The authors thank Aqua-Aerobic Systems, Inc. (Rockford, IL, USA) for providing the granular biofilms studied in this work. The authors also acknowledge the National Science Foundation&#39;s support via Award #210047 and #193729.

1. System setup
<ol>
	<li>Gather the system components which include the commercial OCT system (base unit, stand, imaging head, and computer), waveform generator, transducer, delay/pulse generator, a switch with BNC connections, BNC cables and adapters, optical posts, and clamps.</li>
	<li>Connect the sync signal from the function generator to a switch. Connect the other port of the switch to the delay generator.</li>
	<li>Connect the output of the function generator to the transducer leads.</li>
	<li>Connect the outputs of the delay generator to the trigger channel at the back of the OCT base unit. The output signal from the delay generator is a trigger pulse to initiate the motion of the scanning optics in the OCT system.</li>
	<li>Turn on the system components (OCT base unit, computer, function generator, and delay generator) and launch the OCT software.</li>
	<li>Configure the delay generator to send a transistor-transistor logic trigger signal to the OCT base unit. Refer to the OCT system manual for the trigger signal requirements.</li>
	<li>Position the transducer beneath the OCT lens. The transducer has a 3D-printed wedge tip glued onto one of its ends that serves as a line source for elastic waves.</li>
</ol>
2. Image acquisition
<ol>
	<li>On the OCT software, select the Doppler Acquisition Mode and enable the external trigger.</li>
	<li>Place the granular biofilm under the lens in a sample holder and move it towards the tip of the transducer using a translation stage. Ensure that the transducer makes gentle contact with the sample surface, as shown in Figure 2. We used two granular biofilms (also known as granular sludge) with different nominal diameters (4.3 mm and 3.3 mm). This selection was made to investigate the impact of biofilm size on its mechanical properties. These were commercially obtained. 
	NOTE: The sample holder employed in this study consists of a 3D-printed plastic plate with multiple hemispherical indentations. This holder does not allow for measurements under native conditions. Therefore, we introduced water from the natural environment during measurements to prevent the sample from drying.</li>
	<li>Specify the scan region by clicking the Start and Endpoints of the Line of Interest (wave propagation path) in the sample monitor window. Center this line with respect to the transducer tip and ensure it is perpendicular to the edge of the tip.</li>
	<li>Specify the number of pixels along the scan region and the depth of the sample and increase the number of B-scans (2D cross-sectional images) to be recorded to improve the signal-to-noise ratio of the OCE images. The presented results were obtained using 1523 pixels along the scanning path and 1024 pixels along the depth. A total of 50 B-scans were taken.</li>
	<li>Click the Scan button and turn ON the switch. The OCT and OCE images should appear on the screen. Activate the switch within the trigger timeout time and the scan preparation time.</li>
	<li>Ensure that the reference intensity is within the optimum range and position the sample within the focal region of the OCT microscope objective. A properly focused sample should have its top edge close to the top of the image.</li>
	<li>Adjust the phase contour in the OCE image on the display toolbar by increasing the higher value of the left-hand side color bar and decreasing the lower value of the right-hand side color bar. This will increase the fringe contrast.</li>
	<li>Configure the function generator to produce a single-frequency sinusoidal voltage by pressing the Sine button in the front panel and specify the starting excitation frequency for the measurements. The measurements in this study start at 4 kHz and end at 9.6 kHz.&#160;Enable the Output connector by pressing the Output key.</li>
	<li>Set an acceptable voltage for the measurement. This value should maximize fringe visibility but also avoid phase wrapping. For the biofilms in this study and the frequency range of the measurements, a voltage between 5 and 10 V typically results in a phase map with good contrast.</li>
	<li>Acquire the OCT and OCE images by clicking the Record button.</li>
	<li>Repeat the measurements at different frequencies to obtain cross-sectional images of the elastic wave field with different wavelengths (or fringe periods).</li>
</ol>
3. Image Analysis
<ol>
	<li>Obtain the physical size of the pixels. The physical pixel size in x is obtained by dividing the field of view in the x direction by the image size in the x direction and then multiplying by a factor of two.&#160;The physical pixel size in z is obtained by dividing the field of view in the z direction by the image size in the z direction. The field of view and image size values are stored in the structure array with the image information which can be accessed with the OCTFileOpen function provided in the MATLAB SDK in the ThorImageOCT package.</li>
	<li>Obtain the OCT and OCE matrices using the OCTFileGetIntensity and OCTFileGetPhase functions, respectively, and take the average of the recorded frames. These functions are provided in the MATLAB SDK in the ThorImageOCT package.</li>
	<li>Obtain the pixel locations of the top edge of the sample by binarizing the image and detecting the white pixels from top to bottom for each column.</li>
	<li>Extract the phase distribution of the OCE image along this edge using the improfile function and compute the cumulative arc length in real dimensions. Compute the arc length by taking the cumulative sum of the norm of scaled differences between consecutive points in the x and z directions.</li>
	<li>Compute the spatial fast Fourier transform of the measured OCT phase distribution (i.e., from the OCE images) with respect to the cumulative arc length using the plomb function.</li>
	<li>Determine the location of the peak in the spectrum. This location represents the spatial frequency of the wave. Calculate the wave speed (or phase velocity) from the ratio of the transducer excitation frequency (units of Hz) and the spatial frequency (unit of inverse length).</li>
</ol>

