A simple method for multiday imaging of slice cultures.

Area of Science:

• Neuroscience
• Cell Biology
• Microscopy

Background:

• Organotypic slice cultures are crucial for neuroscience research.
• Repeated live imaging of these cultures over extended periods is often necessary but challenging.
• Existing methods may cause tissue damage or photobleaching during long-term imaging.

Purpose of the Study:

• To develop a simple and effective method for long-term, repeated live imaging of organotypic slice cultures.
• To enable multi-day imaging of individual cells within slice cultures without compromising tissue integrity.
• To facilitate advanced neuroscience experiments requiring longitudinal observation of neural tissue.

Main Methods:

• Prepared organotypic slice cultures from mouse auditory brainstem (P3-P4) and maintained them in vitro for up to 4 weeks.
• Transfected single auditory brainstem cells with fluorescent protein plasmids via electroporation.
• Developed a custom-built insulator for a perfusion chamber on an inverted wide-field microscope to maintain controlled temperature and perfuse with oxygenated ACSF for repeated imaging over multiple days.

Main Results:

• Successfully imaged fluorescently labeled single cells in organotypic slice cultures repeatedly over multiple days.
• The imaging method did not cause noticeable damage to the tissue.
• No significant photobleaching was observed during the extended imaging sessions.

Conclusions:

• The described method provides a simple, inexpensive, and non-damaging approach for long-term live imaging of organotypic slice cultures.
• This technique supports longitudinal studies in neuroscience, allowing for repeated observation of cellular dynamics in neural tissue.
• The method is suitable for various applications requiring extended imaging of cultured nervous tissue.