all the liquid. Washing and centrifugation (approximately 847,200 RCF, > 10 min) with ddH₂O were performed several times before the supernatant was filtered through a 0.22 μm microporous membrane. The pH (7.4) of the resulting black suspension was adjusted with NaOH to obtain the N-GQDs.

GQDs doped with N and functionalized with amino groups (amino-N-GQDs): The as-prepared N-GQDs were mixed with ammonia, stored in a Teflon-lined stainless steel autoclave, and reacted at 180 °C for 5 h. The resulting mixture was washed with ddH₂O, centrifuged several times, and subsequently dried in an oven at 50 °C overnight to obtain the amino-N-GQDs.

The 0.1 mg mL⁻¹ or 1.0 mg mL⁻¹ stock solutions were prepared for the following experiments.

2.2 Femtosecond laser optical system (for fluorescence lifetime imaging microscopy, FLIM)

The self-made femtosecond Ti-sapphire laser (a repetition rate of 80 MHz; Mai Tai with the optical parametric oscillators, Spectra-Physics, USA; Scheme S1) was used and FLIM were used according to the previous studies (Kuo et al., 2020, Kuo et al., 2020). The lifetime data and parameter are generated using the triple-exponential equation fitting [3 exp fitting model: a0*exp(a1x)+a2*exp(a3x)+a4*exp(a5x)+a6] while monitoring the emission under TPE (Ex: 980 nm).

All materials and methods for this article can be found in the Supplementary Material.

3.1 Characterization of materials

Graphene oxide sheets were prepared by a modified Hummers method to fabricate GQDs via an ultrasonic shearing reaction (Hummers et al., 1958). Nitrogen (N) was used to dope the GQDs, which were then functionalized with an amino group to produce amino-N-GQDs. Low-magnification transmission electron microscopy (TEM; Fig. 1a) and high-magnification high-resolution TEM (HR-TEM; Fig. 1b) were used to determine the mean size of the amino-N-GQDs in the lateral dimension (7.1 ± 0.6 nm) as well as their size distribution (Table S1). The interlayer spacing of the derived amino-N-GQDs was 0.213 nm (Fig. 1b), which could be attributed to the d-spacing {1  $1^{-}$ 0 0} present in graphene lattice fringes. The height profiles of the materials were determined through atomic force microscopy (AFM), indicating a single 1.00 ± 0.03-nm-thick layer of amino-N-GQDs (Fig. S1a). Hydroxyl, amino, and epoxy groups were exposed on the amino-N-GQDs, as revealed by Fourier-transform infrared spectroscopy (Fig. S1b). Therefore, the surface charge was approximately 13.6 mV, as evidenced by zeta-potential spectroscopy. An absorption peak was identified at approximately 225 nm corresponding to the aromatic C=C bonds’ π–π* transition. The C–N and C=O shoulder’s n–π* transitions appeared at ∼323 nm, indicating π electron transition in the oxygen-containing amino-N-GQDs. This also confirmed the N doping of the dots, as revealed by ultraviolet–visible (UV–Vis) spectroscopy (Fig. S1c). The D- and G-band (located at ∼1384 and 1606 cm⁻¹, respectively) integrated intensity ratio (I_D/I_G) in the Raman spectrum was approximately 0.90, indicating high-quality samples. The amino-N-GQDs had higher disorder and distortion (Fig. 1c; Fig. S1d) than graphite (0.104) (Wu et al., 2012). The I_D/I_G values were used to determine the GQD specimens’ mean sp²-domain size (Sheka et al., 2020), which was similar in the Raman and HR-TEM calculations but the Raman calculations (∼6.9 nm) slightly underestimated the size because the Raman estimation (Kuo et al., 2020) [Equations (S10-S11), Supplementary Material] ignores the oxygenated regions. X-ray diffraction (XRD) showed the diffraction angle of amino-N-GQDs peaked broadly at ∼24.3°, which means an interlayer distance of 0.360 nm (Fig. S1e). XPS to analyze the deconvoluted C(1 s) spectra revealed C–C/C=C (285.8 eV), C–N (286.8 eV), C–O (287.2 eV), and C=O (288.1 eV) bonds in addition to functional groups containing oxygen. The ratio of O(1 s) to C(1 s) was approximately 31%. The deconvoluted N(1 s) spectra revealed relevant details of C–N bonding. The samples treated with ammonia contained pyridinic (398.2 eV), amino (398.9 eV), pyrrolic (399.8 eV), quaternary (400.8 eV), and amide (O=C–N, 401.2 eV) N functional groups (Figs. 1d & e; Fig. S1f). The N functionality composition [5.3% of N(1 s)/C(1 s)] was obtained using the deconvoluted N(1 s) spectra, indicating that the hydrothermal ammonia treatment caused N atom substitutional doping forming quaternary, pyrrolic, and pyridinic N functionalities (Fig. 1e; Fig. S1f) and some carbonyl and epoxy group moieties were converted into amide and amino functionalities, respectively. In graphene oxide-based materials, electron orbital resonance patterns are affected by N functionalities. Some aromatic rings may be damaged by the ammonia annealing treatment, so when graphene oxide is subjected to ultrasonic exfoliation and cutting in nitric acids, such aromatic ring damage may have engendered defective peripheral carbonyl and pyrrolic groups. The product was subsequently treated with hydrothermal ammonia, which may have converted carbonyl groups to amide groups on the amino-N-GQDs. Fig. S1g presents a conceptual diagram of an amino-N-GQD. These experimental details confirm the successful preparation of amino-N-GQDs.

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Fig. 1

(a) Low-magnified TEM image [inset: Quantitative size distribution was determined by DLS. Cell type: DTS1060C; Measurement duration: 30 number of runs, 20 run duration (sec)]; (b) HR-TEM image of a single amino-N-GQD with d-spacing of 0.213 nm. (c) Amino-N-GQD Raman spectrum decomposed and fitted to D-band (gray line) and G-band (brown line) peaks (at ∼1384 and ∼1606 cm⁻¹, respectively; pink line: decomposed spectrum; black line: raw data). (d and e) Deconvoluted XPS spectra of C(1 s) with peaks for C–C/C=C, C–N, C–O, and C=O. Spectra and XPS peaks for N(1 s) of pyridinic N, amino N (NH₂), pyrrolic N, quaternary N, and amide N (O=C–N). The Gaussian function was used to fit the peaks. Other XPS findings are presented in Fig. S1f (Supplementary Material). Material dose: 1 μg mL⁻¹.

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3.2 Nitrogen dopant, amino functionalization, and polymer conjugation

For phototherapy, the intrinsic properties of GQD-based materials can be modified with N dopants to markedly change the carrier density, resulting in electrical and optical properties that are completely distinct from their intrinsic counterparts. N-doped GQD-based materials have remarkable electrochemical, photochemical, and electrocatalytic activities which are advantageous in optoelectronic and biomedical applications (Kuo et al., 2020). The efficient engineering of graphene structure band gaps and local chemical features can be achieved by doping them with heteroatoms to modify their optical properties and electronic characteristics (Zhang et al., 2023). Some promising contrast agents for bioimaging have limited practical applicability because carbon-based materials have an insufficient QY for such applications. However, the QY can be efficiently increased by heteroatom doping, surface passivation, or both. Polystyrene sulfonate (PSS) and polyethylenimine (PEI) were used to paint the amino-N-GQD surfaces in the present study. These GQDs were N-doped as well as amino-functionalized through an electrostatic interaction with the PSS and PEI to establish whether the PL and QY could be enhanced. Some details of the successful characterizations of amino-N-GQD-PSS-PEI (or amino-N-GQD-polymer composites) are illustrated in Figs. S2–S5. There were approximately 11,642 PSS and approximately 25,991 PEI coated on the surfaces of each amino-N-GQD and amino-N-GQD-PSS, respectively (Fig. S6). For the amino-N-GQDs, the relative QY for Cy5.5 in dimethyl sulfoxide [for reference, QY_ref = 0.28 (Li et al., 2012)] was 0.30, whereas the relative QYs for amino-N-free GQDs (0.17) and amino-group-free N-GQDs (0.22) were not favorable (Fig. S7). Similar absolute QYs (Ruhlandt et al., 2020) of 0.16, 0.21 and 0.30 for amino-N-free GQDs, amino-group-free N-GQDs and amino-N-GQDs were obtained. Electrostatic interaction was used to deposit a coating of PSS followed by a coating of PEI to increase the relative and absolute QY to 0.63 and 0.62, respectively.

3.3 Investigation of two-photon properties using a self-developed femtosecond Ti–sapphire laser optical system

To develop a high-QY contrast agent, this study leveraged multiphoton properties and maximized deep-tissue penetration by employing an irradiation source with wavelengths shifting from the visible range to the NIR-I and NIR-II windows that create low photodamage, scattering, energy absorption, and background tissue autofluorescence. The detection limit was enhanced using a nonlinear, coupled multiphoton laser that penetrated less than 1 mm into tissue. This laser was non-invasive in terms of subcellular features. The intensity of two-photon luminescence (TPL), which is non-linear, is proportional to the cross-section of TPE, the square of the excitation power, and the PL QY (Ouzounov et al., 2017). The materials and methods section (Supplementary Material) presents the calculations for the laser power. TPE (159.32 nJ pixel⁻¹, 1.5932 mW) was performed using a self-developed femtosecond Ti-sapphire laser optical system (80-MHz repetition rate; Mai Tai optical parametric oscillators, Spectra-Physics, USA, Scheme S1); the z-axis resolution (full width at half maximum, FWHM) was 948.35 nm (Fig. 2a), and the x−y axis focal spot was 459.85 nm. These settings helped maintain a low laser power and lengthen the excitation wavelength to the NIR-I or NIR-II region, as well as enhancing TPL visibility in two-photon imaging (TPI). In deep-tissue NIR investigations, as illustrated in Fig. 2b and Fig. S8a, the most efficient excitation wavelength in TPE was 980 nm, probably because of the involved interband transitions (Ouzounov et al., 2017). This phenomenon is also called two-photon absorption (TPA). Since the observed intrinsic- and defect-state emissions may have involved a PL mechanism, the amino-N-GQD-polymer composite could be used as a contrast probe under TPE. Figure 2c illustrates the amino-N-GQD-polymer composites indicating strong TPL under the TPE of a short-pulse laser. The amino-N-GQD-polymer composite had a TPL spectrum ranging from 500 to 900 nm that peaked at 845 nm (Ex: 980 nm); this corresponded to 846 nm in the emission spectrum under OPE [continuous-wave (CW) laser diode, Ex: 808 nm; Fig. 2d]. A two-photon process was observed with an exponent of approximately 2.00 (Fig. 2e; approximately 1.99 for amino-N-GQD, Fig. S6b), and the PL intensity quadratically depended on the TPE excitation power (Ex: 980 nm) (Kuo et al., 2020). A large cross-section is useful for the monitoring of molecular events and the amino-N-GQD-polymer composites had an estimated absolute TPE cross-section of 60,158 Goeppert–Mayer units (GM; 1 GM = 10⁻⁵⁰ cm⁴ s photon⁻¹ and was Rhodamine B according to Kuo et al. (2020) (Table 1; Fig. S9 and Table S1). The amino-N-GQD had an estimated cross-section of 51,031 GM. An extremely low TPE cross-section generally results in a weak TPL intensity. In the present study, the amino-N-GQD-polymer composites’ absolute TPE cross-section was at least 443 times those of conventional fluorophores and semiconductor quantum dots (Table 1). Since intrinsic fluorophores and enhanced localized excitation power result in a high QY as well as a large TPE cross-section, they can enhance TPL signals (Ouzounov et al., 2017). The observed lifetimes of the amino-N-GQD-PSS-PEI samples were 0.15, 0.87, and 3.87 ns. The amino-N-GQD-polymer composites had an estimated average lifetime of 1.00 ns, determined using time-correlated single-photon counting and a triple-exponential function to fit the results (Fig. 2f and Table 2), compared to the estimated average lifetime of 1.28 ns for amino-N-GQDs (Table 2; Fig. S8c). The increase in TPL occurred because of enlargements in the QY, and the TPL could be confirmed according to lifetime variations. The radiative decay rate was 6.30 × 10⁸ s⁻¹ for the amino-N-GQD-polymer composites, and the nonradiative decay rate was 3.70 × 10⁸ s⁻¹; the ratio of these two rates was approximately 1.70. The amino-N-GQDs had corresponding rates of 2.19 × 10⁸ and 5.63 × 10⁸ s⁻¹ and a ratio of ∼0.39, which was notably smaller. After TPE, as the QY increased and the lifetime decreased, the amino-N-GQD-polymer composites primarily decayed through the radiative pathway.

