biophysical modeling of forward scattering from bacterial colonies using scalar diffraction theory

$: Biophysical modeling of forward scattering from bacterial colonies using scalar diffraction theory$
Biophysical modeling of forward scattering from bacterialcolonies using scalar diffraction theory

Euiwon Bae,1,* Padmapriya P. Banada,2 Karleigh Huff,2 Arun K. Bhunia,2 J. Paul Robinson,3,4

and E. Daniel Hirleman1

1School of Mechanical Engineering, Purdue University, West Lafayette, Indiana 47906, USA2Molecular Food Microbiology Laboratory, Department of Food Science, Purdue University,

West Lafayette, Indiana 47906, USA3Department of Basic Medical Science, Purdue University, West Lafayette, Indiana 47906, USA

4Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana 47906, USA

*Corresponding author: [email protected]

Received 22 November 2006; revised 7 February 2007; accepted 8 February 2007;posted 13 February 2007 (Doc. ID 77298); published 18 May 2007

A model for forward scattering from bacterial colonies is presented. The colonies of interest consist ofapproximately 1012–1013 individual bacteria densely packed in a configuration several millimeters indiameter and approximately 0.1–0.2 mm in thickness. The model is based on scalar diffraction theory andaccounts for amplitude and phase modulation created by three macroscopic properties of the colonies:phase modulation due to the surface topography, phase modulation due to the radial structure observedfrom some strains and species, and diffraction from the outline of the colony. Phase contrast and confocalmicroscopy were performed to provide quantitative information on the shape and internal structure of thecolonies. The computed results showed excellent agreement with the experimental scattering data forthree different Listeria species: Listeria innocua, Listeria ivanovii, and Listeria monocytogenes. Theresults provide a physical explanation for the unique and distinctive scattering signatures produced bycolonies of closely related Listeria species and support the efficacy of forward scattering for rapid detectionand classification of pathogens without tagging. © 2007 Optical Society of America

OCIS codes: 290.0290, 170.0170, 110.0110.

1. Introduction

Bacterial contamination in food and other productscan be harmful or even fatal. It is therefore imperativeto quickly detect and identify bacterial contaminationas early in the product life cycle as possible, as high-lighted by recent outbreaks. For the food and health-care industries methods for rapid identification andisolation of bacteria such as pathogenic Listeria mono-cytogenes and Escherichia coli in products are veryimportant [1]. While the dangers of Escherichia coliare well known, Listeria monocytogenes has muchgreater lethality. Recently developed selective enrich-ment and plating methods for Listeria detection havesignificantly reduced the time needed for identifica-tion, but even so these still require 3–6 days to confirmsuspect colonies on agar growth-medium plates by bio-chemical and serological tests [2,3].

Various types of noninvasive optical methods havebeen applied to classify and differentiate the constitu-ents of biological samples. Some sensors utilize singlebacteria detection while earlier methods generallymeasured scattering by ensembles of bacteria dis-persed in liquid phase and used modified Mie scatter-ing theory for spherical and cylindrical particles toanalyze the measurements [4–8]. Laser spectroscopictechniques and polarized differential light scatteringhave been also investigated for characterizing bacte-rial cells in suspension. These conventional methodsrequire an extra sample preparation step in the ex-periments or are limited to modeling scattering froma single bacterium in a liquid. In recent work Guo andco-workers [9,10] investigated the possibility of usinga light-scattering method for differentiating the solidstate of a bacterial colony using both reflection andtransmission types of scatterometers. It was foundthat the transmission (forward-scattering) configura-tion proved most effective in differentiating severalgenera and species.

0003-6935/07/173639-10$15.00/0© 2007 Optical Society of America

10 June 2007 � Vol. 46, No. 17 � APPLIED OPTICS 3639

In this paper we propose a biophysical model of thebacteria colony and then use scalar diffraction theoryto predict the forward-scattering pattern. In realitythis is a multiscale problem, i.e., a combination ofscattering at the microscale (by individual bacteria)and at the macroscale (by the geometric form of thecolony). Here we take the macroscopic approachwhereby the colony is modeled as a 2D amplitude andphase object. Theoretical explanations are presentedand compared with the experimental results.

Section 2 discusses the mathematical descriptionof the forward-scattering phenomena, and Section 3will present scattering signatures, phase contrastmicroscope images, and confocal microscope images.Section 4 discusses the calibration experiment from areference sample and a comparison of the model pre-dictions with experimental results. Section 5 providesthe biophysical interpretation of the scattering imagefor three Listeria species.

