Method and apparatus for measuring biofilm thickness and topology

Patent metadata

Field	Value
Publication number	US-2016272933-A1
Application number	US-201615071921-A
Country	US
Kind code	A1
Filing date	Mar 16, 2016
Priority date	Mar 18, 2015
Publication date	Sep 22, 2016
Grant date	—

Abstract

An apparatus and method of measuring biofilm and biological activity on a surface is disclosed. The apparatus includes a biofilm, which includes one or more microorganisms, grown on a substrate. A viewing window is placed on a surface of the biofilm and a gas bubble is introduced between the viewing window and the surface of the biofilm. The space between the substrate and the viewing window may be enclosed in a casing that has an inlet and an outlet, forming a flow cell. A microscope system, such as a white light interferometer, captures data of the biofilm in situ and non-destructively. The 3D images of biofilm surface have high resolution while maintaining a large field of view. This invention will be useful for fundamental studies of biofilms, biomedical and environmental screening, and many other applications in biology and the life sciences.

First claim

We claim:

1 . An apparatus for measuring biofilm surface properties with a microscope system comprising:
a. a biofilm including one or more microorganisms grown on a substrate; b. a viewing window placed on a surface of the biofilm; and c. a gas bubble introduced between the viewing window and the surface of the biofilm.

2 . The apparatus of claim 1 wherein the substrate includes a reflective surface.

3 . The apparatus of claim 2 wherein the substrate comprises one of the following materials: glass, metal, ceramic and plastic.

4 . The apparatus of claim 1 wherein a needle is used to supply the gas bubble.

5 . The apparatus of claim 1 wherein the microscope system is a three-dimensional profile microscope system.

6 . The apparatus of claim 5 wherein the three-dimensional profile microscope system is a white light interferometer which generates and captures interference fringes of the biofilm.

7 . The apparatus of claim 1 wherein the viewing window is a cover slip.

8 . The apparatus of claim 1 wherein the space between the substrate and the viewing window is enclosed in a casing that has an inlet and an outlet, forming a flow cell.

9 . The apparatus of claim 8 wherein the flow cell is coupled to one or more pumps that provides a supply of liquid media and gas bubble to the biofilm.

10 . The apparatus of claim 9 wherein the one or more pumps includes a first pump for providing the liquid media to a region between the substrate and the window, and a second pump for introducing the gas bubble to the biofilm.

11 . The apparatus of claim 7 wherein the apparatus comprising the flow cell includes a plurality of individual flow cells.

12 . The apparatus of claim 1 wherein the apparatus monitors changes in topology and thickness of the biofilm in response to changes in a local environment, wherein the local environment is the structure, composition, and chemistry of materials of the viewing window and the substrate.

13 . The apparatus of claim 1 wherein the apparatus is used for biomedical and environmental screening and/or to measure effects of chemicals or drugs on the biofilm.

14 . An apparatus for measuring biofilm surface properties with a microscope system comprising:
a. a flow cell including an inlet, an outlet, a window, and a substrate; b. a biofilm including one or more microorganisms grown on the substrate of the flow cell; and c. a gas bubble introduced between the window and a surface of the biofilm.

15 . The apparatus of claim 14 wherein the flow cell is coupled to one or more pumps that provide a supply of liquid media and gas bubble to the biofilm.

16 . The apparatus of claim 15 wherein the one or more pumps includes a first pump for providing the liquid media to a region between the substrate and the window, and a second pump for introducing the gas bubble to the biofilm.

17 . The apparatus of claim 14 wherein the apparatus comprising the flow cell includes a plurality of individual flow cells.

18 . The apparatus of claim 14 wherein the microscope system is a three-dimensional profile microscope system.

19 . The apparatus of claim 18 wherein the three-dimensional profile microscope system is a white light interferometer.

20 . A method of measuring biofilm surface properties comprising:
a. growing one or more microorganisms on a substrate to form a biofilm; b. placing a viewing window on a surface of the biofilm; c. introducing a gas bubble between the viewing window and the surface of the biofilm; and d. capturing data of the biofilm using a microscope system.

21 . The method of claim 20 wherein the substrate includes a reflective surface.

22 . The method of claim 21 wherein the substrate comprises one of the following materials: glass, stainless steel, and plastic.

23 . The method of claim 20 wherein a syringe needle is used to supply the gas bubble.

24 . The method of claim 20 further comprising enclosing the space between the substrate and the viewing window in a casing that has an inlet and an outlet, forming a flow cell.

25 . The method of claim 20 wherein the viewing window of the flow cell is a cover slip.

26 . The method of claim 25 wherein the flow cell is coupled to one or more pumps that provides a supply of liquid media and gas bubble to the biofilm.

27 . The method of claim 26 wherein the first of one or more pumps provides the liquid media to a region between the substrate and the window, and a second pump introduces the gas bubble to the biofilm.

28 . The method of claim 27 further comprising the use of a plurality of individual flow cells.

29 . The method of claim 20 further comprising monitoring changes in topology and thickness of the biofilm in response to changes in a local environment, wherein the local environment is the structure, composition, and chemistry of the viewing window and the substrate.

30 . The method of claim 20 wherein the method is used for biomedical and environmental screening and/or to measure effects of chemicals or drugs on the biofilm.

31 . An apparatus for measuring biofilm surface properties with a microscope system comprising:
a. a multi-well plate containing a plurality of individual wells, each well having a base; b. a biofilm including one or more microorganisms grown on the base of the wells; and c. a gas bubble localized on a surface of the biofilm.

32 . The apparatus of claim 31 wherein the multi-well plate contains 1, 2, 6, 12, 24, 48, 96, 384, or 1536 individual wells.

33 . The apparatus of claim 31 wherein the base of the well is a flat and transparent material.

34 . The apparatus of claim 31 wherein the plurality of individual wells are in an inverted configuration or orientation.

35 . The apparatus of claim 34 wherein each of the plurality of inverted wells is oriented with its base at the uppermost end of the plate.

36 . The apparatus of claim 31 wherein the microscope system is a three-dimensional profile microscope system.

37 . The apparatus of claim 36 wherein the three-dimensional profile microscope system is a white light interferometer.

38 . The apparatus of claim 31 wherein the microscope system includes a stage to allow the multi-well plate to be inverted automatically.

39 . A method of measuring biofilm surface properties comprising:
a. growing a biofilm including one or more microorganisms on a base of one or more wells of a multi-well plate containing a plurality of individual wells, each well having a base; b. localizing a gas bubble on a surface of the biofilm; and c. capturing data of the biofilm using a microscope system.

40 . The method of claim 39 wherein the microscope system is a three-dimensional profile microscope system.

41 . The method of claim 40 wherein the three-dimensional profile microscope system is a white light interferometer.

42 . The method of claim 39 wherein the plurality of individual wells is inverted.

43 . The method of claim 42 wherein each of the plurality of inverted wells is oriented with its base at the upperm

Assignees

• Battelle Memorial Institute

Inventors

• Larimer Curtis J
• Suter Jonathan D
• Addleman Raymond S
• Bonheyo George T
• Brann Michelle R

Classifications

• C12M23/22

  Transparent or translucent parts (glassware for laboratory use B01L3/00) · CPC title

• C12M41/46 Primary

  of cellular or enzymatic activity or functionality, e.g. cell viability · CPC title

• C12M29/00

  Means for introduction, extraction or recirculation of materials, e.g. pumps (pumps per se F04B) · CPC title

• C12M23/12

  Well or multiwell plates (C12M25/04 takes precedence) · CPC title

• G01N33/48

  Biological material, e.g. blood, urine (G01N33/02, G01N33/26, G01N33/44, G01N33/46 take precedence); Haemocytometers (counting blood corpuscules distributed over a surface by scanning the surface G06M11/02) · CPC title