Fig. 3: Enhanced ratiometric fluorescence imaging with 3D-SpecDIM in live cells.

a Schematic of mitophagy. The mitochondrion was labeled with a pH-sensitive fluorescent probe mGold-HaloTag-JF549. The intensity ratio of mGold to JF549 serves as an indicator of the local environmental pH. As the pH of the local environment decreases, the intensity ratio correspondingly decreases. b, c 3D trajectory of a mitochondrion during mitophagy. The color indicates time (b) or intensity ratio (c). Inserts in (c) show the spectral images in two different time points indicated by arrows, which have different pH ratio values. d Comparison of mitochondrial moving speed in rapamycin-treated cell (0.47 ± 0.07 μm²/s, Mean ± SD) and untreated cell (0.25 ± 0.03 μm²/s, Mean ± SD). For each condition, n = 66 trajectory segments, extracted from mitochondria in multiple independent cells from the same culture. Center line in the violin plot indicates the median; box bounds represent the 25th and 75th percentiles; whiskers extend to the most extreme data points within 1.5 × the interquartile range from the box. e The fluorescence spectral profile changes during mitophagy process. The three spectral peaks from left to right correspond to mGold, JF549, and LysoTracker, respectively. f The pH ratio of pH-sensitive fluorescent probe (blue line) or the intensity of lysosome (yellow line) as a function of time. Note that after 40 s, pH decreased when lysosome strength increased. g Multi-resolution imaging of mitophagy process. The 3D volume image of lysosome is overlayed with spectrally encoded 3D moving trajectory of mitochondrion. The blue-to-yellow color-coded trajectory represents the 3D movement of the mitochondrion, with the color encoding the pH ratio. Meanwhile, the 3D image of the lysosome is color-coded from violet to red, indicating fluorescence intensity. The 3D lysosome volume image was reconstructed by registering the fluorescence photons to laser focus positions in a separated detection channel. Detailed reconstruction information is provided in Supplementary Notes 4. The checkerboard pattern represents transparency, while the alternating color blocks indicate different intensity levels. h The sum intensity projection images of (g) in xy plane (left), xz plane (middle), and yz plane (right).