Rapid and Non-Destructive Characterization of Biofilm Elastic Properties

<ol>
	<li>Mahto, K. U., Das, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+biofilm+and+extracellular+polymeric+substances+in+the+moving+bed+biofilm+reactor+for+wastewater+treatment:+A+review.">Bacterial biofilm and extracellular polymeric substances in the moving bed biofilm reactor for wastewater treatment: A review.</a> Bioresour Technol. 345, 126476 (2022).</li><li>Pholchan, M. K., Baptista, J. d. e. C., Davenport, R. J., Curtis, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+study+of+the+effect+of+operating+variables+on+reactor+performance+and+microbial+diversity+in+laboratory-scale+activated+sludge+reactors.">Systematic study of the effect of operating variables on reactor performance and microbial diversity in laboratory-scale activated sludge reactors.</a> Water Res. 44 (5), 1341-1352 (2010).</li><li>Briones, A., Raskin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+and+dynamics+of+microbial+communities+in+engineered+environments+and+their+implications+for+process+stability.">Diversity and dynamics of microbial communities in engineered environments and their implications for process stability.</a> Curr Opin in Biotechnol. 14 (3), 270-276 (2003).</li><li>Sanchez-Huerta, C., Sanchez Medina, J., Wang, C., Fortunato, L., Hong, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+role+of+sorption+and+biodegradation+in+the+removal+of+organic+micropollutants+by+membrane+aerated+biofilm+reactor+(MABR)+with+different+biofilm+thickness.">Understanding the role of sorption and biodegradation in the removal of organic micropollutants by membrane aerated biofilm reactor (MABR) with different biofilm thickness.</a> Water Res. 236, 119935 (2023).</li><li>Wang, H. F., Hu, H., Yang, H. Y., Zeng, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+anaerobic+granular+sludge+using+a+rheological+approach.">Characterization of anaerobic granular sludge using a rheological approach.</a> Water Res. 106, 116-125 (2016).</li><li>Ma, Y. J., Xia, C. W., Yang, H. Y., Zeng, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rheological+approach+to+analyze+aerobic+granular+sludge.">A rheological approach to analyze aerobic granular sludge.</a> Water Res. 50, 171-178 (2014).</li><li>Lin, X., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microstructure+of+anammox+granules+and+mechanisms+endowing+their+intensity+revealed+by+microscopic+inspection+and+rheometry.">Microstructure of anammox granules and mechanisms endowing their intensity revealed by microscopic inspection and rheometry.</a> Water Res. 120, 22-31 (2017).</li><li>Liou, H. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+mechanical+characterization+of+granular+biofilms+by+optical+coherence+elastography+measurements+of+circumferential+elastic+waves.">Towards mechanical characterization of granular biofilms by optical coherence elastography measurements of circumferential elastic waves.</a> Soft Matter. 15 (28), 5562-5573 (2019).</li><li>Liou, H. C., Sabba, F., Wang, Z., Wells, G., Balogun, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Layered+viscoelastic+properties+of+granular+biofilms.">Layered viscoelastic properties of granular biofilms.</a> Water Res. 202, 117394 (2021).</li><li>Li, C., Guan, G., Reif, R., Huang, Z., Wang, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+elastic+properties+of+skin+by+measuring+surface+waves+from+an+impulse+mechanical+stimulus+using+phase-sensitive+optical+coherence+tomography.">Determining elastic properties of skin by measuring surface waves from an impulse mechanical stimulus using phase-sensitive optical coherence tomography.</a> J R Soc Interface. 9, 831-841 (2012).</li><li>Ramier, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+measurement+of+shear+modulus+of+the+human+cornea+using+optical+coherence+elastography.">In vivo measurement of shear modulus of the human cornea using optical coherence elastography.</a> Sci Rep. 10, 17366 (2020).</li><li>Ramier, A., Tavakol, B., Yin, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+mechanical+wave+speed,+dispersion,+and+viscoelastic+modulus+of+cornea+using+optical+coherence+elastography.">Measuring mechanical wave speed, dispersion, and viscoelastic modulus of cornea using optical coherence elastography.</a> Optics Express. 27 (12), 16635 (2019).</li><li>Crespo, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+determination+of+human+corneal+elastic+modulus+using+vibrational+optical+coherence+tomography.">In vivo determination of human corneal elastic modulus using vibrational optical coherence tomography.</a> Cornea Ext Dis. 11 (7), 1-11 (2022).</li><li>Ambrozinski, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acoustic+micro-tapping+for+non-contact+4D+imaging+of+tissue+elasticity.">Acoustic micro-tapping for non-contact 4D imaging of tissue elasticity.</a> Sci Rep. 6 (38967), 1-11 (2016).</li><li>Pitre, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nearly-incompressible+transverse+isotropy+(NITI)+of+cornea+elasticity:+model+and+experiments+with+acoustic+micro-tapping+OCE.">Nearly-incompressible transverse isotropy (NITI) of cornea elasticity: model and experiments with acoustic micro-tapping OCE.</a> Sci Rep. 10 (12983), 1-14 (2020).</li><li>Lan, G., Aglyamov, S. R., Larin, K. V., Twa, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+human+corneal+shear+wave+optical+coherence+elastography.">In vivo human corneal shear wave optical coherence elastography.</a> Optom Vis Sci. 98, 58-63 (2021).