Optofluidic characterization of marine algae using a microflow cytometer

Abstract

The effects of global warming, pollution in river effluents, and changing ocean currents can be studied by characterizing variations in phytoplankton populations. We demonstrate the design and fabrication of a Microflow Cytometer for characterization of phytoplankton. Guided by chevron-shaped grooves on the top and bottom of a microfluidic channel, two symmetric sheath streams wrap around a central sample stream and hydrodynamically focus it in the center of the channel. The lasers are carefully chosen to provide excitation light close to the maximum absorbance wavelengths for the intrinsic fluorophores chlorophyll and phycoerythrin, and the excitation light is coupled to the flow cytometer through the use of an optical fiber. Fluorescence and light scatter are collected using two multimode optical fibers placed at 90-degree angles with respect to the excitation fiber. Light emerging from these collection fibers is directed through optical bandpass filters into photomultiplier tubes. The cytometer measured the optical and side scatter properties of Karenia b., Synechococcus sp., Pseudo-Nitzchia, and Alexandrium. The effect of the sheath-to-sample flow-rate ratio on the light scatter and fluorescence of these marine microorganisms was investigated. Reducing the sample flow rate from 200 μL/min to 10 μL/min produced a more tightly focused sample stream and less heterogeneous signals.

Figure 1

A schematic of the microflow cytometer. (a) The optical and microfluidics setup. (b) A zoom-in view showing the chevron grooves extending into the PDMS substrate.

Figure 2

Microscopy of the phytoplankton species. The photos show images of (a) Karenia b., (b) Synechococcus sp., (c) Pseudo-Nitzchia, and (d) Alexandrium.

Figure 3

Simulations showing concentration distributions of the sample stream at sheath and sample inlet flow rates of: 800 and 200 μL/min (a and b); and 800 and 10 μL/min (c and d), respectively.

Figure 4

Scatter plots of chlorophyll vs. side scatter signals from phytoplankton using the microflow cytometer at sheath and sample inlet flow rates of 800 and 10 μL/min. Dashed squares represent the boundaries of the cluster regions that were analyzed and described in Table Table II..

Figure 5

Scatter plots of phycoerythryin vs. side scatter signals from phytoplankton using the microflow cytometer at sheath and sample inlet flow rates of 800 and 10 μL/min.

Figure 6

Scatter plots of phycoerythryin vs. chlorophyll signals from phytoplankton using the microflow cytometer at sheath and sample inlet flow rates of 800 and 10 μL/min.