Automatisierte Mikroskopie

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We developed an image acquisition and evaluation system for the automated microscopic evaluation of comparatively simple samples, i.e. the counting of bacterial cells in suspension that are filtered and immobilized on membrane filters. For this we have developed a robust computerized focussing system that combines a proprietary autofocus with custom texture parameters, feedback loops for image contrast maximation, and decisions on image content during acquisition.

The system is capable of excluding inappropriate or badly focussed microscopic fields prior to evaluation without operator interaction with high reliability (>95%), and to evaluate >50 FISH stained samples per day at a total of 2000-3000 counted cells per sample. It consists of two modules:

The first module MPISYS (yellow) allows for the automated scan of the specimen (in this case plankton samples on filter pieces mounted on glass slides) and the acquisition of image series from the sample.

The second module ACMEtool (blue) enumerates cells based on the images taken by MPISYS. A manual inspection of the individual images is possible to ensure a high-quality enumeration of all samples.

General outline of the automated microscopy counting system
Image acquisition and cell enumeration pipeline using MPISYS and ACMEtool.

Instruments

 

- Zeiss Axioimager.Z2m

Fully motorized and software controlled microscope used for automated high-throughput microbial cell enumeration, and tile scan images of large regions or full slides.

Configuration:
- PI piezo driven scanning stage for 8 microscopic glass slides.
- Microscopic image acquisition via Zeiss AxioCam MRm or MRc5 camera.
- LED illumination system (Zeiss Colibri) for fast switching between three fluorescent channels (365 nm, 470 nm, and 590 nm) using a multiband filter set.
- HXP 120 illumination with dedicated filter sets for fluorescent channels not covered by LED illumination, e.g., Cy3 or Cy5.
- Slide overview image acquisition using 1x objective lens.
- Tile scan option with any installed objective lens.

The system is controlled with the Zeiss AxioVision software package and software tools and methods of Zeder et al., 2006 - 2011.
AxioImager.Z2m
© A. Ellrott / MPI-MM

- Zeiss Axioplan 2

High-throughput automatic microbial cell enumeration system for shipboard analyses.

Configurations

2015 - today: Further optimized for automated image acquisition on-board a research vessel.

- Märzhäuser stage EK 14 mot holding a maximum of 2 microscopic glass slides.
- Microscopic image acquisition via Zeiss AxioCam MRm or MRc camera.
- Slide overview image acquisition via Logitech 905 C webcam.
- Three channel LED illumination system Rapp Opto KSL 70 (365, 470, and 590 nm).
- Zeiss AxioVision software package, and software tools and methods of Zeder et al., 2006 - 2011, and Bennke et al., 2016.

The system has been successfully used during the UltraPac cruise in 2015/16 (SO 245), and the Arctic cruise in 2016 (MSM 56).
Axioplan 2, 2015-today
© A. Ellrott / MPI-MM
2012 - 2014: The system and programming has been upgraded and optimized to enable automated image acquisition on-board a research vessel.

- Märzhäuser / Kontron scanning stage holding a maximum of 4 microscopic glass slides.
- Microscopic image acquisition via Hamamatsu ORCA camera.
- Slide overview image acquisition via Logitech 905 C webcam.
- Three channel LED illumination system Rapp Opto KSL 70 (365, 470, and 590 nm).
- Zeiss AxioVision software package, and software tools and methods of Zeder et al., 2006 - 2011, and Bennke et al., 2016.

The system has been successfully used during the Atlantic Meridional Transect cruise in 2012 (AMT 22).
Axioplan 2, 2012-2014
© A. Ellrott / MPI-MM
2002 - 2011: Original setup for laboratory use like described in the corresponding publication of Pernthaler et al., 2003.