</li><li>Rosenthal, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+analysis+of+pore+size+and+connectivity+in+a+thick+mixed-cultured+biofilm.">Morphological analysis of pore size and connectivity in a thick mixed-cultured biofilm.</a> Biotechnol Bioeng. 115, 2268-2279 (2018).</li><li>Liou, H. C., Sabba, F. I., Packman, A., Wells, G., Balogun, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nondestructive+characterization+of+soft+materials+and+biofilms+by+measurement+of+guided+elastic+wave+propagation+using+optical+coherence+elastography.">Nondestructive characterization of soft materials and biofilms by measurement of guided elastic wave propagation using optical coherence elastography.</a> Soft Matter. 15, 575-586 (2019).</li><li>Wagner, M., Taherzadeh, D., Haisch, C., Horn, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+the+mesoscale+structure+and+volumetric+features+of+biofilms+using+optical+coherence+tomography.">Investigation of the mesoscale structure and volumetric features of biofilms using optical coherence tomography.</a> Biotechnol Bioeng. 107 (5), 844-853 (2010).</li><li>Leite-Andrade, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+by+optical+coherence+tomography+for+elucidating+biofilm+formation+by+emergent+Candida+species.">A new approach by optical coherence tomography for elucidating biofilm formation by emergent Candida species.</a> PLoS ONE. 12 (11), e0188020 (2017).</li><li>Blauert, F., Horn, H., Wagner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-resolved+biofilm+deformation+measurements+using+optical+coherence+tomography.">Time-resolved biofilm deformation measurements using optical coherence tomography.</a> Biotechnol Bioeng. 112 (9), 1893-1905 (2015).</li><li>Picioreanu, C., Blauert, F., Horn, H., Wagner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+mechanical+properties+of+biofilms+by+modeling+the+deformation+measured+using+optical+coherence+tomography.">Determination of mechanical properties of biofilms by modeling the deformation measured using optical coherence tomography.</a> Water Res. 145, 588-598 (2018).</li><li>Li, M., Nahum, Y., Matou&#353;, K., Stoodley, P., Nerenberg, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+biofilm+heterogeneity+on+the+apparent+mechanical+properties+obtained+by+shear+rheometry.">Effects of biofilm heterogeneity on the apparent mechanical properties obtained by shear rheometry.</a> Biotechnol Bioeng. 120, 553-561 (2023).</li><li>Karimi, A., Karig, D., Kumar, A., Ardekani, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+of+physical+mechanisms+and+biofilm+processes:+review+of+microfluidic+methods.">Interplay of physical mechanisms and biofilm processes: review of microfluidic methods.</a> Lab Chip. 15 (1), 23-42 (2015).</li><li>Geisel, S., Secchi, E., Vermant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+challenges+in+determining+the+rheological+properties+of+bacterial+biofilms.">Experimental challenges in determining the rheological properties of bacterial biofilms.</a> Interface Focus. 12 (6), 20220032 (2022).</li><li>Winkler, M. K. H., van Loosdrecht, M. C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intensifying+existing+urban+wastewater.">Intensifying existing urban wastewater.</a> Science. 375 (6579), 377-378 (2022).</li><li>Graff, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Wave motion in elastic solids. , (1991).</li><li>Kennedy, B. F., Kennedy, K. M., Sampson, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+optical+coherence+elastography:+Fundamentals,+techniques+and+prospects.">A review of optical coherence elastography: Fundamentals, techniques and prospects.</a> IEEE J. Sel. Top. Quantum Electron. 20 (2), 272-288 (2014).</li><li>Ang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anterior+segment+optical+coherence+tomography.">Anterior segment optical coherence tomography.</a> Prog Retin Eye Res. 66, 132-156 (2018).</li><li>Kirby, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+resolution+in+dynamic+optical+coherence+elastography.">Spatial resolution in dynamic optical coherence elastography.</a> J Biomed Opt. 24 (9), 1-16 (2019).</li><li>Larin, K. V., Sampson, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+coherence+elastography+-+OCT+at+work+in+tissue+biomechanics.">Optical coherence elastography - OCT at work in tissue biomechanics.</a> Biomed Opt Express. 8, 1172-1202 (2017).</li><li>Muhammad, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+species+sorting+and+immigration+on+the+bacterial+assembly+of+different-sized+aggregates+in+a+full-scale+aerobic+granular+sludge.">Importance of species sorting and immigration on the bacterial assembly of different-sized aggregates in a full-scale aerobic granular sludge.</a> Environ Sci Technol. 53 (14), 8291-8301 (2019).</li><li>Candry, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tailoring+polyvinyl+alcohol-sodium+alginate+(PVA-SA)+hydrogel+beads+by+controlling+crosslinking+pH+and+time.">Tailoring polyvinyl alcohol-sodium alginate (PVA-SA) hydrogel beads by controlling crosslinking pH and time.</a> Sci Rep. 12 (1), 20822 (2022).</li><li>Kirby, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+coherence+elastography+in+ophthalmology.">Optical coherence elastography in ophthalmology.</a> J Biomed Opt. 22 (12), 1-28 (2017).</li><li>Liang, X., Boppart, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanical+properties+of+in+vivo+human+skin+from+dynamic+optical+coherence+elastography.">Biomechanical properties of in vivo human skin from dynamic optical coherence elastography.</a> IEEE Trans Biomed Eng. 57 (4), 953-959 (2010).</li></ol>

The authors declare no conflicts of interest.

