H. Suhr

In situ microscopic cytometry enables noninvasive viability assessment of animal cells by measuring entropy states

Current state of the art to determine the viability of animal cell suspension cultures is based on sampling and subsequent counting using specific staining assays. We demonstrate for the first time a noninvasive in situ imaging cytometry capable of determining the statistics of a morphologic transition during cell death in suspension cultures. To this end, we measure morphometric inhomogeneity—defined as information entropy—in cell in situ micrographs. We found that the cells are partitioned into two discrete entropy states broadened by phenotypical variability. During the normal course of a culture or by inducing cell death, we observe the transition of cells between these states. As shown by comparison with ex situ diagnostics, the entropy transition happens before or while the cytoplasmatic membrane is loosing its ability to exclude charged dyes. Therefore, measurement of morphometric inhomogeneity constitutes a noninvasive assessment of viability in real time. Biotechnol. Bioeng. 2011;108: 2884–2893.

In situ microscopy: a perspective for industrial bioethanol production monitoring.

This work reviews the state-of-the-art in image-based in situ methods with regard to their potential use for fermentation of Saccharomyces cerevisiae in sugarcane wine. The integration of real time information from fermentation tanks in the control strategies has high potential to promote better fermentative performance. While several image-based techniques for the measurement of cell concentration have been established, a reliable and consistent viability measurement still remains a challenging task. Reagent-free methods that estimate viability from information contained in micrograph images are reviewed. Nevertheless, the inherent complexity of the sugarcane syrup medium imposes extra challenges regarding its representation in microscopic images and their evaluation by real time image analysis.

On-line and real time cell counting and viability determination for animal cell process monitoring by in situ microscopy

BackgroundTwo of the key parameters to be monitored during cellcultivation processes are cell concentration and viability.Until today, this is very often done off-line by sterilesampling and subsequent counting using a hemocyt-ometer or an electronic cell counter. Cell biology lacks ameasurable quantity by which single cells in suspensioncan be non-invasively diagnosed as dead or alive. How-ever, it would be of significance for process monitoringand in the light of initiatives like PAT if cell density aswell as viability could be determined directly and on-line.Optical measurement of cell density byin situ micro-scopy eliminates the need for sampling and allows forcontinuous monitoring of this key parameter; see e.g.[1,2]; Guez et al. [1] describe an in situ microscope(ISM) which does not use any moving mechanical partswithin or outside the fermentation vessel. It transmits inreal time images taken directly in the stirred suspensionwithin the bioreactor. Image data is processed and eval-uated to provide monitoring of cell-density and mor-phological parameters, e.g. cell size, by means ofassessing the obtained in situ cell-micrographs.Previously, we have extendedin situ microscopytowards viability assessment of suspended cells [3,5].Now, we present new findings on this topic and showthat in cultures of suspended cells, cell-death corre-sponds to measurable changes in morphometric para-meters as e.g. variance, contrast or entropy of thegreyvalues of in situ cell-micrographs. As an example,here we show viability determination via greyvaluedispersion.Material and methodsWe use a custom built high resolution ISM (HS Man-nheim) with water immersion objective, 40x magnifica-tion, numerical aperture 0.75 equipped with optical fiberillumination. Data acquisition is at 0.3– 15 frames persecond, frames have 1293x1040 pixels; primary dataanalysis results in cell micrographs (Figure 1a). We haveapplied the system to bench top and larger bioreactors(see e.g. [4]) and worked mainly with Jurkat, CHO andHybridoma cells.For the experiment presented in Figure 1a/b, cells arecultivated in a Biostat C30 (Sartorius BBI Systems) withthe ISM inserted in one of the existing probe ports. Pro-prietary hybridoma cells (InVivo BioTech Services) arecultured in serum free ISF-1 (InVivo BioTech Services;Biochrom AG) and monitored over the full length ofthe fermentation (not shown).For the experiment presented in Figure 1c, cells arecultivated in a custom built autoclavable steel bench topreactor(HSMannheim)with25mmporttoaccommo-date the ISM and a working volume of 0.7 L. To testreal time and viability determination capabilities of thesystem over a wide range of viabilities in a short time,cells were challenged with 3% Ethanol at 42 hours. Cellcounts (not shown) and viability were determined by insitu microscopy and, as reference, by means of a ViCellcell viability analyser (Beckman Coulter) and Flow Cyto-metry (Partec) using Annexin V / FITC and PI staining.Jurkat cells (DSMZ ACC 282) were cultured in 90%RPMI 1640 + 10% FBS.

In situ microscopy using adjustment-free optics

Abstract. In the past years, in situ microscopy has been demonstrated as a technique for monitoring the concentration and morphology of moving microparticles in agitated suspensions. However, up until now, this technique can only achieve a high resolution if a certain manual or automated effort is established for continuous precise focusing. Therefore, the application of in situ microscopes (ISMs) as sensors is inhibited in the cases where unattended operation is required. Here, we demonstrate a high-resolution ISM which, unlike others, is built as an entirely rigid construction, requiring no adjustments at all. This ISM is based on a specially designed water immersion objective with numerical aperture=0.75 and a working distance of 15  μm. The objective can be built exclusively from off-the-shelf parts and the front surface directly interfaces with the moving suspension. We show various applications of the system and demonstrate the imaging performance with submicron resolution within moving suspensions of microorganisms.

In situ microscopy as a tool for the monitoring of filamentous bacteria: a case study in an industrial activated sludge system dominated by M. parvicella

The present study demonstrates the application of in situ microscopy for monitoring the growth of filamentous bacteria which can induce disturbances in an industrial activated sludge process. An in situ microscope (ISM) is immersed directly into samples of activated sludge with Microthrix parvicella as dominating species. Without needing further preparatory steps, the automatic evaluation of the ISM-images generates two signals: the number of individual filaments per image (ISM-filament counting) and the total extended filament length (TEFL) per image (ISM-online TEFL). In this first version of the image-processing algorithm, closely spaced crossing filament-segments or filaments within bulk material are not detected. The signals show highly linear correlation both with the standard filament index and the TEFL. Correlations were further substantiated by comparison with real-time polymerase chain reaction (real-time PCR) measurements of M. parvicella and of the diluted sludge volume index. In this case study, in situ microscopy proved to be a suitable tool for straightforward online-monitoring of filamentous bacteria in activated sludge systems. With future adaptation of the system to different filament morphologies, including cross-linking filaments, bundles, and attached growth, the system will be applicable to other wastewater treatment plants.

Advanced In Situ Microscopy for On-Line Monitoring of Animal Cell Culture

Cell concentration is one of the key parameters to be monitored during cell cultivation processes. This is very often done off-line by sterile sampling and subsequent counting using a hemocytometer or an electronic cell counter. A direct optical measurement of cell density via an in situ microscope (ISM) eliminates the need for sampling and allows for continuous monitoring of this key parameter. Two such systems have been described in the literature, one of them has been developed at Mannheim University of Applied Sciences. This system has the advantage of not using any moving mechanical parts within or outside the fermentation vessel. Here we show two examples of advanced applications of a new version of this ISM with unprecedented resolution and frame rate: Adaptation to double glass jacket equipped bench top reactors and longer term application in a perfused 30 L steel reactor. Results in both cases show the performance of the ISM, the comparability of cell culture data obtained by ISM and traditional methods and the potential for further development of the ISM.

Optical Sampling in-situ Microscopy for on-line Monitoring of Animal Cell Cultures

Cell concentration and cell vitality are key parameters to be monitored during cell cultivation processes. Common techniques used for these purposes are often based on sterile sampling and subsequent off line measurements. Extraction and preparation of samples is labour-intensive and risk-entailing. These disadvantages are avoided if the cell culture is directly inspected by using an in-situ technique, e.g. an in-situ microscope (ISM). An ISM delivers a wealth of image data which can be evaluated so as to provide automatic monitoring of the cell density and of morphological parameters as the cell-size. In-situ microscopy can either employ periodic opening and closing of a probe chamber inside the reactor or, alternatively, flash illumination and optical depth of field in order to define a virtual probe zone. Here, we describe optics and software of an advanced version of such an ISM with unprecedented resolution and frame rate. Fast collection of online-galleries of individual cell-portraits even at low cell concentrations enables online morphological analysis without sample extraction. Cell density data obtained by the ISM and traditional counting are shown in comparison, revealing the advantage of the ISM with respect to statistic deviations.

Flow morphometry to assess the red blood cell storage lesion

We present a novel automated system for morphology analysis of red blood cells (RBC) under flow. RBC concentrates collected by blood banks for transfusions are stored for periods of up to several weeks, during which time a number of changes occur, collectively termed the storage lesion. Typically the extent of hemolysis is the defining criterion to determine the acceptability of the RBCs for transfusions. Morphological changes are related with biochemical alteration during the storage of RBCs. The typical blood smear procedure for determining such changes is a labor‐intensive and potentially biased manual process. The advantage of the flow morphometry system presented here is that it provides fully automated morphological classification of RBCs with large sample numbers in a short time. Our system uses a commercially available flow cell and flow conditions that prevent adhesion of RBCs, thus eliminating the need for blocking agents such as albumin that affect the distribution of cell shapes. Our morphometry results are validated by comparison with standard biochemical assays (hemolysis, ATP) for blood from 17 donors stored under blood bank conditions for 13 weeks. We show that the percentage of spherocytes present can be used to estimate the status of RBC concentrates.