Method for Passive Droplet Sorting after Photo-Tagging

Abstract

We present a method to photo-tag individual microfluidic droplets for latter selection by passive sorting. The use of a specific surfactant leads to the interfacial tension to be very sensitive to droplet pH. The photoexcitation of droplets containing a photoacid, pyranine, leads to a decrease in droplet pH. The concurrent increase in droplet interfacial tension enables the passive selection of irradiated droplets. The technique is used to select individual droplets within a droplet array as illuminated droplets remain in the wells while other droplets are eluted by the flow of the external oil. This method was used to select droplets in an array containing cells at a specific stage of apoptosis. The technique is also adaptable to continuous-flow sorting. By passing confined droplets over a microfabricated trench positioned diagonally in relation to the direction of flow, photo-tagged droplets were directed toward a different chip exit based on their lateral movement. The technique can be performed on a conventional fluorescence microscope and uncouples the observation and selection of droplets, thus enabling the selection on a large variety of signals, or based on qualitative user-defined features.

Keywords: droplet array; droplet microfluidics; microfluidics; passive sorting; photo-tag; sorting.

Figure 1

Linear array of droplets with increasing irradiation times from left to right. (A) Fluorescence from blue excitation (436 nm). (B) Fluorescence from violet excitation (395 nm). pH of droplets as determined from blue to violet fluorescence intensity ratio based on calibration curve as shown in Supplemental Figure S2. (C) Droplet pH with increasing irradiation time with fit to a linear regression.

Figure 2

(A) Array device channel geometry. The location of the droplet array is highlighted by a dashed red square. (B) Bright-field image of droplet array. All wells are occupied with droplets that are approximately the same size as the wells. (C) Fluorescence from blue excitation. Irradiated droplets have lower fluorescence intensity. (D) Droplet array after the elution of non-irradiated droplets. Fluorescence excitation light was used in combination with bright-field image to increase droplet visibility.

Figure 3

(A) Rail device channel geometry. The location of the images in (B) are shown. Location of droplet irradiation is indicated by a blue dashed rectangle and the sorting rail is highlighted by a dashed red rectangle. (B) Droplets containing fluorescent beads, circled in green, were previously irradiated with light. Irradiated droplets have lower pH and hence higher interfacial tension. They follow the rail upwards and leave at a higher lateral position toward the selected exit. Other droplets are immediately pushed off the rail by the flow of oil toward the unselected exit. Inset: A droplet circled in green was irradiated prior to sorting. Neighboring droplets are also partially irradiated. The red arrow indicates a fluorescent bead.

Figure 4

(A) Bright-field image of droplet array. Droplets containing cells were classified as viable (green circle), early apoptosis (yellow diamond), late apoptosis (red square), and necrosis (black square). (B) Fluorescence image with blue excitation. The droplet with an early apoptosis cell was irradiated and displays lower fluorescence intensity. (C) Droplet array after the elution of non-irradiated droplets. The only droplet remaining in the array contains the early apoptosis cell.