2. Modeling

A. System Description

The forward-scattering measurement platform usedhere is known as bacteria rapid detection using opti-cal scattering technology (BARDOT), which encom-passes several design improvements over the originalsystem of Guo [9,10]. The measurement system con-sists of a laser source with a wavelength in the visiblerange, bacterial colonies grown on an agar plate, anda sensor to image the scattering pattern as shown inFig. 1. The bacterial colonies are grown on top of theagar plate, and their diameters are 1–2 mm. The rig-orous approach to treat wave propagation is to use

Maxwell’s equations and vector diffraction theory.However, since the bacterial colonies are large com-pared to the wavelength, and the diffracted or scat-tered fields are observed far away from the aperture,the problem can be solved with more simplified scalardiffraction theory [11].

B. Modeling of Bacterial Colony Scattering

Optical theory indicates that the diffraction field inthe image plane is the Fourier transform of the fieldin the object plane. The object plane for these pur-poses is considered as the plane just to the right of(i.e., downstream of) the bottom of the petri dish asshown in Fig. 2(a). We define coordinate systems forthe source, the bacterial colony, and the image planeas Xs, Ys, Xa, Ya, and Xi, Yi, respectively. Assuming aTEM00 mode of the laser beam centered on the z axiswith its waist on the z � 0 location, the electric fieldon the source plane can be expressed as

E�xs, ys, z� � E0� w0

w�z�exp��

�xs2 � ys

2�w2�z� ��

� exp��ikz � tan�1� zz0��

��exp�ikxs

2 � ys2

2R�z� ��, (1)

where xs, ys are the points on the source plane, k is thewave vector, and E0 is the on-axis field strength. Thethree bracketed terms account for variations of am-plitude of field, longitudinal phase, and radial phase,respectively. The variation of beam waist w(z) andthe radius of the wavefront R(z) are expressed as [12]

Fig. 1. Schematic of bacterial rapid detection using optical scattering technology (BARDOT) platform.

3640 APPLIED OPTICS � Vol. 46, No. 17 � 10 June 2007

w2�z� � w02�1 � � z

z02�, R�z� � z�1 � �z0

z 2�, (2)

where z0 is defined as the z location where 1�e2 radiushas expanded to 2w0. Since z0 �� z, the field incidenton the colony plane can be expressed as

E1�xa, ya, z1� � E0 exp��xa

2 � ya2�

w2�z1� exp�ikz1�

� exp�ik�xa

2 � ya2�

2R�z1� �, (3)

where xa and ya are the coordinates in the aperture,and w�z1� and R�z1� are the beam waist and radius ofthe wavefront in the colony plane �z � z1�. When there

is a series of physical objects through which an inci-dent beam passes, the wavefront modification isobtained by multiplying the amplitude and phase mod-ulation caused by each independent object. We char-acterize the modulated field in the image plane byconsidering the transmission of the agar plate, thecolony core, and an edge region in some cases consist-ing of spokes. Applying the Huygens–Fresnel principlein rectangular coordinates, this can be expressed as

E2�xi, yi� �1i�

�

t�xa, ya�E1�xa, ya�exp�ik��xa, ya��

� exp�ikrai

rai�cos �dxadya, (4)

Fig. 2. (a) Coordinates for source, colony, and image plane. (b) Side and top view and geometric parameter of the bacteria colony.


where xi, yi are points in the image plane, � denotesthe colony surface, t�xa, ya� is the 2D transmissioncoefficient, ��xa, ya� is the 2D phase modulation fac-tor, rai is the distance from the aperture plane to theimage plane, and � is the wavelength. As shown inFig 2(b), R is the radius of curvature of the bacteriacolony, wb is the 1�e2 beam radius and n2, 2 and n3,3 are the refractive index and thickness of the brain–heart infusion (BHI) agar and the Petri dish, respec-tively. xe stands for the size of the total computationaldomain. Based on diffraction theory, assuming itsvalidity criteria [11] are met, the diffracted field onthe image coordinates can be approximated as

E2�xi, yi� � C1 �

t�xa, ya�exp��xa

2 � ya2�

w2�z1� �� exp�ik

�xa2 � ya

2�2R�z1� �exp�ik

�xa2 � ya

2�2z2

�� exp�ik��xa, ya�� exp��i2�fxxa � fyya��dxadya, (5)

where C1 is a constant that is not related to theintegration variable, and fx and fy are spatial frequen-cies defined as

