	US 12,241,901 B2
	Systems and methods for accurate optical pH sensing of biofilms
Eric J. Seibel, Seattle, WA (US); Leonard Y. Nelson, Seattle, WA (US); Manuja Sharma, Seattle, WA (US); and Jasmine Graham, Seattle, WA (US)
Assigned to The University of Washington, Seattle, WA (US)
Appl. No. 17/260,957
Filed by University of Washington, Seattle, WA (US)
PCT Filed Jul. 19, 2019, PCT No. PCT/US2019/042665 § 371(c)(1), (2) Date Jan. 15, 2021, PCT Pub. No. WO2020/018946, PCT Pub. Date Jan. 23, 2020.
Claims priority of provisional application 62/700,873, filed on Jul. 19, 2018.
Prior Publication US 2021/0263051 A1, Aug. 26, 2021
Int. Cl. G01N 21/64 (2006.01); G01N 33/52 (2006.01); G01N 33/84 (2006.01)

CPC G01N 33/84 (2013.01) [G01N 21/64 (2013.01); G01N 33/52 (2013.01)]

18 Claims

1. A method of measuring an extracellular pH level using multiple wavelengths emitted by a fluorescent substance, the method comprising:

exciting the fluorescent substance at an excitation wavelength;

measuring a first fluorescence intensity at a first wavelength of a fluorescence emission;

measuring a second fluorescence intensity at a second wavelength of the fluorescence emission, wherein the second wavelength is different from the first wavelength; and

determining the extracellular pH level based on the first fluorescence intensity at the first wavelength and the second fluorescence intensity at the second wavelength,

wherein the fluorescent substance is a fluorescein, and

wherein the method further comprises:

calibrating the fluorescence emission of an anion component and a dianion component of the fluorescein by:

subtracting ambient emission from the anion component and the dianion component; and

dividing the anion component and the dianion component by a peak spectral intensity to normalize the anion component and the dianion component.