Method Article

In Situ Microscopy for Real-time Determination of Single-cell Morphology in Bioprocesses

DOI:

10.3791/57823

December 5th, 2019

In This Article

Summary

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A photo-optical in situ microscopy device was developed to monitor the size of single cells directly in the cell suspension. The real-time measurement is conducted by coupling the photo-optical sterilizable probe to an automated image analysis. Morphological changes appear with dependence on the growth state and cultivation conditions.

Abstract

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In situ monitoring in microbial bioprocesses is mostly restricted to chemical and physical properties of the medium (e.g.,pH value and the dissolved oxygen concentration). Nevertheless, the morphology of cells can be a suitable indicator for optimal conditions, since it changes with dependence on the growth state, product accumulation and cell stress. Furthermore, the single-cell size distribution provides not only information about the cultivation conditions, but also about the population heterogeneity. To gain such information, a photo-optical in situ microscopy device1 was developed to enable the monitoring of the single-cell size distribution directly in the cell suspension in bioreactors. An automated image analysis is coupled to the microscopy based on a neural network model, which is trained with user-annotated images. Several parameters, which are gained from the captures of the microscope, are correlated to process relevant features of the cells, like their metabolic activity. Until now, the presented in situ microscopy probe series was applied to measure the pellet size in filamentous fungi suspensions. It was used to distinguish the single-cell size in microalgae cultivation and relate it to lipid accumulation. The shape of cellular particles was related to budding in yeast cultures. The microscopy analysis can be generally split into three steps: (i) image acquisition, (ii) particle identification, and (iii) data analysis, respectively. All steps have to be adapted to the organism, and therefore specific annotated information is required in order to achieve reliable results. The ability to monitor changes in cell morphology directly in line or on line (in a by-pass) enables real-time values for monitoring and control, in process development as well as in production scale. If the off line data correlates with the real-time data, the current tedious off line measurements with unknown influences on the cell size become needless.

Introduction

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Morphological features of cells are often related to the physiological state, a connection between form and function exists for many applications. The morphology of a single cell is influenced by the state of growth, the cell's age, osmotic and other potential cell stresses or product accumulation. Morphological changes of cells are often a measure of the growth vitality of a culture. Intracellular product synthesis, lipid accumulation in algae and inclusion body formation in bacteria, among others, are related with the cell size as well. Cell agglomeration can be another factor that is worth investigating as summarized recently2.

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Protocol

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NOTE: The following steps are necessary to adapt the parameters to the respective microorganism and culture conditions. The adjustment of probe settings lasts about 20 min for an experienced user. A detailed description of tools and steps is given in the corresponding probe manual from SOPAT GmbH. In general, the tools that are presented in the following protocol are needed: (i) Probe Controller for probe adjustments and image acquisition; (ii) Fiji (ImageJ) for annotations on acquired images; (iii) SOPAT support for artificial neural network (ANN) training and workflow creation; (iv) Batcher for data batch processing using already ....

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Results

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The cell size detection in yeast cultures with the ISM and automated image detection to distinguish between budding and non-budding cells was successfully conducted. Both the stroboscope intensity and the choice of the measurement gap have a range of tolerance, in which the particle identification is not affected. For example, S. cerevisiae cells were measured with various stroboscope intensities within a variation range of 11% at a dry biomass concentration of 4 g L-1.......

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Discussion

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ISM as presented here with the same or very similar devices was used to measure morphologic dynamics of fungi, microalgae, and yeast cells, which enabled the determination of growth activity, and in case of algae, intracellular product accumulation. The sensor has no movable parts and is directly applicable in any standard stirred tank bioreactor, either through a standard port or in a sterilizable by-pass. Since yeast is much smaller than algae, the reduction in cell size required some recent hardware adaptions like a h.......

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Disclosures

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The authors have nothing to declare.

Acknowledgements

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The authors are thankful for the support of the German Federal Ministry of Economics and Energy within the framework ZIM-Koop, project "Smart Process Inspection", grant no. ZF 4184201CR5.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Sensor MM 2.1 - MFCSOPAT GmbH, Germanyn.a.Inline Monocular Microscopic probe Version 2.1 with a Mirco Flow Cell
Sofware version v1R.003.0092SOPAT GmbH, Germanyn.a.
Thickness gaugen.n.It can be any supplier, DIN 2275:2014-03
Ethanol 70%n.n.It can be any supplier
SOPAT manual Version 2.0.5SOPAT GmbH, Germany
Optical lense paperVWR470150-460
Fiji, ImageJopen source
50 mL conical centrifuge tubesIt can be any supplier

References

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  1. Maaß, S., Rojahn, J., Hänsch, R., Kraume, M. Automated drop detection using image analysis for online particle size monitoring in multiphase systems. Computers & Chemical Engineering. 45, 27-37 (2012).
  2. Lemoine, A., Delvigne, F., Bockisch, A., Neubauer, P., Junne, S.

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Tags

In Situ MicroscopySingle Cell MorphologyBioprocess MonitoringAutomated Image AnalysisNeural Network ModelParticle IdentificationData AnalysisCell Size DistributionMorphological AssessmentReal time Measurement

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