When a convex shape for the colony is assumed andexpressed as a phase modulation, Eq. (5) is simpli-fied as

E2�xi, yi� � C1 �

t�xa, ya�exp��xa

2 � ya2�

w2�z1� �� exp�ik��xa, ya�� exp��i2�fxxa � fyya��dxadya, (7)

where ��xa, ya� is the total phase modulation, whichhas contributions from the shape (quadratic phase)and internal structure of the colony. We model thecalculation domain as comprising three regions: (1)the core of the colony, characterized by a dome-shaped structure with radius of curvature and refrac-tive index ��1�; (2) an edge region characterized by adifferent refractive index in some cases and by iso-lated fingers or spokes of bacterial growth on the bareagar substrate ��2�; and (3) a bare agar region outsidethe radius of the colony ��3� as shown Fig. 2(b). Based

on this division of the computational domain, Eq. (7)can be separated as

E2�xi, yi� � C1 �1

t1 exp��xa

2 � ya2�

w2�z1� �exp�ik�1��xa, ya��

� exp��i2�fxxa � fyya��dxadya

� C1 �2

t2 exp��xa

2 � ya2�


� exp��i2�fxxa � fyya��dxadya

� C1 �3

t3 exp��xa

2 � ya2�


� exp��i2�fxxa � fyya��dxadya, (8)

where t1, t2, and t3 are the transmission coefficientsfor the colony core, edge, and agar areas, respectively,and �1��xa, ya�, �2��xa, ya�, and �3��xa, ya� are the phasemodulations for the �1, �2, and �3 areas. This factorcould be divided by the phase modulation from theconvex shape with a different radius of curvature,thus resulting in different focal lengths as

�1��xa, ya� � ��xa2 � ya

2�2 � 1

R�z1��

1z2

�1f1

� �1s�xa, ya�,

�2��xa, ya� � ��xa2 � ya

2�2 � 1

R�z1��

1z2

�1f2

� �2s�xa, ya�,

�3��xa, ya� � ��xa2 � ya

2�2 � 1

R�z1��

1z2

1f1

� �n11 � 1�� 1R1

�1

R�,

1f2

� �n12 � 1�� 1R2

�1

R�, (9)

C1 �

E0 exp�ikn22�exp�ikn33�exp�ik�z1 � z2��exp� ik2z2

�xi2 � yi

2��i�z2

,

fx �xi

�z2, fy �

yi

�z2. (6)


where n11 and n12 are the refractive indices of thecenter and edge areas of the bacterial colony, R1 andR2 are the radii of curvature of the convex surface ofthe center and edge areas, R� is the radii of curvaturefor the flat surface, f1 and f2 are the two effective focallengths caused by the different radii of curvature andrefractive indices and �1s and �2s are the phasescaused by the radial spokes, which will be discussedin Section 5. The final intensity of the scattering im-age was determined by calculating the modulus of thefield in the image plane.

3. Experiment

A. Material and Methods

Listeria innocua, Listeria ivanovii, and Listeriamonocytogenes cultures were selected for our experi-ments. Each bacteria was grown in BHI broth at37 °C for 15–18 h. Respective dilutions were platedon the surface of the BHI agar, so as to obtain 30–50colonies per plate, and were incubated at 37 °C. Foreach species of Listeria, colony measurements weredone using the phase contrast microscope (LeicaMicrosystems, Bannockburn, Illinois, USA) and thescatterings of the colonies using BARDOT were per-formed at every 6 h of incubation, for up to 46 h withan average of 10–15 colonies per plate. For BARDOTthe corresponding scattering image was recorded,while for the phase contrast microscope the 2D phasemap and the diameter of the bacterial colony weremeasured.

