Time-Lapse Imaging of Cortical Neuron Radial Migration in Transduced Mouse Embryonic Brain Slices

Samenvatting

Source: Wiegreffe, C., et. al., Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation. J. Vis. Exp. (2017)

This video demonstrates time-lapse confocal microscopy to observe cortical neuron radial migration from the subventricular zone to the cortical plate via radial glial scaffolds in transduced mouse embryonic brain slices.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Time-Lapse Imaging

1. Microscope Setup
   1. Set a climate chamber placed onto the stage of an inverted confocal microscope to 37 °C and 5% carbon dioxide atmosphere. Use a hybrid detector to reduce laser intensity and improve cell viability. For detailed analysis, use an extra-long working distance 40X dry objective with a numerical aperture of 0.6 or higher.
      Note: All components of the microscope need to be pre-warmed to 37 °C for 2 - 3 h before imaging to provide a stable focus during the imaging process.
2. Slice Imaging
   1. Select a brain slice for imaging from the 6-well plate containing the membrane inserts using an inverted fluorescence microscope.
      Caution: Avoid prolonged exposure time to ultraviolet light as this may cause cellular photodamage.
      NOTE: Select a slice with bright single neurons in the upper SVZ (Subventricular zone) migrating radially towards the pial surface of the slice. Fine fluorescent processes of radial glial cells that span the entire CP (Cortical plate) indicate an intact radial glial scaffold, which is used by radially migrating cortical neurons.
   2. Transfer the membrane insert with the slice, which was selected for time-lapse imaging, into a 50 mm diameter glass bottom dish containing 2 ml of slice culture medium with the help of forceps. Place the dish into the climate chamber of the confocal microscope.
   3. Set the resolution to 512 by 512 pixels and increase the scan speed from 400 Hz to 700 Hz in order to increase the frame rate from about 1.4 to about 2.5 frames per second, respectively. Use no more than two times averaging. Define a z-stack through the electroporated region (usually 400-100 μm) with a step size of 1.5 μm. Collectively, these settings allow for a sufficient resolution and brightness of the image while keeping photodamage low during acquisition. Start the time-lapse series by taking a z-stack every 30 min.
      NOTE: Prolonged exposure of the slice to laser light will induce photodamage reducing the viability of the cells. Adjust the exposure time to laser light accordingly.
   4. Analyze time-lapse series using ImageJ software (https://imagej.nih.gov/iij/) or equivalent.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
6-well plate	Corning	351146
Inverted fluorescence tissue culture microscope	Leica	DM IL LED
Confocal laser scanning microscope	Leica	TCS SP5II
Hybrid detector	Leica	HyD
Objective, 40x/0.60 NA	Leica	11506201
Microscope temperature control system	Life Imaging Services	Cube, Brick & Box
Cell culture insert	Millipore	PICM0RG50
Microgrinder	Narishige	EG-45	Use 38° angle for beveling
Microinjector	Parker Hannifin	052-0500-900	Picospritzer III
Tweezers with 5 mm platinum disk electrodes	Sonidel	CUY650P5
Micropipette puller	Sutter Instrument	P-97
Box filament	Sutter Instrument	FB255B	2.5 mm x 2.5 mm
Micro-spoon spatula	VWR	231-0191	185 mm x 5 mm
Glass bottom dish, 50 mm	World Precision Instruments	FD5040-100