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Fig. 2

(a) Laser system z-axis resolution (FWHM; ∼948.35 nm; fitting function: Gaussian function). The second harmonic generation signal at different positions was measured through a z-axis scan of a thin gold film. (b) Amino-N-GQD-polymer composites relative TPA spectra. TPE executed at 118.32 nJ pixel⁻¹ as a function of wavelength (900–1000 nm) was applied for signal monitoring. (c) Amino-N-GQD-polymer composite TPL image (gray level) obtained through TPE at 118.32 nJ pixel⁻¹ over 30 scans (total effective exposure time: ∼0.20 s; scan rate: 6.80 ms per scan; scan area: 200 × 200 μm²; calculations presented in the materials and methods section, Supplementary Material). (d) Relative PL spectra of amino-N-GQD-polymer composites exposed to 10 mW of excited power under OPE (808-nm CW laser diode; Ex/Em: 808 nm/ 846 nm) and TPE (Ex/Em: 980 nm/ 845 nm; power: 159.32 nJ pixel⁻¹; cutoff: 900 nm, as executed using cascading filters). (e) TPL intensity dependence (slope: 2.00) on the amino-N-GQD-polymer composites’ excitation power (logarithm); TPE exposure from 1183.20 to 4732.80 nJ pixel⁻¹ (Ex: 980 nm; R² > 0.999). (f) Material time-resolved room-temperature PL decay profiles (Ex: 980 nm; power: 118.32 nJ pixel⁻¹). Material dose: 1 μg mL⁻¹.

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3.4 Stability, physiological, and reactive oxygen species (ROS) assays

A contrast agent for the bioimaging of pathological changes or diseased tissue should be biocompatible, photostable, and efficient uptake in acidic environments. The uptake was assessed in A431 skin cancer cells using a conjugated anti-epidermal growth factor receptor antibody (Ab_EGFR) and amino-N-GQD-PSS-PEI to increase the efficiency and specificity of targeting cells whose surfaces overexpressed epidermal growth factor receptor. As presented in Fig. 3a, there was a burst uptake rate of 51.5% for the basic material within 5 h but the initial profile of material-Ab_EGFR was approximately 84.8%. Since the antibody had highly specific binding, considerable quantities of material-Ab_EGFR were either adsorbed onto the surface or incorporated into the cells, thereby affecting the burst and rapid rates. The sites that could absorb material-Ab_EGFR were saturated from the 4th to the 10th h, therefore the uptake rate plateaued but the rate for the material alone did not plateau until the 8th hour. The amino-N-GQDs (Fig. S8d) behaved similarly to the amino-N-GQD-polymers. Although both amino-N-GQD-polymer composite-Ab_EGFR and amino-N-GQD-Ab_EGFR demonstrated acceptable biocompatibility (Fig. S8e) and favorable stability in physiological environments (Table S2), the cancer cells subjected to TPE exhibited non-ROS-dependent oxidative stress (Tables S3-S5), which was consistent with the singlet oxygen (¹O₂) phosphorescence signal from the amino-N-GQD-polymer composites at 1270 nm (Fig. 3b). Additionally, the estimated amino-N-GQD and amino-N-GQD-polymer ¹O₂ QYs (Φ_Δ) were 0.52 and 0.03, respectively [for reference, Φ_Δ = 0.64 for meso-tetra(4-sulfonatophenyl)porphine dihydrochloride in deuterium oxide (Kuo et al., 2022)], therefore the polymer coating prevented the release of the generated ROS, so amino-N-GQD-polymers are more suitable than amino-N-GQDs for imaging probes. After the TPL intensity was determined in 6 min (approximately 52,941 scans) of photoexcitation under TPE (4732.80 nJ pixel⁻¹; Ex: 980 nm), sustainable two-photon photostability and a diminished photobleaching effect were observed for the amino-N-GQD-polymer composites (Fig. 3c). The amino-N-GQD-polymer composites’ two-photon characteristics are effective for producing marked changes in two-photon inquiries. In acidic environments (e.g., specimens with cancerous cells), the amino-N-GQD-polymer composites could produce high TPL because they had suitable photostability (Fig. 3d).