B. BARDOT

The BARDOT system was designed with (1) a laserdiode module of 635 nm wavelength, (2) an XY stageto move the sample, (3) a Z manual stage with a40 mm travel length to vertically move the camera,(4) an adjustable Petri dish holder, which could holdsamples of up to 100 mm wafers, (5) a CCD imagesensor to acquire the scattering images, and (6) astage controller and computer. The laser diode ismounted on a stage for alignment and generates acollimated beam light of 1�e2 diameter of approxi-mately 1 mm that is impinged on the bacterial colonyand passes through the substrate of the bacterialagar media. One achievement of the newly designedsystem is a reduction of the scattering measurementtime by adapting the XY and Z stages. To accommo-date the variety of samples, a custom designed plat-form was manufactured and attached to the top of theXY stage. This 150 mm long aluminum mountingplatform has 3.2 mm stepped edges on one end forsample attachment and a 50 mm slot providing anadjustable sample holder to accommodate from a sil-icon wafer to a 100 mm Petri dish. The imager is a7.4 �m unit pixel size IEEE-1394 camera from Mi-cropix, London, UK, (model M-640) with 640 � 480resolution. The image sensor is monochromatic, incontrast to a red-green-blue (RGB) color image sensorin the previous instrument [10]. This design decisionwas made to maximize the quantum efficiency of the

system. The CCD image sensor was attached to a Zstage, which enabled observation and capture of thescattering pattern as a function of Z. The forward scat-tering from a blank agar plate was negligible com-pared with the scattering of the bacterial colonies.

C. Phase Contrast Microscope

Phase contrast microscopy was used to provide qual-itative information of 2D phase variation and toquantify the diameter of the bacterial colony. Thephase contrast microscope separates the diffractedand refracted waves and recombines them at the im-aging plane [13]. Since optical phase is defined as theproduct of wavenumber and optical path length,phase contrast microscope images provide qualitativeinformation of the specimen. Compositional differ-ences, which create the variation of refractive index,are captured, and the contrast is enhanced such thata 2D contrast map can be constructed. The phasecontrast microscope images of the 30 h Listeria in-nocua, 36 h Listeria ivanovii, and 36 h Listeria mono-cytogenes are shown in Fig. 3. The overall diametersof the colonies were 1001, 1520, and 1148 �m, respec-tively, as measured by the built-in calibrated functionof the Leica microscope with a 10� objective. Theimage for Listeria innocua shows a dark center re-gion that gradually diminishes until the edge area.Listeria ivanovii and Listeria monocytogenes showcharacteristic features such as circular spots or arc-shaped features. These features will be used tomodel the optical response of these species in latersections.

D. Confocal Microscope

A bacterial colony is a 3D object, which is similar to aspheroid shape. A single bacterium appears as a shortrod, approximately 1–1.5 �m long and 0.5–0.8 �m indiameter. When a bacterium multiplies, it producesproteins and metabolic byproducts that become partof the microscopic structure. Along with macroscopicshape and thickness, compositional information suchas interbacterial distance and volume concentra-tion play a role in determining the optical response ofa colony. The confocal microscope is widely used inbiology to image subsurface structure without de-stroying the sample. The sample is tagged with afluorophore, which emits photons when illuminatedwith a characteristic wavelength of light. The confo-cal microscope uses an aperture to reject out-of-focuslight, which enables imaging of planes at differentdepths into the sample [14]. This technique is com-bined with scanning mirrors, which raster the inci-dent beam to create a 2D XY data set. In this studygreen-fluroescent-protein- (GFP-) producing Listeriamonocytogenes was allowed to grow and form colonieson a BHI agar plate. The laser scanning confocalmicroscope used in this experiment was the BioRadMRC 1024 that uses filters and Kr-Ar lasers with488, 568, and 647 nm excitation wavelengths. Theemission wavelength was set to 510 nm as appropri-ate for the fluorophore used in the bacterial sample


and measured with a photomultiplier tube. The XYZscanning step was 2 �m � 2 �m � 2 �m, and 50slices of 512 � 512 pixel images were taken. Afterstacking the z sliced image of the bacteria colony, a

maximum intensity projection (MIP) was performedto create a single representative image of the 3Dcolony. The MIP shows the existence of internalstructure as shown in Fig. 4(a). Figure 4(b) shows thecross section of the 3D stack of z sliced images of thebacterial colony. The contour of the bacterial colonywas fitted to a circle to calculate a radius of curva-ture, which in turn resulted in effective focal lengthusing Eq. (9). The R value was estimated to be ap-proximately 1.4–2.6, which resulted in an effectivefocal length of 4.2–6.9 mm, assuming R� as zero.

4. Results

A. Comparison for Reference Samples

To provide some reference experimental data forstandard cases, we have fabricated transmissionmeshes with various apertures and stops. Patternedchrome masks as developed by our group [15,16] forcalibration and validation of forward-scattering in-struments are widely used and are written intoAmerican Society for Testing and Materials and In-ternational Organization for Standardization stan-dard test methods. The patterns were designed with

Fig. 3. (Color online) Phase contrast microscopic images of colo-nies of (a) Listeria innocua, (b) Listeria ivanovii, (c) Listeriamonocytogenes.