The attainable imaging depth in the OCT system is determined by the degree of light penetration from the light source, which depends on the wavelength of the source. Moreover, the wavelength determines the axial resolution. Longer wavelengths can penetrate more deeply into the sample but at the expense of reduced axial resolution compared to shorter wavelengths. Transverse&#160;resolution, on the other hand, is dependent on both the numerical aperture of the system and the wavelength, with shorter wavelengths delivering ...

In wastewater treatment and water resource recovery, beneficial biofilms in attached growth reactors are increasingly employed to enable microbes to convert undesirable pollutants, such as organic matter, nitrogen, and phosphate, into stabilized forms that can be easily removed from the water1. In these systems, the biofilm&#39;s emergent function, namely biochemical transformations, is closely associated with the diversity of microbes residing in it and the nutrients these microbes receive2. Accordingly, ongoing biofilm growth can pose a challenge to maintaining consistent reactor functionality because the new biofilm growth may alter the biofilm&#39;s overall metabolic processes, mass transfer characteristics, and community composition. Stabilizing the biofilm environment as much as possible can protect against such changes3. This includes ensuring a consistent flow of nutrients and keeping the structure of the biofilm stable with a steady thickness4. Monitoring the biofilm&#39;s stiffness and physical structure would enable researchers to gain insight into the overall health and functioning of the biofilm.
Biofilms exhibit viscoelastic properties5,6,7. This viscoelastic nature results in a combination of an instantaneous and slow, time-dependent deformation in response to external mechanical forces. One unique aspect of biofilms is that, when they are subjected to substantial deformation, they respond like viscous liquids. Conversely, when subjected to minor deformation, their response is comparable to solids5.&#160;Moreover, within this small-deformation region, there is a deformation range under which biofilms exhibit a linear force-displacement relationship5,6,7. Deformations within this linear range are optimal for assessing biofilm mechanical characteristics because these yield reproducible measurements. Several techniques can quantify the elastic response within this range. Optical coherence elastography (OCE) is an emerging technique that is being adapted for analyzing biofilms in this linear range (strains on the order of 10-4-10-5)8,9.
OCE&#39;s most established application so far is in the biomedical field, where the technique has been applied to characterize biological tissues that only require superficial optical access.&#160;For example, Li et al. used OCE to characterize the elastic properties of skin tissue10. Other authors characterized the anisotropic elastic properties of porcine and human corneal tissues and how they are affected by intraocular pressure11,12,13,14,15,16. Some advantages of the OCE method for studying biofilms are that it is non-destructive and provides mesoscale spatial resolution, it does not require any sample preparation, and the method itself is rapid; it provides co-registered measurements of physical structure&#160;and elastic properties (e.g., porosity, surface roughness, and morphology)8,9,17,18.
The OCE method measures the local displacement of propagating elastic waves in a specimen using phase-sensitive optical coherence tomography (OCT). OCT is a low-coherence optical interferometer that transforms local changes in the sample displacement into an intensity change that is recorded with an optical spectrometer. The OCT technique has also been utilized in biofilm research for the characterization of mesoscale structure, porosity distribution in three dimensions, and biofilm deformation17,19,20,21. In addition, Picioreanu et al. estimated biofilm mechanical properties using fluid-structure interaction inverse modeling of OCT cross-sectional deformation images22.
On the other hand, OCE measurements, coupled with inverse elastodynamic wave modeling, yield the wave speed of elastic waves in the sample, which enables the characterization of the elastic and viscoelastic properties of the sample. Our group adapted the OCE technique for quantitative measurement of biofilm elastic and viscoelastic properties8,9,18 and validated the technique against shear rheometry measurements in agarose gel plate samples18. The OCE approach provides precise and reliable estimates of the biofilm properties since the measured elastic wave speed is correlated with the elastic properties of the sample. Furthermore, the spatial decay of the elastic wave amplitude can be directly correlated with the viscoelastic properties due to viscous effects in the material. We have reported OCE measurements of viscoelastic properties of mixed culture bacterial biofilms grown on coupons in a rotating annular reactor (RAR) and granular biofilms with complex geometries using elastodynamic wave models18.
The OCE technique is also a powerful alternative to traditional rheometry18which is used for viscoelastic characterization. Rheometry methods are best suited for samples with planar geometry. As such, granular biofilms, which have arbitrary shapes and surface morphologies, cannot be accurately characterized on a rheometer8,23. In addition, unlike OCE, rheometry methods may be challenging to adapt for real-time measurements, for example, during biofilm growth in flow cells24,25.
In this paper, we show that OCE measurements of the frequency-independent wave speed of surface waves can be used to characterize the biofilm elastic properties without the need for complicated models. This development will make the OCE approach more accessible to the broader biofilm community for studying the biofilm mechanical properties.
Figure 1 shows a schematic illustration of the OCT system used in this study. The system incorporates several instruments, including a commercial spectral-domain phase-sensitive OCT system, a delay generator, a function generator, and a piezoelectric transducer. The OCT system operates on the principle of interferometry by employing a broadband light source with a center wavelength of 930 nm. The collected light intensity, which is correlated with intricate structural details in the sample, is analyzed in the postprocessing unit and then converted to a cross-sectional image of the sample - commonly referred to as an OCT image. The OCT imaging depth depends on the severity of the optical scattering in the sample that stems from local variation in the refractive index and is limited to 1- 3 mm in biological tissues and biofilms. Since the optical phase in the sample and the interference intensity are modulated by motion, the OCT can be used to detect the local sample displacement. We leverage the displacement sensitivity of the OCT in the OCE method to track the steady state displacement field of elastic waves in the sample. Specifically, the function generator outputs a sinusoidal voltage to drive the piezoelectric transducer. The transducer, in turn, stretches and contracts with an oscillatory time history. The oscillatory displacement of the transducer imparts a sinusoidal force on the sample surface through a 3D-printed wedge tip at the apex of the transducer, leading to the generation of harmonic elastic waves in the sample. The wedge tip makes light contact with the sample, such that the sample remains intact after the actuator is retracted from the sample surface. To record the local displacement in the sample, adjacent depth scans separated by a fixed time delay are acquired at each pixel in the sample. The optical phase difference between consecutive scans at each pixel point is proportional to the local vertical displacement at the same point. Synchronization between the displacement of the transducer and the scanning optics in the OCT system is achieved through a trigger pulse that originates from the function generator and is delayed in the delay generator. This synchronization step facilitates the acquisition of consistent cross-sectional images of the local optical phase distribution in the sample. These images are directly proportional to the local vertical harmonic displacement in the sample and are known as the OCE image. OCE images are acquired at different transducer actuation frequencies to obtain the elastic wavelength and wave speed as a function of frequency. The wave speeds measured are analyzed with an elastodynamic model to determine the elastic properties of the sample.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3D printed sample holder</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3D printed wedge tip</td>
			<td></td>
			<td></td>
			<td>3 mm width</td>
		</tr>
		<tr>
			<td>BNC cables</td>
			<td>Any brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Delay generator</td>
			<td>Stanford Research Systems</td>
			<td>DG535</td>
			<td>DG535 Digital delay/ Pulse Generator&#160;</td>
		</tr>
		<tr>
			<td>Function generator</td>
			<td>Agilent Technologies</td>
			<td></td>
			<td>33250A 80 MHz Function / Arbitrary Waveform Generator</td>
		</tr>
		<tr>
			<td>Granular biofilm</td>
			<td>Aqua-Aerobic Systems</td>
			<td></td>
			<td>Obtained from an Aerobic Granular Sludge reactor (Aqua-Aerobic Systems, Inc.)</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>MathWorks</td>
			<td></td>
			<td>Release 2022a (MATLAB 9.12)</td>
		</tr>
		<tr>
			<td>Piezoelectric transducer</td>
			<td>Thorlabs</td>
			<td>PK2JUP1</td>
			<td>Discrete Piezo Stack, 75 V, 30.0 &#181;m Displacement</td>
		</tr>
		<tr>
			<td>SD-OCT System</td>
			<td>Thorlabs</td>
			<td></td>
			<td>Ganymede II, LSM03 scan lens</td>
		</tr>
		<tr>
			<td>ThorImageOCT</td>
			<td>Thorlabs</td>
			<td></td>
			<td>Version: 5.5.5</td>
		</tr>
	</tbody>
</table>

