9.23 Micro-PIV in Life Science

Contributed by:

A. Delgado, H. Petermeier, W. Kowalczyk

Table 9.25. PIV recording parameters for micro-PIV in life science.

Flow geometry vicinity of the surface of Granular ActivatedSludge (GAS)

Maximum in-plane velocity Umax = 40µm/sField of view 602× 505µm2

Interrogation area 12× 6µm2

Observation distance z0 = 100–300µmRecording method double frame/single exposureRecording medium CCD cameraRecording lens objective Zeiss “Epiplan” 20× /0.40 HD

objective Zeiss “Epiplan” 10× /0.20 HDIllumination light build in microscopePulse delay 40msSeeding material yeast cells Saccharomyces cervisiae

dp = 3–10µm)

9.23.1 Introduction

Most of the biological processes in Life Sciences are connected to convectivephenomena on the micro-scale. For making progress towards a better under-

9.23 Micro-PIV in Life Science 385

standing of natural phenomena being optimized by evolution in nature, abiologically oriented fluid mechanics has to overcome totally new challengesimposed by the new studies of biological effects on the micro-scale. In thiscontext the availability of powerful optical whole field systems such as PIV orPTV is of crucial importance. This is especially the case in microorganismicflows, that is flows induced by living microorganisms are studied and discussedin the present contribution. However, any measuring method has to strictlyfulfil the requirements imposed by its biocompatibility with respect to thebiological system in aspects according to the prevention of systematic errors.On the other hand this imposes considerable restrictions on the experimentalsetup and the flow evaluation methods. Till this date in the literature thistopic has been treated poorly, hence this contribution focuses on the corre-sponding aspects which must be kept in very similar ways to a large numberon application in Life Sciences.

The microorganismic flow studied here by using Micro-PIV and Micro-PTV is generated by the peritriche ciliate Opercularia Asymmetrica (typicaldimension of zooids amounts approx. 30µm), which plays a vital role not onlyin nature but also in biofilm reactors used for water purification. These ciliatesgenerate a flow field to access the nutrients in the surrounding fluid by ciliarybeating, see figure 9.108.

The generated flow pattern shows a vortex ring inducing strong elonga-tional and shear effects in the fluid, see figure 9.109. This influences the masstransport to the biofilm and thus also the nutrition of the biofilm [424], [425].The main components of the experimental test rig used are an invert phasecontrast microscope (magnification factors 10-, 20- and 40-fold) with micro-scope object holder consisting of glass plates both without and with coverplates (distance 200 and 300µm) as well as a CCD camera with a macro-zoom objective allowing a maximum speed of 500 frames/s, which are recordedon the computer. Obtained frames have a resolution of 860 × 1024 pixel (i.e.602µm × 505µm). The determination of the flow field is carried out basi-cally with the PIVview2C which employs a statistical correlation algorithm.The frames are interrogated with a Fast Fourier Transform accelerated inter-

Fig. 9.108. Pho-tographs (40× mag-nification) of theciliary kinematics ofthe investigated cil-iates (OperculariaSymmetrica) at threedifferent horizon-tal planes. The dis-tance between themamounts about 5µm.

386 9 Examples of Application

Fig. 9.109. Illustration of the biological milieu of the ciliates (left) as well as adetailed picture of single ciliates (middle; magnification 20) and the correspond-ing evaluation of the flow field (right). The latter show the model based GPU-reconstruction of the velocity field after a regularization based on a predictor-corrector method [429].

rogation algorithm. The interrogation window size is 32 × 32 pixel and theinterrogation grid is 16× 16 pixel.

9.23.2 Biocompatible µPIV/µPTV

Biocompatibility means avoiding from any changes in the environment of theciliates, since the ciliates react very sensitive to them. As a consequence ofthis a large number of measures are required which may result in a peculiardesign of the image delivering system and, even, in influencing negatively theexperimental determination of the flow field.

For the microorganismic motion generated by the ciliates biocompatibililtycan only be achieved by employing biocompatible tracer particles, an illumi-nation not altering the microbiological milieu and, last but not least, an ade-quate observation volume with negligible impact on the microorganismic flowfield. Additionally, it must be taken into consideration that the microorganis-mic flow generated is dominated by viscous forces (Stokes region). Therefore,special consideration must be devoted to the viscous effects on the seedingparticles for avoiding departure from the flow path due to possible propermotion.

Hence, it is obvious that the choice of seeding fulfilling the wide spectrumof restrictions imposed by biocompatibility is of essential importance. In fact,it has been observed, that usual artificial seeding (polystyrene particles of4.8µm diameter from Microparticles GmbH, Germany) is detected (obviouslyby chemotaxis) and rejected by the ciliates. It results in their artificial motionand, thus, in systematic deviations from the natural motion to be observed.These systematic deviations can be avoided by using biotic particles whichare accepted by the ciliates as natural nutrient. However, this imposes thedemand to operate a bioreactor [423] as the seeding source.

