A Molecular Logic Gate Enables Unconventional Super-resolution Same-Day Imaging of Lysosome Membrane in Live Cells

Abstract

Lysosomes are acidic membrane-bound organelles that aid digestion, excretion, and cell renewal. The lysosomal membranes are essential for maintaining lysosomal functions and cellular homeostasis. In this work, we developed a molecular "NOR" logic gate, SIATFluor-580L, by introducing malachite green into the spirocyclic rhodamine. SIATFluor-580L requires restriction of molecular rotation of the malachite green motif (Input 1, tight membrane structure) and a large amount of H⁺ ions to convert the spirocyclic rhodamine into the zwitterionic form (Input 2, acidic environment) to produce a fluorescent product (Output), providing a fluorogenic probe to visualize the lysosomal membrane dynamics in living cells with subdiffraction resolution by using HyVolution (also known as Lightning), an unconventional and inexpensive super-resolution imaging approach based on a basic confocal optical system.

Figure 1

Working mechanism of SIATFluor-580L. (A) Schematic illustration of “NOR”-type logic gate. (B) Fluorescence spectra of SIATFluor-580L (10 μM) in buffer solution with different pH/viscosity (η) combinations: pH 7, η = 1.01 cP; pH 7, η = 219.00 cP; pH 2, η = 1.01 cP; pH 2, η = 219.00 cP. The inset shows photos of different combinations of inputs under 520 nm light irradiation. (C) Column diagram of the fluorescence intensities. The inset shows the truth table of the NOR gate with rotation (yes or no) and spirolactone (yes or no) as inputs.

Figure 2

(A, B) Fluorescence spectra of SIATFluor-580L (10 μM) in neutral/acidic buffer with different fractions of glycerol. (C) Fluorescence intensity plot of SIATFluor-580L (10 μM) at 580 nm in pH 2/7 solutions with different viscosities (η). The inset shows the molecular structure of SIATFluor-580L in acidic condition. (D, E) Fluorescence spectra of SIATFluor-580L (10 μM) in nonviscous/viscous buffer with different pH values. (F) Fluorescence intensity plot of SIATFluor-580L (10 μM) at 580 nm in η = 1.0 cP/η = 219 cP solutions with different pH values. The inset shows the molecular structure of SIATFluor-580L in viscous condition.

Figure 3

Cellular images of SIATFluor-580L. (A) Confocal fluorescence images of SIATFluor-580L. HeLa cells were treated with SIATFluor-580L (5 μM) and different organelle trackers (LysoTracker, ER Tracker and MitoTracker, respectively). Scale bars: 20 μm. (B) Wash-free fluorescence imaging of HeLa cells treated with SIATFluor-580L/LysoTracker. Scale bar: 10 μm. (C) Photostability evaluation of SIATFluor-580L/LysoTracker Green. Irradiation time: 250s. Concentration: 5 μM. Scale bars: 5 μm. (D) Kinetic curves of average fluorescence signal of the HeLa cells treated with SIATFluor-580L (red) or LysoTracker (white).

Figure 4

Lysosomal membrane imaging. (A) Comparison of confocal and HyVolution imaging. Scale bar: 2 μm. (B, C) Enlarged images of regions of interest in the HyVolution and confocal images. Scale bar: 1 μm. (D) Fluorescence intensity distribution analysis of the yellow line regions in (B) and (C). (E) Dynamic process of lysosomes in living cells. (F). Fluorescence intensity distribution analysis of the yellow line regions in (E).