quantifying elastic properties of environmental biofilms using optical coherence elastography

Biofilms are complex biomaterials comprising a well-organized network of microbial cells encased in self-produced extracellular polymeric substances (EPS). This paper presents a detailed account of the implementation of optical coherence elastography (OCE) measurements tailored for the elastic characterization of biofilms. OCE is a non-destructive optical technique that enables the local mapping of the microstructure, morphology, and viscoelastic properties of partially transparent soft materials with high spatial and temporal resolution. We provide a comprehensive guide detailing the essential procedures for the correct implementation of this technique, along with a methodology to estimate the bulk Young's modulus of granular biofilms from the collected measurements. These consist of the system setup, data acquisition, and postprocessing. In the discussion, we delve into the underlying physics of the sensors used in OCE and explore the fundamental limitations regarding the spatial and temporal scales of OCE measurements. We conclude with potential future directions for advancing the OCE technique to facilitate elastic measurements of environmental biofilms.

In this study, we used granular biofilms (also known as granular sludge), which were commercially obtained. Granules are spherical biofilms that form through self-aggregation, meaning that they do not require a carrier or surface on which to grow26. Figure 3A shows a representative cross-sectional OCT image that arises due to the spatial variation of the local refractive index in a granular biofilm. The biofilm has a nominal diameter of 3 mm. Some of ...

Watch this Scientific Journal Video about Quantifying Elastic Properties of Environmental Biofilms using Optical Coherence Elastography at JoVE.com

Author Spotlight: Characterizing Environmental Biofilm Mechanics Using Optical Coherence Elastography and its Applications in Wastewater Treatment

This paper highlights the optical coherence elastography (OCE) technique's efficacy in rapidly and non-destructively characterizing biofilm elastic properties. We elucidate critical OCE implementation procedures for accurate measurements and present Young's modulus values for two granular biofilms.