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Fig. 3

(a) Uptake of amino-N-GQD-polymer composites with and without Ab_EGFR by A431 cells at 37 °C. (b) PL spectral measurements of amino-N-GQDs and amino-N-GQD-polymer composites at 1270 nm. (c) Two-photon stability of amino-N-GQD-polymer composites. TPL spectrum of the material under TPE at 4732.80 nJ pixel⁻¹ for detecting materials with irradiation. Peak located at 845 nm under TPE (Ex: 980 nm). The integrated area maintained nearly constant relative intensity as a function of TPE time (0–6 min; number of scans: approximately 0–52941 scans) and at 500–900 nm, demonstrating high photostability. (d) Amino-N-GQD-polymer composite TPL emissions under 4732.80 nJ pixel⁻¹ TPE (Ex: 980 nm) were pH dependent. Integrated-area TPL intensity as a function of pH (1–11) from 500 to 900 nm. The amino-N-GQD-polymer composites’ TPL intensity values were high from pH 1 to 11. Material dose: 1 μg mL⁻¹. Data presented as mean ± SD (n = 6).

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3.5 Deep and noninvasive TPI observed under the TPE wavelength in the NIR-I or NIR-II window

The amino-N-GQDs had a favorable absolute cross-section, absorption, luminescence, QY, and stability under TPE, thus are suitable contrast agents for the non-invasive detection of deep and nonplanar biological specimens at a TPE wavelength in the NIR-I or NIR-II range. As indicated in Fig. 2b and Fig. S8a, a 980-nm excitation wavelength resulted in the most intense TPL signal from amino-N-GQD-polymer composites, and the 980-nm excitation wavelength resulted in the most intense two-photon autofluorescence (TPAF) signal in specific cancer cells. These cancer cells have biological molecules that caused the signal in the TPAF image (Feng et al., 2021, Kuo et al., 2020). In vivo assays cannot be performed using our developed, optically inverted microscopy system, so cells were embedded in a collagen matrix resembling epithelial tissue to simulate TPL in deep tissue through TPI (Ouzounov et al., 2017) with TPL images of the material-Ab_EGFR-treated cells recorded at depths of 120–240 μm under TPE (Ex: 980 nm; Fig. 4). To achieve a constant TPL intensity, both the amino-N-GQD-Ab_EGFR-treated cells (Figs. 4a–c) and amino-N-GQD-polymer composite-Ab_EGFR-treated cells (Figs. 4d–f) in TPI required TPE power increases of 35% and 27%, respectively, after 160 scans (total effective exposure time: ∼1.09 s, 6.80 ms per scan, 200 × 200-μm² scan area; calculations presented in the materials and methods section, Supplementary Material). The detection power for the TPI of the cells treated with amino-N-GQD-polymer composite-Ab_EGFR was nearly 8, 8.5, and 9 times less than the emission of the cells treated with amino-N-GQD-Ab_EGFR. Conversely, the cells treated with amino-N-GQD-polymer composites demonstrated notably less polymer attachment to their surfaces and remarkably little polymer internalization (800 scans, approximately 5.44 s of total exposure time, Fig. 4g). Emission intensity is quadratically dependent on incident power, therefore, for equal excitation power levels, the excitation ratio between the TPI modalities of the cells treated with the amino-N-GQDs and amino-N-GQD-polymer composites-Ab_EGFR