3D optical coherence tomography for analysis of biofilm architecture


Marine biofouling has serious negative effects on the environment, the economy and the energy sector. The scientific community has recognized the non-stick and antimicrobial qualities of carbon nanostructures, such as diamond, fullerenes, graphene and carbon nanotubes (CNTs).

Carbon nanotubes (CNTs)

CNTs are promising nanomaterials for various applications, particularly in the medical, environmental and industrial fields. This is due to their structural stability, high thermal conductivity and exceptional tensile strength.

CNTs have already been tested in composite materials that come into contact with salt water, creating antifouling surfaces to prevent biofouling, primarily to protect ship hulls. CNTs have also been shown to affect the structure of marine biofilms and the colonization of macrosalient organisms.

Single-walled carbon nanotubes (SWCNT) and multi-walled carbon nanotubes (MWCNT)

Single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) are two different types of CNTs. These carbon nanomaterials have a concentric cylindrical shape, with a length of several microns which can be increased up to a few millimeters and a diameter of the order of a nanometer which varies according to the number of walls.

Marine biofouling

The main effects of marine biofouling are caused by the attachment of macrofouling, such as hard-fouling calcareous animals (tube worms, mussels and barnacles) and soft-fouling species (hydradians, tunicates, anemones, sponges and non-calcareous algae).

Additionally, microfouling such as diatoms, cyanobacteria, and bacteria can inhibit adhesion and biofilm formation, slowing the progression of biofouling to later stages. A better understanding of biofilm behavior and its interaction with the environment will create effective methods to prevent biofouling and reduce its harmful effects.

Optical Coherence Tomography (OCT)

Technical advancement in the field of biofilm research has been facilitated by molecular biology techniques, biochemical processes and new imaging technologies. Unlike some time-consuming and damaging approaches to biofilm research, such as some microscopic techniques, optical coherence tomography (OCT) is an intriguing modality.

In addition to tedious sample preparation, most relevant microscopic methods used to search for biofilms require costly staining or the use of fluorochromes, which can affect local biofilm characteristics.

Also, some only provide low resolution photographs with a limited field of view (FOV). Due to its ease of use, low cost, lack of sample preparation and/or staining requirements, and ability for in situ, noninvasive, real-time imaging without modifying the structure biofilm, OCT offers several advantages over conventional microscopic approaches.

Development of new analysis parameters from 3D OCT imaging

This study aims to create new analytical criteria based on 3D OCT imaging to assess biofilm. The study is all the more relevant since OCT is an in situ, non-destructive method that can be used in various fields. This is the first investigation into how CNT-modified surfaces affect the behavior of cyanobacterial biofilms in an in vitro environment that closely resembles the hydrodynamic conditions seen in real marine habitats.

Two control surfaces and a CNT composite were used to evaluate surface antifouling effectiveness on cyanobacterial biofilm formation. CNT composites, epoxy resin and glass surfaces were evaluated for their wettability by measuring their contact angles with water.

Bruker Catalyst contact microscopes were used to conduct AFM experiments. The surface roughness of two samples of each material was evaluated using a random sample area at room temperature. SEM was used to assess surface morphology with nanometer precision.

Assessment of biofilm formation

Biofilm formation was assessed on 12-well microtiter plates stirred under parameters optimized for cyanobacterial biofilm growth to simulate the hydrodynamic conditions prevailing in marine habitats.

First, transparent double-sided tape was applied in the wells to secure the coupons. After sterilizing all coupons and plates using ultraviolet light, the sterile coupons were fixed. Every seven days, two coupons from each surface were tested for the presence of biofilm.

Cyanobacteria biofilms were photographed and examined. Each coupon underwent two-dimensional (2D) and three-dimensional (3D) imaging with a minimum of two fields of view (FOV) to ensure the accuracy and reliability of the acquired results.

Important Study Findings

A set of unique structural metrics derived from OCT imaging was constructed to measure marine biofilm structure over time and on three distinct surface materials, one known to have antifouling activity. The maturity stage of cyanobacterial biofilm was delayed by CNT-treated surfaces.

Compared to biofilms that grew on epoxy resin and glass, those that grew on composite had lower biovolume, thickness and wet weight and were less porous and smoother.

A better understanding of the process of biofilm growth in many environments, especially the marine environment, is made possible by the analysis of unique features derived from OCT imaging.


Maria J. Romeu, Marta Lima, Luciana C. Gomes, ed. D. de Jong, João Morais, Vítor Vasconcelos, Manuel FR Pereira, Olívia SGP Soares, Jelmer Sjollema and Filipe J. Mergulhão (2022) The use of 3D optical coherence tomography to analyze the architecture of cyanobacterial biofilms formed on a composite of carbon nanotubes. Polymers. https://www.mdpi.com/2073-4360/14/20/4410

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