9.23 Micro-PIV in Life Science 387

Comprehensive studies have shown yeast cells (Saccharomyces cerevisiae)to have the best suitability. They have an ellipsoidal shape with a typicallength scale of 3–10µm, a density deviating only slightly from that of wa-ter (ρ = 1050 kg/m3) with good refractive properties. Thus, they fulfil basicrequirements for seeding. But additionally, in order to keep biocompatibil-ity, i.e. to prevent from an excessive feeding of the ciliates, only a moderateyeast cell concentration is suitable. As a consequence of the minimum spa-tial information density required by usual PIV-correlation algorithms is oftennot achieved. Thus, using PTV may represent an adequate alternative. How-ever, as a rule, the natural milieu of the ciliate contains biotical and abioticalstructures which increase substantially the difficulties of evaluating the flowimages provided the optical system. This in connection to the required sparseseeding leads to image artefacts. In [428, 429] a system based on a neuronu-merical hybrid based on the synergetic use of an Artificial Neuronal Networksupported by a priori knowledge applying a numerical approach is suggestedfor suppressing images artefacts and correcting spurious velocity vectors au-tomatically. In this context the model based reconstruction of the flow fieldon the graphical processor unit (GPU) and the interactive visualization of theflow topology results in considerable advancements [429, 430].

Regarding the viscous effects on the seeding particles mentioned above themost prominent are Brownian motion, a motion as a consequence of a devia-tion from sphericity as well as orbital drift due to the availability of a shear orextensional gradient, a curvature of the velocity profile and sedimentation ofthe particles. Fortunately, these influences seem to be negligible for the yeastcells employed [426].

The impact of illumination on the microorganismic flow induced by theciliates is not totally understood, yet. However, the sensitivity of microbio-logical systems on light radiation (due to phototaxis, photokinetics or, even,scotophobotaxis) is well described in literature. Thus, this prohibits the useof a laser light sheet as done in PIV generally. At first glance, fluorescencemicroscopy, that is marking the yeast cell by genetic modification delivers analternative as the intensity of the illumination could be kept at low level. But,on the other hand, the ciliates have been proven to be very sensitive to thewave lengths (ultraviolet) of the fluorescence exciting radiation required [426].More crucial, even employing light transmission microscopy appears to stressthe ciliates. In contrast to this, the used invert phase contrast microscopyseems to have the smallest impact on biocompatibility. Hereby, the depth ofsharpness (approx. 11µm) determined the thickness of the measuring plane.A photorefractive novelty filter microscope (NFM) [427, 428] has been alsoproven to fulfil the restrictions imposed by biocompatibility in an excellentmanner.

As mentioned above, realizing biocompatibility required even detailed con-siderations on an adequate observation volume which prevent the viscousforces exerted by the walls of the microscope object holders from influenc-ing the behaviour of the ciliates and distorting the flow field generated by

388 9 Examples of Application

them. In this context the ratios of the typical length dimension of the zooidand flow field in relation to the distance of the glass plates of the object holderare of essential importance. From a comparison of the results obtained by us-ing microscope object holders with and without a glass plate cover, it can beconcluded, that the influence of the viscous forces on the biological systembecomes negligible when the distance of the glass plate achieves the order ofmagnitude of the wave length of the vortex ring induced by the microorgan-isms which takes on nearly the 10-fold value of the typical dimension of thezooid (i.e. approx. 300µm).

9.23.3 Experimental Results

The most prominent features of the microorganismic motion found are (a)three different scenarios of motion and rest, (b) pairs of motion eddies witha wave length which corresponds to the distance between two neighboringciliates, and (c) a synergistically emphasized transport of nutrients by twociliates which are intermittently active.

Figure 9.109 illustrates the flow induced by the ciliates in their activephase, the biological milieu available and the determination of the velocityfield by Micro-PIV.

A more detailed evaluation of the flow field shows that dissipation effectsof the microorganismic convection induced by the ciliates takes on an orderof magnitude of only 1% of the kinetic energy of the flow. This indicates thatthe natural evolution has conducted to a strategy of highly efficient energyconversion. Thus, survival of the ciliates is guaranteed by an incredibly favor-able balance of the energy they employed to get access to the nutrient andthe energy contain of the latter. Furthermore, the intermittent ciliary activityof neighboring individuals increases substantially mixing effect and, thus, theprobability to access nutrients from the surroundings.