Fig. 4. Confocal microscope image for the colony of a green-fluorescence-protein- (GFP-) expressing Listeria monocytogenesstrain. (a) Maximum intensity projection, (b) cross section alongthe Y axis of a 3D stack of confocal microscope images.


IC station software and fabricated on a 4 in. �100mm� chrome mask. One mask contained circular ap-ertures, and the other contained circular stops withdiameters from 0.05 up to 2 mm in steps of 0.05 mm.The calibration meshes were placed in the sampleholder of BARDOT, and measurements were made atvarious Z positions. If circular symmetry exists Eq.(4) can be simplified as


0

r0

J0�krira�z2�exp�ik�ra

2 � 1z1

�1z2�radra,

(10)

where ra and ri are radial coordinates, which areequal to xa

2 � ya2 and xi

2 � yi2, respectively. In the

case of an aperture, the integral limit is set to r0,which is the aperture radius. For comparison with ageneral case, Eq. (10) was also computed with 2D fastFourier transform (FFT). A comparison of these pre-dictions and experiment for an 800 �m aperture isshown in Fig. 5(a). The distance from aperture todetector was set to 57 mm. The solid curve is the FFT,the dotted curve is the Bessel function, and the ex-perimental data are shown as circles. The patternshows a typical result from a Fresnel diffraction. Theother case modeled is when there exists a circularblock. In this case Eq. (10) is changed to


r0

�

J0�krira�z2�exp�ik�ra

2 � 1z1

�1z2�radra.

(11)

A comparison of the circular stop case is shown in Fig.5(b) for a stop diameter of 800 �m. The distance fromaperture to detector was set to 57 mm. The scatteringimage showed a Possison spot [17] at the origin andedge diffraction at ri at 0.4 mm of the aperture size.However, in this case the Gaussian incident beamtends to limit the beam extent so that the intensityfluctuation exponentially decreased when radius waslarger than 0.4 mm.

B. Comparison for Listeria Species

In this section, the proposed model is applied to pre-dict the optical response assuming that the coloniesact as random amplitude and phase modulators. Forthe amplitude modulation, the Gaussian beam profileis multiplied by the transmission coefficient of thebacterial colony. For phase modulations, based onthe observation and theoretical modeling, we havecategorized possible physical aspects, which createan amplitude–phase modulation. They are the spher-ical surface, concentric rings, radial spokes, andrandomly positioned circular spots. The amplitudemodulation is modeled as the product of Gaussianbeam amplitude and the transmission coefficient,which was set to 0.3 for radial coordinates less than0.7 of colony radius, 0.7 for 0.7 to 1.0 of colony radius,

and 0.9 for the agar area. The phase modulation wasmodeled with Eq. (9) with z2 set to 40 mm and f1 andf2 set to 7 and 8 mm, respectively.

For Listeria innocua, Figs. 6(a) and 6(b) show a com-parison of the experiment and computational modelpredictions. The scattering signatures of Listeriaivanovii revealed a pattern similar to the previousListeria innocua but with distinctive speckle or ran-dom spots in the center. According to the phase con-trast microscope image from Fig. 3(b), the texture ofthe Listeria ivanovii colony shows random grainy fea-tures. Inside the 300 �m core diameter area we ob-serve random circular spots with a variation ofrefractive index or in transmission coefficient, i.e.,inclusions. To simulate this phenomenon we createda phase map of a spherical surface with randomlypositioned circular spots (50 spots with diameters of20–40 �m), which randomly modulates the phase be-tween � to � and modeled with �1s�xa, ya�. Figures6(c) and 6(d) show a comparison of the experimentand computational model results for Listeria ivano-vii. The last species studied is Listeria monocyto-genes, which showed the most complicated scatteringpattern among the three species. The scattering im-ages show concentric rings similar to Listeria innocuaand Listeria ivanovii as well as multiple bright ringsformed at the maximum intensity caused by the knifeedge pattern. In addition, there are radial spokes atvarious angular positions. This is consistent with thephase contrast image in Fig. 3(c), which reveals acomplicated phase map for Listeria monocytogenes.Figure 3(c) shows an arc-shaped dark area with anarc length of approximately 50–100 �m. Radialspoke features are clearly seen in the MIP of theconfocal microscope images of Listeria monocyto-genes shown in Fig. 4. Therefore, in addition to thephase profile caused by the spherical surface, twomore phase modulation terms are added: a concen-tric circular gratinglike structure with a spacingof 10 �m ��1s�xa, ya�� and spokes of approximately100 �m in length ��2s�xa, ya��. These additional phaseterms were randomly distributed uniformly between� to �. Figures 6(e) and 6(f) show a comparison ofthe experiment and computational model for Listeriamonocytogenes.