Quantifying Elastic Properties of Environmental Biofilms using Optical Coherence Elastography

Biofilms are complex biomaterials comprising a well-organized network of microbial cells encased in self-produced extracellular polymeric substances ...

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Research

JoVE Journal

Environment

876 Views.  Northwestern University. This paper highlights the optical coherence elastography (OCE) technique's efficacy in rapidly and non-destructively characterizing biofilm elastic properties. We elucidate critical OCE implementation procedures for accurate measurements and present Young's modulus values for two granular biofilms.

Video: Author Spotlight: Characterizing Environmental Biofilm Mechanics Using Optical Coherence Elastography and its Applications in Wastewater Treatment

Waste Water Derived Electroactive Microbial Biofilms: Growth, Maintenance, and Basic Characterization

The growth of anodic electroactive microbial biofilms from waste water inocula in a fed-batch reactor is demonstrated using a three-electrode setup controlled by a potentiostat. Thereby the use of potentiostats allows an exact adjustment of the electrode potential and ensures reproducible microbial culturing conditions. During growth the current production is monitored using chronoamperometry (CA). Based on these data the maximum current density (jmax) and the coulombic efficiency (CE) are discussed as measures for characterization of the bioelectrocatalytic performance. Cyclic voltammetry (CV), a nondestructive, i.e. noninvasive, method, is used to study the extracellular electron transfer (EET) of electroactive bacteria. CV measurements are performed on anodic biofilm electrodes in the presence of the microbial substrate, i.e. turnover conditions, and in the absence of the substrate, i.e. nonturnover conditions, using different scan rates. Subsequently, data analysis is exemplified and fundamental thermodynamic parameters of the microbial EET are derived and explained: peak potential (Ep), peak current density (jp), formal potential (Ef) and peak separation (&#916;Ep). Additionally the limits of the method and the state-of the art data analysis are addressed. Thereby this video-article shall provide a guide for the basic experimental steps and the fundamental data analysis.

Chronoamperometric growth of anodic electroactive microbial biofilms in a fed-batch reactor using a three-electrode setup, controlled by a potentiostat, is demonstrated. The extracellular electron transfer is characterized using cyclic voltammetry in the presence of the electron donor and in the absence of the electron donor. Fundamental data analysis is demonstrated.

The growth of anodic electroactive microbial biofilms from waste water inocula in a fed-batch reactor is demonstrated using a three-electrode setup ...

The Benthic Exchange of O2, N2 and Dissolved Nutrients Using Small Core Incubations

The measurement of sediment-water exchange of gases and solutes in aquatic sediments provides data valuable for understanding the role of sediments in nutrient and gas cycles. After cores with intact sediment-water interfaces are collected, they are submerged in incubation tanks and kept under aerobic conditions at in situ temperatures. To initiate a time course of overlying water chemistry, cores are sealed without bubbles using a top cap with a suspended stirrer. Time courses of 4-7 sample points are used to determine the rate of sediment water exchange. Artificial illumination simulates day-time conditions for shallow photosynthetic sediments, and in conjunction with dark incubations can provide net exchanges on a daily basis. The net measurement of N2 is made possible by sampling a time course of dissolved gas concentrations, with high precision mass spectrometric analysis of N2:Ar ratios providing a means to measure N2 concentrations. We have successfully applied this approach to lakes, reservoirs, estuaries, wetlands and storm water ponds, and with care, this approach provides valuable information on biogeochemical balances in aquatic ecosystems.

The Benthic Exchange of O2, N2 and Dissolved Nutrients Using Small Core Incubations

Here, we present small core incubations for the measurement of sediment-water gas and solute exchange. These will provide reliable measurements of sediment-water exchange that assess the role of sediment in influencing biological and biogeochemical processes in aquatic ecosystems.

The measurement of sediment-water exchange of gases and solutes in aquatic sediments provides data valuable for understanding the role of sediments in ...

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic pathway of dimethylarsinic acid (DMAV) thiolation, have been recently found in the environment as well as human organs. DMMTAV and DMDTAV can be quantified to determine the ecological effects of dimethylated thioarsenicals and their stability in environmental media. The synthesis method for these compounds is unstandardized, making replicating previous studies challenging. Furthermore, there is a lack of information about storage techniques, including storage of compounds without species transformation. Moreover, because only limited information about synthesis methods is available, there may be experimental difficulties in synthesizing standard chemicals and performing quantitative analysis. The protocol presented herein provides a practically modified synthesis method for the dimethylated thioarsenicals, DMMTAV and DMDTAV, and will help in the quantification of species separation analysis using high performance liquid chromatography in conjunction with inductively coupled plasma mass spectrometry (HPLC-ICP-MS). The experimental steps of this procedure were modified by focusing on the preparation of chemical reagents, filtration methods, and storage.

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

This article presents modified experimental protocols for dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV) synthesis, inducing dimethylarsinic acid (DMAV) thiolation through mixing of DMAV, Na2S, and H2SO4. The modified protocol provides an experimental guideline, thereby overcoming limitations of the synthesis steps that could have caused experimental failures in quantitative analysis.

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic ...

