Automated Microscopy and Cell Counting

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.

Instruments
> Zeiss Axioimager.Z2m
> Zeiss Axioplan 2

Software Tools
> Automated Cell Counting (ACMEtool)
> Overview Image Acquisition of Microscopic Slides via Web Camera
> Device driver for MCP4 stage controller

References

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

Configurations

2020 - today:

- PI piezo driven scanning stage for 8 microscopic glass slides.
- Microscopic image acquisition via Zeiss AxioCam 702 mono, MRm or MRc5 camera.
- LED illumination system (Zeiss Colibri 7) for fast switching between fluorescent channels (385, 469, 555 or 590, and 631 nm) in combination with a multiband filter set.
- Slide overview image acquisition using 1x objective lens.
- Windows 10 and 64bit programming upgrade.

The system is controlled with the Zeiss AxioVision software package and software tools and methods of Zeder et al., 2006 - 2011, and Bennke et al., 2016.

2006 - 2019:

- 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.

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

Configurations

2020 - today: LED, camera and software upgrade

- Märzhäuser stage EK 14 mot holding a maximum of 2 microscopic glass slides.
- Microscopic image acquisition via Zeiss AxioCam 702 mono.
- Slide overview image acquisition via Logitech 905 C webcam.
- Multi channel LED illumination system Zeiss Colibri 7 (385, 469, 555 or 590, and 631 nm).
- Zeiss AxioVision software package, and software tools and methods of Zeder et al., 2006 - 2011, and Bennke et al., 2016.
- Windows 10 and 64bit programming upgrade

2015 - 2019: 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).

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.

Automated cell counting on microscopic images is done by using the software ACMEtool.

ACMEtool (Automated Cell Measuring and Enumeration Tool) has been developed by M. Zeder (www.technobiology.ch).

It is a program that allows for image analysis using multiple fluorescence channels. It was developed to analyse aquatic bacterial cells concentrated on filter membranes and stained usually with DAPI and FISH. It can also be used for simple cell counting, or more complex experiments, such as microautoradiograpy combined with FISH or geneFISH. It can yield cell shape, signal intensities, and it can also be applied to other objects.

The program requires 8 bit grayscale images that follow a specific naming scheme, explained in the first video tutorial.

Since 2018 the MPI-Bremen takes care of new software developments and bug-fixing.

The program is available for free for any non-commercial purpose. The usage of the software is at the user's own risk. There is no warranty whatsoever, and there is no guarantee that the obtained results are correct. For any question, contact: A. Ellrott.

Software download: