OPTIMIZATION OF 3D NEURON-SURFACE INTERFACE IMAGING BY SEM/FIB MICROSCOPE

Introduction

In the last decade there is increasing in the use of SEM/FIB for imaging and studying the neural-surface^1,2,34 or neural-electrode^5,6,7,8. Studying the cell interface is important for various applications such as tissue culture engineering, electrical stimulation and more. However, it is of critical importance to preserve the cellular morphology during the fixation and dehydration process. The aim of the current research is to optimize drying, membrane staining and imaging techniques for the study of the neuron-electrode or neuron-surface interface.

Materials and Methods

Retinal Pigmented Epithelium (ARPE) and photoreceptor precursors (PRP) cells were cultured on glass for 24hrs. We evaluated 4 different drying techniques (critical point drying machine (CPD)^9,10, hexamethyldisilazane (HMDS) ¹¹, Resin¹² and OTOTO¹³ air dry and four different staining techniques (Osmium¹⁴, Osmium+salts, OTOTO, osmium + uranil^15,16,17) the cell morphology was investigated by the FIB/SEM technique. The ARPE are characterized by fine protrusions, which are easily broke in response to change in cellular morphology and thus can serve for evaluating the drying and fixation effect on cellular structures. Scanning electron microscope (SEM) imaging was used to measure the preservation of the cell-surface interface by analyzing the percentage of broken protrusions. In addition, Ga⁺ Focus Ion beam (FIB) milling, SEM and HR-SEM imaging were used to evaluate the cellular membrane structure contrast when stained by the various staining techniques. Furthermore, we studied the cell interface with a micro-well scaffold fabricated by SU-8 photolithography and with a gold mushroom-shaped electrodes printed by a 3D two-photon based nano-printing (Nanoscribe GmbH, Germany).

Results and Discussion

The percentage of broken cellular extensions in the resin drying technique (22.57%,±13.3) and OTOTO air dry (27.74%,±14) were significantly lower as compared with CPD (59.73%, ±10.3) and HMDS (61.42%,±13.7) (mean, STDEV, p < 0.001). Furthermore, the inner cellular structures contrast was enhanced by the resin drying and OTOTO staining accompanied with a higher membrane contrast and with the lowest shrinkage (less artifacts).

Conclusion

We optimized the fixation and staining protocols and studied cells interface with different surfaces including 3D surfaces and structures. Our results reveal that the resin and OTOTO air dry technique significantly preserve cellular structure as compared with CPD or HMDS techniques. The OTOTO staining yields the most detailed images of the cell, enables the visualization of cell organelles and membrane. Finally, this study highlights that a proper drying and fixation method is vital for the preservation of natural cellular morphology which is of great importance for the study of cell-surface interface.

References

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