Development of New Methods for Quantifying Fish Density Using Underwater Stereo-video Tools

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and estimating fish abundance. We developed and implemented a rotating stereo-video camera tool that covers a full 360 degrees of sampling, which maximizes sampling effort compared to stationary camera tools. A variety of studies have detailed the ability of static, stereo-camera systems to obtain highly accurate and precise measurements of fish; the focus here was on the development of methodological approaches to quantify fish density using rotating camera systems. The first approach was to develop a modification of the metric MaxN, which typically is a conservative count of the minimum number of fish observed on a given camera survey. We redefine MaxN to be the maximum number of fish observed in any given rotation of the camera system. When precautions are taken to avoid double counting, this method for MaxN may more accurately reflect true abundance than that obtained from a fixed camera. Secondly, because stereo-video allows fish to be mapped in three-dimensional space, precise estimates of the distance-from-camera can be obtained for each fish. By using the 95% percentile of the observed distance from camera to establish species-specific areas surveyed, we account for differences in detectability among species while avoiding diluting density estimates by using the maximum distance a species was observed. Accounting for this range of detectability is critical to accurately estimate fish abundances. This methodology will facilitate the integration of rotating stereo-video tools in both applied science and management contexts.

We describe a new method for counting fishes, and estimating relative abundance (MaxN) and fish density using rotating stereo-video camera systems. We also demonstrate how to use distance from camera (Z distance) to estimate species-specific detectability.

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and ...

Characterization of Aquatic Biofilms with Flow Cytometry

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond quickly to environmental changes and can be thus used as indicators of water quality. Currently, biofilm assessment is mostly based on integrative and functional endpoints, such as photosynthetic or respiratory activity, which do not provide information on the biofilm community structure. Flow cytometry and computational visualization offer an alternative, sensitive, and easy-to-use method for assessment of the community composition, particularly of the photoautotrophic part of freshwater biofilms. It requires only basic sample preparation, after which the entire sample is run through the flow cytometer. The single-cell optical and fluorescent information is used for computational visualization and biological interpretation. Its main advantages over other methods are the speed of analysis and the high-information-content nature. Flow cytometry provides information on several cellular and biofilm traits in a single measurement: particle size, density, pigment content, abiotic content in the biofilm, and coarse taxonomic information. However, it does not provide information on biofilm composition on the species level. We see high potential in the use of the method for environmental monitoring of aquatic ecosystems and as an initial biofilm evaluation step that informs downstream detailed investigations by complementary and more detailed methods.

Flow cytometry in combination with visual clustering offers an easy-to-use and fast method for studying aquatic biofilms. It can be used for biofilm characterization, detection of changes in biofilm community structure, and detection of abiotic particles embedded in the biofilm.

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond ...

Composition and Distribution Analysis of Bioaerosols Under Different Environmental Conditions

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, we described a protocol for multiple analyses of the biological compositions in environmental PM. Five experiments are presented: (1) PM number monitoring by using a laser particle counter; (2) PM collection by using a cyclonic aerosol sampler; (3) PM collection by using a high-volume air sampler with filters; (4) culturable microbes collection by the Andersen six-stage sampler; and (5) detection of biological composition of environmental PM by bacterial 16SrDNA and fungal ITS region sequencing. We selected hazy days and a livestock farm as two typical examples of the application in this protocol. In this study, these two sampling methods, cyclonic aerosol sampler and filter sampler, showed different sampling efficiency. The cyclonic aerosol sampler performed much better in terms of collecting bacteria, while these two methods showed the same efficiency in collecting fungi. Filter samplers can work under low temperature conditions while cyclonic aerosol samplers have a sampling limitation for temperature. A solid impacting sampler, such as an Andersen six-stage sampler, can be used to sample bioaerosols directly into the culture medium, which increases the survival rate of culturable microorganisms. However, this method mainly relies on culture while more than 99% of microbes cannot be cultured. DNA extracted from the culturable bacteria collected by the Andersen six-stage sampler and samples collected by cyclonic aerosol sampler and filter sampler were detected by bacterial 16S rDNA and fungal ITS region sequencing.All the methods above may have wide application in many fields of study, such as environmental monitoring and airborne pathogen detection. From these results, we can conclude that these methods can be used under different conditions and may help other researchers further explore the health impacts of environmental bioaerosols.

Understanding the biological composition of environmental particulate matter is important for the study of its significant impacts on human health and disease spread. Here, we used three types of bioaerosol sampling methods and a biological analysis of airborne microbes to better explore airborne microbial communities under different environmental conditions.

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, ...

