Quantify the Accumulation of Fluorescent Drugs or Nanoparticles in Endo-Lysosomes by Local pH

Wang J, MacEwan SR, Chilkoti A*. Quantitative mapping of the spatial distribution of nanoparticles in endo-lysosomes by local pH. Nano Letters, 2016, 17, 1226-1232.

Materials:

    Lysosensor Yellow/Green (Invitrogen)

    35 mm glass-bottom dishes (MatTek)

    MES calibration buffer (pH 4 to pH 7.5 at intervals of 0.5 pH units)

        MES buffer contains 5 mM NaCI, 115 mM KCI, 1.2 mM MgSO4, 25 mM MES, 10 μM monensin, and 10 μM nigericin (Adjust pH by HCI or NaOH).

    AF488-labeled nanoparticle as a representative fluorophore to demonstrate how to study their endo-lysosomal distribution by local pH.

Equipment:

    Spinning disk confocal microscope with EMCCD

Cell treatment and imaging:

    1) Seed adherent cells on 35 mm glass-bottom dishes and incubate overnight to allow their attachment to the dish bottom.

    2) Rinse live cells with PBS thrice, incubate with 2 μM Lysosensor Y/G at 37°C for 30 min, and then rinse cells with PBS thrice.

    3) Image live cells under the spinning disk confocal microscope, capture images at three fluorescence channels and also the DIC channel.

        Lysosensor-Blue  : Ex = 405 nm, Blue Em filter (447 ± 30 nm)

        Lysosensor-Green: Ex = 405 nm, Green Em filter (525 ± 15 nm)

        AF488-labeled nanoparticle: Ex = 488 nm, Green Em filter (525 ± 15 nm)  

    4) Save images as 512 pixels × 512 pixels tiff files.

Quantitative analysis using ImageJ and MATLAB (MATLAB Code):

Figure. Flowchart of image analysis process for ratiometric fluorescence imaging. Three fluorescence images, including the fluorescence image of AF488-labeled nanoparticles (fluorescent nanoparticle image) as well as the blue and green fluorescence images of Lysosensor (Lysosensor-Blue image and Lysosensor-Green image) were acquired during ratiometric fluorescence imaging. The calibration curve of Lysosensor’s Blue-to-Green ratio against pH was obtained in the calibration experiment. Image analysis was performed with ImageJ and MATLAB softwares. The MATLAB code to group pixels with the same pH value and to plot the average AF488 fluorescence intensity against pH is shown at the end of the page.

Plot calibration curve of Lysosensor Blue-to-Green against pH

    1) Seed adherent cells on 35 mm glass-bottom dishes and incubate overnight to allow their attachment to the dish bottom.

    2) Rinse live cells with PBS thrice, incubate with 2 μM Lysosensor Y/G at 37°C for 30 min, and then rinse cells with PBS thrice.

    3) Equilibrate live cells with MES buffers with different pH values for 2 min, then image the cells under the microscope and capture Lysosensor-Blue and Lysosensor-Green images, save then as 512 pixels × 512 pixels tiff files.

    4) Distinguish endo-lysosomal area by ImageJ, calculate Lysosensor Green-to-Blue ratio by ImageJ, then plot the calibration curve with MATLAB.

Relative data published in Nano Letter, 2017

Figure. Method of studying spatial distribution of fluorescent nanoparticles as a function of local pH using ratiometric fluorescence imaging of Lysosensor and pixel-by-pixel analysis. (A) Fluorescence emission spectra of Lysosensor at pH 4 and pH 7 excited at 405 nm and the fluorescence emission spectrum of AF488 excited at 488 nm. Lysosensor exhibits two pH-dependent emission peaks at 440 nm and 530 nm measured using a blue filter (447 ± 30 nm) and green filter (525 ± 15 nm), respectively, while the fluorescence of AF488 is measured using a green filter (525 ± 15 nm). The blue and green bars indicate the emission filters used for fluorescence emission measurements. (B) Spinning disk confocal fluorescence images of Lysosensor-Blue, Lysosensor-Green and AF488 peaks captured in live cells. The white scale bar indicates 2 μm. (C) All fluorescence images are analyzed as 512 pixels × 512 pixels grids where the origin (0, 0) is set at the bottom-left and every pixel is assigned a coordinate (x, y). Fluorescent endo-lysosomal areas (shown as grey pixels) are identified and distinguished from the largely non-fluorescent cytosol using ImageJ software. (D) The ratio of Lysosensor’s two emission peaks (IBlue/IGreen) in endo-lysosomal compartments shows a linear relationship with pH in live cells equilibrated with a series of calibration buffers ranging from pH 4 to pH 7.5. According to this IBlue/IGreen ratio versus pH curve, the IBlue/IGreen ratio of each pixel can be converted to a pH value. (E) Using this method to map endo-lysosomal pH, normal cells show endo-lysosomal pH ranging from pH 4 to pH 7 while chloroquine-treated cells show increased endo-lysosomal pH. The black scale bar indicates 2 μm and the color bar indicates pH value.