나노 온도계를 위한 차세대 재료로서의 탄소 점:검토

Bioimaging in Living Cells (Thermal Imaging)

In literature, only a few articles explored the temperature-responsive fluorescent properties of CDs in biological imaging. Prior to such experimentations, in vitro cytotoxicity analysis is crucial for CDs because they make it possible to estimate the CD’s toxicity in living subjects. In vitro cytotoxicity analysis evaluates the effect or influence of the nanomaterial on cultured cells [89].

Yang et al. [94] verified that the CDs could be used as an effective thermometer in living cells; HeLa cells were washed with PBS after treatment with CDs for 6 h. Bright-blue fluorescence of the CDs in HeLa cells is observed when the temperature is 25 °C as shown in Fig. 17a. With the temperature increasing to 37 °C, the blue fluorescence becomes weaker (Fig. 17b), the fluorescence of N-CDs is recovered when the temperature was decreased to 25 °C (Fig. 17c). As a thermos-imaging in vivo, the fluorescent images of mice were collected immediately after being injected with N-CDs at different temperatures. By setting the fluorescence intensity at 28 °C as the reference (I _오 ), 나 /나 _오 of the area where CDs were injected varies from 1.0 to 0.87 with the increase of temperature from 28 to 34 °C (Fig. 17d, e). With temperature further increasing to 43 °C, I /나 _오 declines to 0.52 and the fluorescence becomes nearly undetectable (Fig. 17f). And I /나 _오 can be reversibly enhanced back from 0.66 to 1.0 with the temperature decreases from 39 to 28 °C (Fig. 17g–i). All of these results indicated that N-CD could be used as an effective in vitro and in vivo nanothermometer [94].

a–c Fluorescent images of a single Hela cell at 25, 37, and 25 °C after treatment with N-CDs, respectively. d –f Fluorescent photographs of a mouse given an injection of N-CDs at increasing temperatures. 지 –나 Fluorescent photographs of a mouse given an injection of N-CDs at decreasing temperatures. ( Reproduced from reference [94])

In an exploratory experiment, Kalytchuk et al. tested the capacity of CDs for intracellular temperature monitoring in human cervical cancer HeLa cells. Figure 18 shows the measured intracellular temperatures of HeLa cells incubated with CDs (500 μg/mL). The CDs’ PL decay curves at each temperature were highly reproducible and could be fitted with a single-exponential function at all recorded temperatures. In Fig. 18a, the recorded PL decay curves for temperatures between 25 and 50 °C (with a step size of 5 °C) are indicated by symbols, while the corresponding single-exponential fits are represented by solid lines. They were able to confirm that the PL signal in these PL lifetime measurements was derived exclusively from the CDs for the CD concentration of ≥ 100 μg/mL. The intracellular temperature in each measurement was determined from the calibration curve between PL lifetimes as the temperature increases from 2 to 80 °C. The temperatures determined in this way (T _meas ) are plotted as functions of the PL lifetime in Fig. 18b. Independently, the temperature of the cell solution was determined using a calibrated reference thermometer (shown in Fig. 18b as T_set ). The temperatures reported by the luminescent CD probe and the reference detector are in good agreement. These results show that the PL lifetime of nanoprobes based on CDs can be reliably used to measure intracellular temperatures [89].

In vitro intracellular PL lifetime thermal sensing using CDs. 아 PL emission decays of HeLa cells incubated with CDs (500 μg/mL) at different temperatures (T _set ). ㄴ Temperatures determined using the calibration (T _meas ) and set temperatures (T _set ) plotted against the PL lifetime. ㄷ –f Applicability of CDs for long-term remote intracellular temperature monitoring. ㄷ PL lifetimes extracted from PL transients recorded every 15 min for 24 h of HeLa cells incubated with CDs (500 μg/mL). d Temperatures determined using the calibration curve. 이 Temperatures measured with a reference thermometer (T _ref ). 에 Histogram showing the distribution of temperature differences between the obtained and reference temperatures; the solid line is the distribution curve. (Reproduced from reference [89])

To further evaluate the potential of luminescent CD nanoprobes for long-term real-time temperature monitoring, PL decay profiles of HeLa cells incubated with CDs (500 μg/mL) every 15 min for 24 h were recorded. The PL lifetimes extracted from the measured PL decay values during this period were then plotted as functions of time, as shown in Fig. 18c. Using these results, the temperature variation over time was calculated using the calibration curve, as shown in Fig. 18d. In addition, the sample’s temperature at each measurement point was determined using a reference thermometer with a temperature reproducibility of 60 mK. The temperature determined with the reference thermometer is plotted in Fig. 18e, which shows that there was excellent agreement between the measured and reference temperatures. The high accuracy of the PL-based temperature measurements is further demonstrated by statistical analysis of the differences between the measured and real (reference) temperatures (Fig. 18f). Using these data, the absolute average accuracy of temperature detection by the presented method was calculated to be 0.27 °C. This experiment confirms the potential of CD-based thermal probes in biological systems [89].

Macairan et al. [29] displayed that the prepared CDs can be used for thermal sensing inside cells using intensity and ratiometric approaches. HeLa cells treated with CDs were allowed to equilibrate at 32, 37, and 42 °C (Fig. 19). Excitation at 640 nm was used to selectively monitor the red fluorescence of the CDs in the cells. The thermal changes could be due to changes in intracellular concentration or not correlate with a change in intensity (λex + 640 nm). This could be due to changes in intracellular concentration or localization of the CDs at higher temperatures. Thus, simply relying on changes in fluorescence intensity leads to accurate intracellular thermal sensing.

Fluorescence microscopy images of CD-treated HeLa cells. Fluorescence signals from the CDs (λ_ex =640 nm; left and 405 nm; right) fluorescence ratios are 1.8 at 32 °C, 2.0 at 37 °C, and 2.3 at 42 °C. The control shows untreated HeLa cells at 42 °C with no fluorescence signal as expected. (Reproduced from reference [29])

In contrast, these limitations are excluded using the ratiometric approach. The CDs maintain dual blue and red fluorescence in cells following excitation at 405 nm, as previously observed for the colloidal dispersions (Fig. 19). The red-to-blue ratio increases with increasing temperature, with values of 1.8 at 32 °C, 2.0 at 37 °C, and 2.3 at 42 °C. The ratiometric relationship of the red-to-blue fluorescence of the CDs highlights the advantage of ratiometric temperature sensing in the development of fluorescent nano-thermometry probes. The relative red-to-blue emission ratio remains unaffected, regardless of the amount of CDs taken up by the cells, which can be affected by various factors such as confluency and is not concentration-dependent. Lastly, the CDs have shown fluorescence reversibility with respect to changes in intracellular temperature. Following incubation in HeLa cells, they were subjected to a heating/cooling cycle from 32/42/32 °C. This emphasizes the robustness of the proposed CD-nanothermometer and these findings further demonstrate the fluorescence reversibility [29].

Confocal laser scanning microscopy was used to thermal image colon cancer cell HT-29 using N, S, and I-doped CDs, as shown in Fig. 20 [100]. The fluorescent spots were temperature-dependent as shown in Fig. 20g–i, as the most intense is at 15 °C, while the weakest point was at 35 °C. Interestingly, the fluorescence spots were reversible, and the spots were very photostable after 20 min of continuous excitation [100]. Shin et al. [96] also used confocal laser scanning microscopy for Hela cells, shown in Fig. 20e–g.

a–d Are confocal microscopy images of N, S, I-CDs-colon cancer cell HT-29 with corresponding fluorescence field at 15, 25, 35, and 15 °C, respectively. (Reproduced from reference [100]). 이 –지 Confocal microscopy images of N, S-CDs-stained cells with corresponding fluorescence field at 25, 35, and 25 1C, respectively. (Reproduced from reference [96])

Li et al. 103 prepared a nanocomposite composed of MnOx-CDs to be used as a nano thermos responsive fluorophore for biological environments. HepG2 cells were incubated with MnOx-CDs at different culture temperatures. As illustrated by confocal laser scanning microscopy (excited at 405 nm), the blue luminescence of the MnOx-CDs in the HepG2 cells is weak when the environmental temperature was 40 °C (Fig. 21a), and the blue luminescence of the MnOx-CDs in HepG2 cells enhances as the temperature decreased to 30 °C (Fig. 21b) even to 20 °C (Fig. 21c). Due to the temperature-responsive properties, the as-synthesized MnOx-CDs can be readily applied in the biomedical fields like bioimaging and photothermal therapy in cancer treatment [103].

Confocal laser scanning microscopy images (excited at 405 nm) of MnOx-CDs in HepG2 cells at 40 °C (a ), at 30 °C (b ), and 20 °C (c ). (Reproduced from reference [103])