Journal: bioRxiv

Article Title: Photothermal Dye-based Subcellular-sized Heat Spot Enabling the Modulation of Local Cellular Activities

doi: 10.1101/2021.12.19.471878

Figure Lengend Snippet: Characterization of nanoHT with the ability of heat release and temperature sensing. A) Schematic illustrations of nanoHT and its controlled heating inside a cell. B) Excitation and fluorescence spectra of C102 and EuDT, and absorption spectrum of V-Nc in nanoHT . C) DLS measurement of nanoHT . The average of diameter: 153 ± 51 nm (mean ± SD). D) The normalized fluorescence intensity (FI) values of C102 and EuDT were plotted against temperature as the 1 st axis, and the ratio value (EuDT/C102), which is normalized to that of 37 °C, as the 2 nd axis. Error bars, SD (n=3). E) The evaluation of the heating ability of nanoHT suspension in the cuvette by the irradiation of a NIR laser (808 nm). Error bars, SD (n=3).

Article Snippet: Poly(methyl methacrylate- co -methacrylic acid)(PMMA-MA)(Mw: 34,000), Vanadyl 2,11,20,29-tetra-tert-butyl-2,3-naphthalocyanine (V-Nc), and Coumarin 102 (C102) were purchased from Sigma-Aldrich.

Techniques: Fluorescence, Irradiation

Journal: bioRxiv

Article Title: Photothermal Dye-based Subcellular-sized Heat Spot Enabling the Modulation of Local Cellular Activities

doi: 10.1101/2021.12.19.471878

Figure Lengend Snippet: Validation of nanoHT with the ability of heat generating. A) Schematic image of a means to validate the temperature sensing ability of nanoHT under the microscope with a NIR infrared laser (980 nm). Scale bar: 5 μm. B) The mean fluorescence intensities of C102 and EuDT at each ROI as shown in A) were plotted every 0.56 sec in the time course (5 sec NIR laser stimulation). C) The calibration curve of nanoHT against temperature obtained under the microscopic observation. Error bars, SD (n=3). D) The normalized ratio (EuDT/C102) was converted to the temperature increment profile (Δ T ) using the calibration curve. E,F) Validation of the heat generating ability of nanoHT using an 808 nm laser. A NIR laser was only illuminated to a white circle spot. The mean fluorescence intensity at each ROI was plotted in the time course while a NIR laser stimulation is performed during 5 sec at different laser powers (0.98-11 mW). G) The average of temperature increment provided by nanoHT (error bars, SD. n = 10) was plotted at each 808 nm laser power. Solid line showed the linear fit.

Article Snippet: Poly(methyl methacrylate- co -methacrylic acid)(PMMA-MA)(Mw: 34,000), Vanadyl 2,11,20,29-tetra-tert-butyl-2,3-naphthalocyanine (V-Nc), and Coumarin 102 (C102) were purchased from Sigma-Aldrich.

Techniques: Microscopy, Fluorescence

Journal: bioRxiv

Article Title: Photothermal Dye-based Subcellular-sized Heat Spot Enabling the Modulation of Local Cellular Activities

doi: 10.1101/2021.12.19.471878

Figure Lengend Snippet: Validation of nanoHT in HeLa cells. A,B) Validation of the heat generating ability of nanoHT using an 808 nm laser. The mean fluorescence intensity of nanoHT in A) was plotted in the time course while a NIR laser stimulation was performed during 5 sec at different laser powers (0.98-11 mW). Scale bar: 10 μm. C) The average of temperature increment provided by nanoHT (Error bars, SD. n = 12) were plotted at varying 808 nm laser powers. Solid line showed the linear fit. D) The different temporal pattern of the temperature increment created by nanoHT . The mean of normalized ratio of nanoHT with SD (n=3) was plotted in the time course. E) Colocalization test with a lysosome tracker in the upper panel (Red: C102, Green: lysosome tracker to stain acidic organelles). Enlarged view of the region surrounded by a dashed square before, during and after heating. Scale bar: 10 μm.

Article Snippet: Poly(methyl methacrylate- co -methacrylic acid)(PMMA-MA)(Mw: 34,000), Vanadyl 2,11,20,29-tetra-tert-butyl-2,3-naphthalocyanine (V-Nc), and Coumarin 102 (C102) were purchased from Sigma-Aldrich.

Techniques: Fluorescence, Staining

Journal: bioRxiv

Article Title: Photothermal Dye-based Subcellular-sized Heat Spot Enabling the Modulation of Local Cellular Activities

doi: 10.1101/2021.12.19.471878

Figure Lengend Snippet: Heat-triggered cell death by nanoHT . A) Dual imaging of Apopxin green (apoptosis marker) and nanoHT (Blue: C102) in a HeLa cell. B) The time course of the normalized ratio of nanoHT and fluorescence of Apopxin green nearby the heat spot (a NIR stimulation during 10 sec). The temperature increments were estimated by the calibration curve. C) Images of HeLa stained with Apopxin green and PI (for detection of necrosis or late stage of apoptosis) after heating (1 and 10 min). D) The correlation between temperature increment of nanoHT and the enhancement of Apopxin green ( F / F 0 ). Laser power was varied from 8.8 to 11.2 mW. E) Dual imaging of Ca 2+ (B-GECO) and Apopxin green in a HeLa cell. Elapsed time is shown to the top left of each image in A) and E). Scale bars: 10 μm.

Article Snippet: Poly(methyl methacrylate- co -methacrylic acid)(PMMA-MA)(Mw: 34,000), Vanadyl 2,11,20,29-tetra-tert-butyl-2,3-naphthalocyanine (V-Nc), and Coumarin 102 (C102) were purchased from Sigma-Aldrich.

Techniques: Imaging, Marker, Fluorescence, Staining

Journal: bioRxiv

Article Title: Photothermal Dye-based Subcellular-sized Heat Spot Enabling the Modulation of Local Cellular Activities

doi: 10.1101/2021.12.19.471878

Figure Lengend Snippet: The evaluation of intracellular ATP dynamics during local heating. A) The morphological change of mitochondria occurs after heating at the temperature above the threshold to induce the cell death. Scale bar: 20 μm. B) Fluorescence image of HeLa cell expressing MaLionG (cytoplasmic ATP) and mitoMaLionR (mitochondrial ATP) with nanoHT (C102). Scale bar: 20 μm. C) The time course of mitoMaLionR nearby nanoHT (the local area of the cell shown in B)). D) The ATP dynamics in cytoplasm (MaLionG) and mitochondria (mitoMaLionR) at ROI1 and 2 of the same cell in B). The heating is 1 min. E) The analysis of mitochondrial ATP dynamics nearby the heat spot like ROI1 in B) in 4 cells at different temperature increments (3.6 to 10.1 °C below the threshold of the cell death). F) The thick lines of MaLionG and mitoMaLionR represent the average of 7 cells with SD at different temperatures.

Article Snippet: Poly(methyl methacrylate- co -methacrylic acid)(PMMA-MA)(Mw: 34,000), Vanadyl 2,11,20,29-tetra-tert-butyl-2,3-naphthalocyanine (V-Nc), and Coumarin 102 (C102) were purchased from Sigma-Aldrich.

Techniques: Fluorescence, Expressing