Light-addressable measurements of cellular oxygen consumption rates in microwell arrays based on phase-based phosphorescence lifetime detection

Abstract

A digital light modulation system that utilizes a modified commercial digital micromirror device (DMD) projector, which is equipped with a UV light-emitting diode as a light modulation source, has been developed to spatially direct excited light toward a microwell array device to detect the oxygen consumption rate (OCR) of single cells via phase-based phosphorescence lifetime detection. The microwell array device is composed of a combination of two components: an array of glass microwells containing Pt(II) octaethylporphine (PtOEP) as the oxygensensitive luminescent layer and a microfluidic module with pneumatically actuated glass lids set above the microwells to controllably seal the microwells of interest. By controlling the illumination pattern on the DMD, the modulated excitation light can be spatially projected to only excite the sealed microwell for cellular OCR measurements. The OCR of baby hamster kidney-21 fibroblast cells cultivated on the PtOEP layer within a sealed microwell has been successfully measured at 104 6 2.96 amol s À1 cell À1. Repeatable and consistent measurements indicate that the oxygen measurements did not adversely affect the physiological state of the measured cells. The OCR of the cells exhibited a good linear relationship with the diameter of the microwells, ranging from 400 to 1000 lm and containing approximately 480 to 1200 cells within a microwell. In addition, the OCR variation of single cells in situ infected by Dengue virus with a different multiplicity of infection was also successfully measured in real-time. This proposed platform provides the potential for a wide range of biological applications in cell-based biosensing, toxicology, and drug discovery. V C 2012 American Institute of Physics.

Metabolic profile analysis of a single developing zebrafish embryo via monitoring of oxygen consumption rates within a microfluidic device

“…The system is equipped with a UV light-emitting diode (LED) as a light modulation source and spatially directs excited light toward a microwell array device to measure the oxygen consumption rate (OCR) of single cells via phase-based phosphorescence lifetime detection. 9 The in situ OCR variation of single cells infected by Dengue virus with different multiplicities of infection was also successfully measured in real time. However, the mitochondrial function in cell lines, tissues, and embryos was not monitored continually over long periods by sequentially adding pharmacological inhibitors of bioenergetic pathways in our previous work.…”

“…The impermeability of the sealed microwells to the surroundings was previously confirmed. 9 To Fig. 5(a).…”

“…For detailed descriptions of the fabrication of the microfluidic module, refer to our previous work on cellular OCR measurements. 9 The aqueous solution (denoted with a blue dashed line) and air pressure (denoted with a red dashed line) can be introduced from layer 1 to layer 2 via six holes. Finally, the glass substrate with 2 Â 2 microwells (layer 4) was adhered to the microfluidic module using double-sided adhesive (410 M, 3 M, USA) for ease of operation.…”

“…After the embryos settle down, a microfluidic module with pneumatically actuated lids was set above the microwells to controllably seal the microwells. An air-pressure system, described in our previous work, 9 was used to pneumatically actuate the glass lids (5 mm in diameter) to controllably seal the microwells. A regulated compressed-air source was connected to multiple three-way solenoid valves (Lee Inc., United States), where each valve was controlled by a custom LabVIEW program (National Instruments, Inc.) to switch rapidly between atmospheric and input pressure.…”

“…Detailed descriptions of the facility setup, data acquisition, and the phase-based phosphorescence lifetime detection have been reported in our previous work. 9,11 The phosphorescence lifetime was calculated by measuring the phase shift (h) between the reference LED signal (modulated excitation light) and the detected phosphorescence signal to calculate the luminescence lifetime (s) via phase-based phosphorescence lifetime detection. The relationship between the phase shift (h) and the lifetime (s) can be approximated by the following:…”