Imaging of microdroplets and image analysis

Protocols for Image Analysis for water-in-oil microdroplet

1. Preparation of aqueous microdroplet containing salt for confocal microscopy
   1. To prepare water-in-oil microdroplets, microfuge tubes containing 10:1 mixture of oil:aqueous solution containing each 1-µM fluorescent dye were placed in a bath sonicator (Elmasonic S 10; Elma Schmidbauer GmbH, Germany) and sonicated for 2 minutes at ‘sweep’ function in room temperature.
   2. Because microdroplets formed by the sonication started fusing after 10 minutes, confocal images of microdroplets were taken within 10 minutes so that we could get the images of desirable sized microdroplets ranging from a few to tens of microns in diameter and avoid the fusion of microdroplets. 
   3. Different types of oils were used, including immersion oil (type A; 1,4-dimethyl-2-(1-phenylethyl)-benzene; Nikon Inc., Japan), triolein oil (glyceryl trioleate; CAS No. 122-32-7, Sigma-Aldrich, MO), olive oil (CAS No. 8001-25-0, Sigma-Aldrich, MO), or silicone oil (CAS No. 63148-52-7, Sigma-Aldrich, MO). 
   4. The prepared oil-solution emulsion was casted onto a slide glass and imaged with a confocal microscope (LSM 780; Carl Zeiss, GmbH, Germany). 
   5. Images were taken for water microdroplets freely floating in oil at least 10 µm above the glass surface to prevent any interference originated from the glass surface. In order to minimize the drift of microdroplets while taking images, we waited typically for one minute until the initial rapid drift caused by mechanical shock of mounting the glass slide onto a microscope stage subsided.
   6. The fluorescence signals were recorded with excitation laser beams (561 nm for AF555 and Mitotracker; 633 nm for AF647) and their pre-programmed emission windows. The acquired images were processed with ZEN software suite (Carl Zeiss GmbH, Germany) and ImageJ software (NIH, Bethesda, MD). 
2. Data analysis in polar coordinate system
   1. The size of droplet ranged from 3 to 24 µm in diameter. The fluorescence intensities of the observed microdroplets were numerated in the terms of pixels and their positional information was transformed in polar coordinates so that we can present them in distances from the center of the droplet.
   2. The transformation of coordinate system has been done in ImageJ software and Microsoft Excel. First, open the image taken from the microscope with ImageJ and select the area of interest in ‘Oval’ shape, because microdroplet is circle. Second, save the pixel data in .csv format text. (Menu > Analyze > Tools > Save XYcoordinates) Then, the saved files were opened by Excel so that we could convert the XY coordinate into the polar coordinate system through a simple conversion equation of your own.
   3. After converting the positional information into the ‘distance from the center of circle’, we plotted the fluorescence intensities against the distance. The detailed process in each experiment was written in the main manuscript.

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