- Märzhäuser / Kontron scanning stage holding a maximum of 4 microscopic glass slides.
- Microscopic image acquisition via Hamamatsu ORCA camera.
- Classical mercury lamp illumination (Zeiss HBO 100) and optical filter for two fluorescent channels (DAPI and Fluos).
- Zeiss KS400 software package.
Axioplan 2, 2002-2011
© A. Ellrott / MPI-MM

Software Tools

 

- Automated Cell Counting

Automated cell counting on microscopic images is done by using the software ACMEtool (Automated Cell Measuring and Enumeration Tool) from M. Zeder.

- Overview Image Acquisition of Microscopic Slides via Web Camera

In-house development of a system for overview image acquisition of microscopic slides via web camera.
Supplementary material to the corresponding publication of Zeder et al., 2011.

Description and software download.

System overview with description of the used hard- and software. Guide for setup and calibration.

CapCam - A software to capture high resolution images from a web camera. Executable of the program, general information and needed dll's.

- Device driver for MCP4 stage controller

Our Axioplan 2 system consists of a Märzhäuser scanning stage operated via the Zeiss MCP4 stage controller from Kontron. Designed for the outdated Zeiss KS400 software package, it lacks support for current software, e.g., Zeiss AxioVision. The only possibility to operate the stage with AxioVison VBA was to program a device driver.
Here we provide a dll, which holds all needed functions to operate a scanning stage via the MCP 4 controller from Kontron.
After registration of the dll to the operation system and referencing it in the VBA-Module of AxioVision, the functions can be used to control the stage via an AxioVision VBA-program.
Driver for Zeiss MCP4 stage controller from Kontron. Package holding the dll, a description of the dll functions and a Visual Basic programming example.

References

  • Alonso C., Zeder M., Piccini C., Conde D., Pernthaler J. 2009. Ecophysiological differences of betaproteobacterial populations in two hydrochemically distinct compartments of a subtropical lagoon. Environ. Microbiol. 11:867-876. [PMID:19040452]
  • Bennke C.M., Reintjes G., Schattenhofer M., Ellrott A., Wulf J., Zeder M., Fuchs B.M. 2016. Modification of a High-Throughput Automatic Microbial Cell Enumeration System for Shipboard Analyses. Appl. Environ. Microbiol. 82:3289-96. [PMID:27016562]
  • Pernthaler J., Pernthaler A., Amann R. 2003. Automated enumeration of groups of marine picoplankton after fluorescence in situ hybridizationMicrobiol. 69:2631-2637. [PMID:12732531]
  • Salcher M.M., Pernthaler J., Zeder M., Psenner R., Posch T. 2008. Spatio-temporal niche separation of planktonic Betaproteobacteria in an oligo-mesotrophic lake. Environ. Microbiol. 10:2074-2086. [PMID:18430016]
  • Schattenhofer M., Fuchs B.M., Amann R., Zubkov M.V., Tarran G.A., Pernthaler J. 2009. Latitudinal distribution of prokaryotic picoplankton populations in the Atlantic Ocean. Environ. Microbiol. 11:2078-2093. [PMID:19453607]
  • Zeder M., Ellrott A., Amann R. 2011. Automated sample area definition for high-throughput microscopy. Cytometry A. 79:306-310. [PMID:21412981]
  • Zeder M., Kohler E., Pernthaler J. 2010. Automated quality assessment of autonomously acquired microscopic images of fluorescently stained bacteria. Cytometry A. 77:76-85. [PMID:19821518]
  • Zeder M., Pernthaler J. 2009. Multispot live-image autofocusing for high-throughput microscopy of fluorescently stained bacteria. Cytometry A. 75:781-788. [PMID:19658173]
  • Zeder M., Peter S., Shabarova T., Pernthaler J. 2009. A small population of planktonic Flavobacteria with disproportionally high growth during the spring phytoplankton bloom in a prealpine lake. Environ. Microbiol. 11:2676-2686. [PMID:19601962]
  • Zeder M., van den Wyngaert S., Köster O., Felder K.M., Pernthaler J. 2010. Automated quantification and sizing of unbranched filamentous cyanobacteria by model-based object-oriented image analysis. Appl. Environ. Microbiol. 76:1615-1622. [PMID:20048059]
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