Measurement of Aerosols Optical Thickness of the Atmosphere using the GLOBE Handheld Sun Photometer

Here, we describe the measurement of aerosol optical thickness using the GLOBE handheld sun photometer. Aerosol optical thickness (AOT) was measured at Xavier University of Louisiana (XULA, 29.96&#176; N, 90.11&#176; W and 3 m above sea level). The measurements were done at two different wavelengths, 505 nm and 625 nm. AOT measurements were done 6 times a day (7 AM, 9 AM, 11 AM, solar noon, 3 PM and 5 PM). The data shown in this paper are the monthly average AOT values taken at solar noon. During each measurement time; at least five values of the sunlight voltage V and the dark voltage Vdark are taken for each channel. The mean for these five measurements is taken as the average for that measurement time. Other meteorological data such as temperature, surface pressure, rainfall and relative humidity are also measured at the same time. The whole protocol is completed within a time span of 10&#8211;15 min. The measured AOT values at 505 nm and 625 nm are then used to extrapolate the AOT values for wavelengths 667 nm, 551 nm, 532 nm and 490 nm. The measured and extrapolated AOT values were then compared with values from the nearest AERONET station at Wave CIS site 6 (AERONET, 28.87&#176; N, 90.48&#176; W and 33 m above sea level), which is about 96 km south of XULA. In this study we tracked the annual and daily variations of AOT for a 12 month period from September 2017 to August 2018. We also compared AOT data from two independently calibrated GLOBE handheld sun photometers at the XULA site. The data show that the two instruments are in excellent agreement.

The goal of the methods presented here is to measure aerosol optical thickness of the atmosphere. The sun photometer is pointed at the sun and the largest voltage reading obtained on an in-built digital voltmeter is recorded. Atmospheric measurements such as barometric pressure and relative humidity are also performed.

Here, we describe the measurement of aerosol optical thickness using the GLOBE handheld sun photometer. Aerosol optical thickness (AOT) was measured at ...

Automated 3D Optical Coherence Tomography to Elucidate Biofilm Morphogenesis Over Large Spatial Scales

Biofilms are a most successful microbial lifestyle and prevail in a multitude of environmental and engineered settings. Understanding biofilm morphogenesis, that is the structural diversification of biofilms during community assembly, represents a remarkable challenge across spatial and temporal scales. Here, we present an automated biofilm imaging system based on optical coherence tomography (OCT). OCT is an emerging imaging technique in biofilm research. However, the amount of data that currently can be acquired and processed hampers the statistical inference of large scale patterns in biofilm morphology. The automated OCT imaging system allows covering large spatial and extended temporal scales of biofilm growth. It combines a commercially available OCT system with a robotic positioning platform and a suite of software solutions to control the positioning of the OCT scanning probe, as well as the acquisition and processing of 3D biofilm imaging datasets. This setup allows the in situ and non-invasive automated&#160;monitoring of biofilm development and may be further developed to couple OCT imaging with macrophotography and microsensor profiling.

Microbial biofilms form complex architectures at interphases and develop into highly scale-dependent spatial patterns. Here, we introduce an experimental system (hard- and software) for the automated acquisition of 3D optical coherence tomography (OCT) datasets. This toolset allows the non-invasive and multi-scale characterization of biofilm morphogenesis in space and time.

Biofilms are a most successful microbial lifestyle and prevail in a multitude of environmental and engineered settings. Understanding biofilm ...

Quantifying Corticolous Arthropods Using Sticky Traps

Terrestrial arthropods play an important role in our environment. Quantifying arthropods in a way that allows for a precise index or estimate of density requires a method with high detection probability and a consistent sampling area. We used manufactured sticky traps to compare abundance, total length (a surrogate for biomass), richness, and Shannon diversity of corticolous arthropods among the boles of 5 tree species. Efficacy of this method was adequate to detect variation in corticolous arthropods among tree species and provide a standard error of the mean that was &#60;20% of the mean for all estimates with sample sizes from 7 to 15 individual trees of each species. Our results indicate, even with these moderate sample sizes, the level of precision of arthropod community metrics produced with this approach is adequate to address most ecological questions regarding temporal and spatial variation in corticolous arthropods. Results from this method differ from other quantitative approaches such as chemical knockdown, visual inspection, and funnel traps in that they provide an indication of corticolous arthropod activity over a relatively long-term, better including temporary bole residents, flying arthropods that temporarily land on the tree bole and crawling arthropods that use the tree bole as a travel route from the ground to higher forest foliage. Furthermore, we believe that commercially manufactured sticky traps provide more precise estimates and are logistically simpler than the previously described method of directly applying a sticky material to tree bark or applying a sticky material to tape or other type of backing and applying that to the tree bark.

We describe a semi-quantitative approach of measuring characteristics of corticolous (bark-dwelling) arthropod communities. We placed commercially manufactured sticky traps on tree boles to estimate abundance, total length (a surrogate to biomass), richness, and Shannon diversity for comparison among tree species.

Terrestrial arthropods play an important role in our environment. Quantifying arthropods in a way that allows for a precise index or estimate of density ...

Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae are a preeminent model for the neurobehavioral study of environmental pollutants. Here, a detailed experimental protocol is provided for the evaluation of the neurotoxicity of environmental pollutants using zebrafish larvae, including the collection of the embryos, the exposure process, neurobehavioral indicators, the test process, and data analysis. Also, the culture environment, exposure process, and experimental conditions are discussed to ensure the success of the assay. The protocol has been used in the development of psychopathic drugs, research on environmental neurotoxic pollutants, and can be optimized to make corresponding studies or be helpful for mechanistic studies. The protocol demonstrates a clear operation process for studying neurobehavioral effects on zebrafish larvae and can reveal the effects of various neurotoxic substances or pollutants.

A detailed experimental protocol is presented in this paper for the evaluation of neurobehavioral toxicity of environmental pollutants using a zebrafish larvae model, including the exposure process and tests for neurobehavioral indicators.

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae ...