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Title: Are those bugs reflective? Non-destructive biofilm imaging with white light interferometry

Abstract

White light interferometry (WLI) is not typically used to image bacterial biofilms that are immersed in water because there is insufficient refractive index contrast to induce reflection from the biofilm’s interface. The soft structure and water-like bulk properties of hydrated biofilms make them difficult to characterize in situ by any means, especially in a non-destructive manner. Here we describe a new method for measuring and monitoring the thickness and topology of live biofilms using a WLI microscope. A microfluidic system was used to create a reflective interface on the surface of biofilms. Live biofilm samples were monitored non-destructively over time. The method enables surface metrology measurements (roughness, surface area) and a novel approach to measuring thickness of the thin hydrated biofilms. Increase in surface roughness preceded observable increase in biofilm thickness, indicating that this measure may be used to predict future development of biofilms. We have also developed a flow cell that enables WLI biofilm imaging in a dynamic environment. We have used this flow cell to observe changes in biofilm structure in response to changes in environmental conditions - flow velocity, availability of nutrients, and presence of biocides.

Authors:
; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1339040
Report Number(s):
PNNL-SA-119531
DOE Contract Number:
AC05-76RL01830
Resource Type:
Conference
Resource Relation:
Conference: Interferometry XVIII, August 28, 2106, San Diego, California. Proceedings of SPIE, 9960:Paper No. 996004
Country of Publication:
United States
Language:
English
Subject:
White light interferometry; biofilm; bacteria; non-destructive; imaging; flow cell; topology; roughness

Citation Formats

Larimer, Curtis J., Brann, Michelle R., Suter, Jonathan D., Bonheyo, George T., and Addleman, Raymond S. Are those bugs reflective? Non-destructive biofilm imaging with white light interferometry. United States: N. p., 2016. Web. doi:10.1117/12.2239375.
Larimer, Curtis J., Brann, Michelle R., Suter, Jonathan D., Bonheyo, George T., & Addleman, Raymond S. Are those bugs reflective? Non-destructive biofilm imaging with white light interferometry. United States. doi:10.1117/12.2239375.
Larimer, Curtis J., Brann, Michelle R., Suter, Jonathan D., Bonheyo, George T., and Addleman, Raymond S. 2016. "Are those bugs reflective? Non-destructive biofilm imaging with white light interferometry". United States. doi:10.1117/12.2239375.
@article{osti_1339040,
title = {Are those bugs reflective? Non-destructive biofilm imaging with white light interferometry},
author = {Larimer, Curtis J. and Brann, Michelle R. and Suter, Jonathan D. and Bonheyo, George T. and Addleman, Raymond S.},
abstractNote = {White light interferometry (WLI) is not typically used to image bacterial biofilms that are immersed in water because there is insufficient refractive index contrast to induce reflection from the biofilm’s interface. The soft structure and water-like bulk properties of hydrated biofilms make them difficult to characterize in situ by any means, especially in a non-destructive manner. Here we describe a new method for measuring and monitoring the thickness and topology of live biofilms using a WLI microscope. A microfluidic system was used to create a reflective interface on the surface of biofilms. Live biofilm samples were monitored non-destructively over time. The method enables surface metrology measurements (roughness, surface area) and a novel approach to measuring thickness of the thin hydrated biofilms. Increase in surface roughness preceded observable increase in biofilm thickness, indicating that this measure may be used to predict future development of biofilms. We have also developed a flow cell that enables WLI biofilm imaging in a dynamic environment. We have used this flow cell to observe changes in biofilm structure in response to changes in environmental conditions - flow velocity, availability of nutrients, and presence of biocides.},
doi = {10.1117/12.2239375},
journal = {},
number = ,
volume = ,
place = {United States},
year = 2016,
month = 8
}

Conference:
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  • In microbiology research there is a strong need for next generation imaging and sensing instrumentation that will enable minimally invasive and label-free investigation of soft, hydrated structures such as in bacterial biofilms. White light interferometry (WLI) can provide high resolution images of surface topology without the use of fluorescent labels but is not typically used to image biofilms because there is insufficient refractive index contrast to induce reflection from the biofilm’s interface. The soft structure and water-like bulk properties of hydrated biofilms make them difficult to characterize in situ, especially in a non-destructive manner. In this report, we build onmore » our prior description of static biofilm imaging and describe the design of a dynamic imaging flow cell that enables monitoring the thickness and topology of live biofilms over time using a WLI microscope. The microfluidic system is specifically designed to create a reflective interface on the surface of biofilms while minimizing disruption of fragile structures. The imaging cell was also designed to accommodate limitations imposed by the depth of focus of the microscope’s objective lens. Example images of live biofilm samples are shown in order to illustrate the ability of the flow cell and WLI instrument to 1) support bacterial growth and biofilm development, 2) image biofilm structure that reflects growth in flow conditions, and 3) monitor biofilm development over time non-destructively. In future work, the apparatus described here will enable surface metrology measurements (roughness, surface area, etc.) of biofilms and may be used to observe changes in biofilm structure in response to changes in environmental conditions (e.g., flow velocity, availability of nutrients, and presence of biocides). Furthermore, this development will open new opportunities for the use of WLI in bioimaging.« less
  • We describe a generic method for using broadband and incoherent light in velocity interferometry. Single frequency lasers are no longer necessary. Compact, powerful and inexpensive light sources previously prohibited due to their incoherence are now suitable for Doppler velocimetry of remote objects through air, including arc lamps, flash lamps, light from detonations, pulsed lasers, chirped frequency lasers and lasers operated simultaneously in several lines. These powerful sources should allow practical line and areal velocimetry. In our technique, the light source is imprinted with a coherent echo having a delay matching the delay in an analyzing interferometer. The technique is genericmore » to all wave phenomena (i.e. radar, ultrasound).« less
  • There is a need for imaging and sensing instrumentation that can monitor transitions in biofilm structure in order to better understand biofilm development and emergent properties such as anti-microbial resistance. Herein, we expanded on our previously reported technique for measuring and monitoring the thickness and topology of live biofilms using white-light interferometry (WLI). A flow cell designed for WLI enabled the use of this non-disruptive imaging method for the capture of high resolution three-dimensional profile images of biofilm growth over time. The fine axial resolution (3 nm) and wide field of view (>1 mm by 1 mm) enabled detection ofmore » biofilm formation as early as three hours after inoculation of the flow cell with a live bacterial culture (Pseudomonas fluorescens). WLI imaging facilitated monitoring the early stages of biofilm development and subtle variations in the structure of mature biofilms. Minimally-invasive imaging enabled monitoring of biofilm structure with surface metrology metrics (e.g., surface roughness). The system was used to observe a transition in biofilm structure that occurred in response to expsoure to a common antiseptic. In the future, WLI and the biofilm imaging cell described herein may be used to test the effectiveness of biofilm-specific therapies to combat common diseases associated with biofilm formation such as cystic fibrosis and periodontitis.« less
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  • An imaging white light velocimeter consisting of two imagine superimposing Michelson interferometers in series with the target interposed is demonstrated. Interferometrically measured 2-D velocity maps can be made of moving surfaces using unlimited bandwidth incoherent and extended area sources. Short pulse and broadband chirped pulse lasers can be used to provide temporal resolution not possible with monochromatic illumination. A 20 m/s per fringe imaging velociemter is demonstrated using an ordinary camera flash for illumination.