Sterile Microscopic Imaging of MCS CMOS MEAs
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1
Tampere University of Technology, Computational Biophysics and Imaging Group, Finland
Motivation
In in vitro electrophysiology, it is crucial to be able to visually observe the living cells and cultures. The recently emerging CMOS MEAs [1,2] offer great spatial electrophysiological measurement resolution. They have mainly been built on printed circuit boards, and are not transparent. Since in long-term studies [1], the cultures need to be kept sterile, microscope imaging is challenging. To this end, I have designed and produced lids which allow sterile imaging of the CMOS MEAs using upright microscopes. Lid designs are provided for both air and water-immersion microscopy.
Material and Methods
Lids were designed for the CMOS MEA models CMOS-MEA16-CC and CMOS-MEA32-CC for the CMOS-MEA5000-System (Multi Channel Systems) using FreeCAD 0.14 (Fig. 1), 3D printable files produced using Cura 15.02.1 (Ultimaker), and 3D printing was done with Ultimaker 2. Working prototypes were printed with PLA (Ultimaker). Round microscope cover glasses (ø25 mm, borosilicate glass, thickness no. 1,5, VWR) were attached to the bottoms of the lids (Fig. 1). Production versions will be printed of Nylon 680 FDA material (taulman3D) and the cover glasses attached with Sylgard 184 (Dow Corning) for biological compatibility. The design is intended to be autoclavable.
The lid for in-air imaging was designed to allow a small amount of cell culture medium to remain in the CMOS MEA well without the medium touching the lid from below. The lid for water immersion imaging was designed so that with a little medium in the CMOS MEA well, the cover glass comes into contact with the medium below the cover glass, and water can be placed on the cover glass in the lid to allow immersion of a microscope objective. Autoclaved sterile lids can be put on MEAs inside a sterile laminar flow hood prior to imaging. The lids were designed to also fit in the CMOS-MEA5000-System so that imaging and measurement can be performed concurrently.
The imaging was done with a Nikon upright Ni-E microscope and a CoolLED pE-4000 light source. In-air imaging for performed using a Nikon Plan Fluor ELWD 20x/0.45 objective (working distance 7.4 mm, with cover glass correction ring) with 700 µm of Milli-Q water in the CMOS MEA well. Water immersion imaging was performed using a Nikon NIR Apo 40x/0.80w objective (working distance 3.5 mm, no cover glass correction ring, for greater image quality, an objective with a cover glass correction ring is recommended), with a little Milli-Q water in the CMOS MEA well and in the lid above the cover glass.
Results
The imaging quality (Fig 1) was found adequate for sterile imaging neuronal cells on CMOS MEAs. The FreeCAD and ready-to-print files Ultimaker 2 files are provided as open source, and can be freely downloaded from GrabCAD (https://grabcad.com/library/cell-culture-well-cover-for-sterile-microscopy-imaging-1).
Discussion
The in-air imaging is aimed at regular long-term culture checkup imaging, and the water immersion imaging for more detailed imaging and fluorescent imaging, thus facilitating visual assessment of CMOS MEAs during long-term culturing. It should be noted in that with reflective lighting via the microscope objective from above with the cells on the dark CMOS surface, the contrast may be low, and lighting strategies should be developed further.
Conclusion
CMOS MEA lids were designed and produced. Imaging of CMOS MEAs was tested with the first working prototypes without cells. The lids functioned as designed and the image quality was found adequate.
References
[1] W. Gong, et al., "Long-term, high-spatiotemporal resolution re-cording from cultured organotypic slices with high-density microelectrode arrays," in Proc. 18th Int. Conf. Solid-State Sensors, Actuators and Microsystems, Anchorage, AK, 2015, pp. 1037-1040. http://dx.doi.org/10.1109/TRANSDUCERS.2015.7181103
[2] G. Bertotti, et al., "A CMOS-based sensor array for in-vitro neural tissue interfacing with 4225 recording sites and 1024 stimulation sites," in Proc. 2014 IEEE Biomedical Circuits and Systems Conference, Lausanne, 2014, pp. 304-307. http://dx.doi.org/10.1109/BioCAS.2014.6981723
Figure Legend
Fig. 1. FreeCAD drawings of the CMOS MEA lids for sterile microscopy imaging seen for the in-air imaging from above (A) and below (B), and for water immersion imaging from above (C) and below (D). Microscope cover glasses are shown attached to the lids for illustration only and do not appear in the FreeCAD designs for 3D printing. (E)-(G) The two different 3D printed CMOS MEA microscopy cover prototypes, the in-air cover on the left and the water immersion cover on the right in each panel. Exemplary images of a CMOS MEA (CMOS-MEA32-CCs (MCS)) with the (H) 20x objective in air and (I) 40x objective in water immersion, using the respective CMOS MEA covers reported in this report.
Acknowledgements
The work has been supported by Jane and Aatos Erkko Foundation, Finland, under the project Biological Neuronal Communications and Computing with ICT.
Keywords:
Microscopy,
Sterile culture,
CMOS MEA,
MEA lid
Conference:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays, Reutlingen, Germany, 28 Jun - 1 Jul, 2016.
Presentation Type:
Poster Presentation
Topic:
MEA Meeting 2016
Citation:
A. Tanskanen
JM
(2016). Sterile Microscopic Imaging of MCS CMOS MEAs.
Front. Neurosci.
Conference Abstract:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays.
doi: 10.3389/conf.fnins.2016.93.00048
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Received:
22 Jun 2016;
Published Online:
24 Jun 2016.
*
Correspondence:
Dr. Jarno M A. Tanskanen, Tampere University of Technology, Computational Biophysics and Imaging Group, Tampere, Finland, tanskanen@ieee.org