Microchip internalization was confirmed by CLSM in living cells (Figure ) as well as cell viability, always over 95%, as estimated using Cell tracker. The fluorescence intensity of internalized SOµC-Bdpy1 and SOµC-Bdpy2 was quantified to assess intracellular GSH concentration. To estimate the GSH concentration in HeLa cells, the ratiometric signal of the internalized SOµC-Bdpy1 and SOµC-Bdpy2 was measured applying the average of eight fluorescence intensity profiles across each microchip. The average fluorescence signal ratio for each internalized SOµC-Bdpy1 was calculated as shown in Figure , and then, the average fluorescence signal ratio for all internalized SOµC-Bdpy1 was converted to GSH concentration by using the ratiometric curve resulting from the SOµC-Bdpy1 in the GSH solution for overnight (Figure ). These calculations manifested that the internalized SOµC-Bdpy1 exhibited the GSH intracellular level of ca. 3.1 ± 0.6 mM. The ratiometric calculation also indicated that ca. 73 % of internalized SOµC-Bdpy1 show intracellular GSH concentration of ca. 2-3 mM while ca. 18 % and 9 % of internalized SOµC-Bdpy1 quantifies the intracellular GSH concentration ca. 3-4 mM and ca. 4-5.8 mM respectively (Table and Figure ). A similar calculation was also done for SOµC-Bdpy2 internalized in HeLa cells (Figure (a) and Figure and Table (Supporting Information)) and average ratiometric fluorescent intensity was converted to GSH concentration by applying the ratiometric curve (Figure ) of these functionalized microchips in the GSH solution. The ratiometric calculations from SOµC-Bdpy2 also indicated that the internalized microchips were able to detect intracellular GSH ca. 3.7 ± 1 mM which is more consistent with the GSH level reported in the literature and it showed that ca. 23 %, ca. 45 %, and ca. 32 % of internalized SOµC-Bdpy2 exhibited the intracellular GSH concentration of ca. 2-3 mM, ca. 3-4 mM, and 4-5.8 mM, respectively (Table and Figure ). On account of the fact that the ratiometric curves resulting from SOµC-Bdpy1 and SOµC-Bdpy2 in the GSH solution are related to the same exposure time of microchips to the GSH solution for all particles, in the case of internalized microchips, the time of internalization and the period of the presence of functionalized microchips inside of the cells may be different during overnight incubation or even intracellular GSH might change during different cell states. For this reason, it could be concluded that the ratiometric curve resulting from the SOµC-Bdpy2 can be more reliable for the intracellular GSH quantification compared to the SOµC-Bdpy1. Moreover, it is concluded that SOµC-Bdpy2 is a good candidate not only for quantifying GSH levels even in individual cells, which is another advantage of these functionalized microchips, but also for distinguishing GSH levels between different cells. HeLa cells were also incubated with SOµC-Bdpy2 for 48 h, and as it can be seen in Figure , the result of the ratiometric calculation signal is ca. 0.96 ± 0.07 (3.8 ± 0.3 mM) which is comparable with the result of internalized SOµC-Bdpy2 during overnight incubation time, demonstrating the potentiality of SOµC-Bdpy2 to yield reliable GSH measurements during different time interval and long-term tracking of intracellular GSH. The sensitivity and reversibility of SOµC-Bdpy2 was also studied toward the variations in GSH concentrations in living cells. For this purpose, living cells were incubated with SOµC-Bdpy2 overnight followed by incubation with 10 mM glutathione ethyl ester (GSHee), which is a cell-permeable derivative of GSH , for 4 h (Figure (b) and Figure ) which exhibited the increment of the ratiometric fluorescent intensity (ca. 2.4 ± 0.3) which was more than this value for cells in the control condition (ca. 0.92 ± 0.2). The performance of SOµC-Bdpy2 in lower intracellular GSH level was also studied with the addition of NEM, which is a GSH scavenger. The difficulty for working with NEM in the biological environment is that the cells cannot tolerate in high concentration of NEM for 4 h incubation even though the reduction in the ratiometric signal is evident as it can be seen in Figure . For this reason, living cells were incubated 24h with 1 mM buthionine sulfoximine (BSO), which reduces levels of glutathione in living cells , and then were incubated with SOµC-Bdpy2 during overnight in presence of 1mM of BSO. As it can be seen in Figure (c), the fluorescence signal in the λem = 595-630 nm is considerably higher than λem = 530-580 nm resulting in the lower ratiometric signal compared to the internalized SOµC-Bdpy2 in the control condition. In this experiment, the fluorescent signal recovery in the range of λem = 530-580 nm was also confirmed by the subsequent addition of GSHee (10 mM, 4 hours of incubation time), as shown in Figure . To investigate the performance of SOµC-Bdpy2 for long-term tracking, HeLa cells were also incubated with SOµC-Bdpy2 for overnight followed by incubation with BSO in the presence of SOµC-Bdpy2 for overnight (Figure As a conclusion, SOµC-Bdpy2 offers several advantages over organic fluorescent probes for accurately measuring intracellular GSH. They are compatible with aqueous suspensions, eliminating the need for organic solvents typically required to dissolve molecular probes. In addition, the precisely defined shape and size of these micro-fabricated microchips, which can be detected using fluorescence microscopy. They are also biocompatible and enable intracellular measurements not only in cell populations but also in individual cells. These microchips present new opportunities for long-term monitoring of biomolecules in living systems, a capability that organic fluorescence probes lack.