5. Discussion

The phase modulation imposed on a laser beam pass-ing through a bacterial colony on an agar substratehas been modeled in Eqs. (1)–(9) and the near-forward diffracted field calculated on that basis usingdiffraction theory. According to the experimental re-sults shown in Figs. 6(a), 6(c), and 6(e), the scatteringimage for Listeria species can be interpreted as thecombination of an Airy ring pattern, a secondarybright ring in the middle of the diffraction pattern, arandom speckle effect, and radial spokes.

Airy ring patterns are created when the incidentlight is diffracted through a small aperture or ob-structed by a circular obstacle. Since the diameterof the incident Gaussian beam exceeds the lateral (inthe Xa, Ya plane) extent of the colony, that portion


Fig. 5. Comparison of intensity distribution across radial direction of proposed model and reference sample at z � 57 mm. (a) Circularaperture with 800 �m diameter, and (b) circular block with 800 �m diameter.


of the beam incident on the colony is attenuated bythe colony, and that portion of the beam outside thecolony is transmitted. This creates a phase- andamplitude-aperture effect, which, in turn, createsAiry rings in the image plane.

The second important characteristic of the scatter-ing pattern is the bright ring in the center. This iscreated by the difference in transmission coefficientin the center and edge areas. Two effects reduce thetransmission in the center of the colony. First, thethickness is greater, and the additional bacteriascatter or absorb more light. Second, the bacteria

nearest the agar surface and in the center are theoldest, and they have excreted the most extracellularmaterial; therefore the mass density is greatest. Con-versely, the edge parts have less bacteria and thushigher transmission coefficients. The resulting effectis similar to the knife edge diffraction phenomenonand was also observed in the reference sample exper-iment for a circular chrome block.

The next factor is a random circular spot observedin the Listeria ivanovii case. After the Listeria ivano-vii colony was incubated approximately 36 h, the cen-ter part of the colony was observed to have numerous

Fig. 6. Comparison of BARDOT image (left column) and proposed model (right column). (a) and (b) Listeria innocua, (c) and (d) Listeriaivanovii, (e) and (f) Listeria monocytogenes.


circular spots, which are not observed in other spe-cies. These circular optical inclusions, which areunique to ivanovii, collectively act as a random phasemodulator and create a cloudy looking scattering im-age. The effect is similar to speckle.

The last factor contributing to the scattering imageis the radial spoke structure of Listeria monocyto-genes. In optical telescopes these kinds of radialspokes are reported and are caused by spider vanes[18,19]. According to the phase contrast microscopeimage, there are number of arc-shaped features,which point outward in the radial direction. In addi-tion, confocal microscopy provided images of the col-ony showing the existence of wavy radial structuresinside the colony. We believe these to be internaldensity fluctuations, related to the multiplication andgrowth characteristics of the bacteria, which in turnaffect the phase modulation in the specified region.

6. Conclusions

A model for optical forward scattering by a bacterialcolony based on scalar diffraction theory was intro-duced. The model treats the colony as an amplitude–phase modulator and suggests macroscopic factorsthat cause the distinctive features shown in theforward-scattering signatures of the three types ofListeria species. Phase contrast and confocal micros-copy were used to provide independent information onthe structure and morphology of the colonies that fixedparameters on the scattering model. A new instrumentwas developed to measure forward-scattering signa-tures from bacteria colonies for rapid identification.The experimental system was validated by using achrome mask reference sample with known diffractionproperties.Finally,distinctivescatteringpatternsmea-sured for three important species of Listeria werefound to show good agreement with the model pre-dictions. The results provide a physical explanationfor the unique and distinctive scattering signaturesproduced by colonies of closely related Listeria spe-cies and support the efficacy of forward scattering forrapid detection and classification of pathogens with-out tagging.

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biophysical modeling of forward scattering from bacterial colonies using scalar diffraction theory

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