was estimated to be 64, 72, and 91 times brighter than the standard. In addition, for TPE in unlabeled cells, the TPAF imaging (800 scans, approximately 5.44 s of total exposure time, Figs. 4h–j) that demonstrated the same intrinsic fluorophore signal level for the cancerous cells was related to a total power 1938.33 nJ pixel⁻¹ (Fig. 4j). However, the TPE power decreased to 23.93 nJ pixel⁻¹ for the cells treated with amino-N-GQD-polymer composites-Ab_EGFR (Fig. 4f) and was approximately 81 times less than the power of the TPAF imaging of the unlabeled cells but reached a similar collected intensity (Fig. 4j), indicating that the TPL intensity was approximately 6561 times brighter than that of the TPAF imaging of the unlabeled cells. Even at the 240-μm depth, the TPI was not degraded by spherical aberrations caused by mismatched refractive indices between the sample and the immersion oil under TPE. However, the emissions of the cells treated with amino-N-GQD-polymer composites-Ab_EGFR at an excitation power of 10 mW under OPE (808 nm CW laser diode) were not effectively detected when the depth was consistently increased (Figs. 4k–m). Furthermore, the lens working distance enabled imaging tissue phantoms of both TPL and TPAF. TPL images of the cells treated with material-Ab_EGFR and unlabeled-cell TPAF images were captured at depths from 270 to 330 μm (at increments of 30 μm) under TPE (Ex: 980 nm) (Fig. 5). The TPI emissions presented in Figs. 5a–c and 5d–f were not detected at a power level of 23.93 nJ pixel⁻¹ (160 scans, total exposure time of ∼1.09 s) or 1938.33 nJ pixel⁻¹ (800 scans, total exposure time of ∼5.44 s). However, images at depths>270 μm contained spherical aberrations that severely degraded their quality; these aberrations were caused by the mismatched refractive indices of the aqueous sample and immersion oil as well as the objective used, detection efficiency, and maximal z depth of the optical laser system.

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Fig. 4

TPL images (gray level) of (a–c) cells treated with amino-N-GQD-Ab_EGFR recorded at depths of 120–240 μm with total TPE power increments of 118.32 to 215.61 nJ pixel⁻¹ over 160 scans. (d–f) Cells treated with amino-N-GQD-polymer composites-Ab_EGFR subjected to TPE at 14.79–23.93 nJ pixel⁻¹ over 160 scans. (g) Cells treated with amino-N-GQD-polymer composites without antibody coating were indistinct under TPE at 23.93 nJ pixel⁻¹ (800 scans) at a 240-μm depth. TPAF images of unlabeled (untreated) cells under TPE at (h) 23.93, (i) 215.61, and (j) 1938.33 nJ pixel⁻¹ (number of scans: 800; EX: 980 nm). (k–m) Images of amino-N-GQD-polymer composites-Ab_EGFR-treated cells at a constant depth and 10-mW excitation power under OPE (808 nm CW laser diode). Material dose: 1 μg mL^−1.

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Fig. 5

(a–c) TPL images (pseudo-color level) of cells treated with amino-N-GQD-polymer composites and (d–f) TPAF images of unlabeled cells at various depths (with a consistent depth increment from 270 to 330 μm) at power levels of 23.93 and 1938.33 nJ pixel⁻¹ (160 and 800 scans, respectively) under TPE (Ex: 980 nm). Material dose: 1 μg mL⁻¹.

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