The quality of cell patterning was assessed 72 hours following cell seeding. Prior to fixation (3.6% PFA for 10 min at ambient conditions), astrocyte cytoplasms were labelled with CMFDA (1 µM, 1 hour at 37 °C and 5% CO2), and astrocyte nuclei were labelled with Hoechst 33258 (1.6 µg mL−1, 20 min at 37 °C and 5% CO2). All imaging was performed on an Olympus BX53 using an 8-bit XC50 camera, mercury bulb illumination, 10xobjective (UMPLFLN, Olympus) and GFP and DAPI filter sets (470–495/550 nm and 360–370/410, Em/Ex).
Umplfln
Manufactured by Olympus
The UMPLFLN is a high-numerical-aperture, long working distance objective lens designed for use in optical microscopy. It is capable of providing high-resolution imaging while maintaining a relatively long working distance. The core function of the UMPLFLN is to serve as an objective lens for various microscopy applications.
Automatically generated - may contain errors
3 protocols using umplfln
1
Astrocyte Cytoplasm and Nucleus Labeling
2
Fluorescence Imaging of Neural Activity
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Wide-field fluorescence imaging was performed on an Olympus Fluoview 1000 system (excitation wavelength at 488 nm, emission filter wavelength centered at 510 nm; Olympus, Corp.). The fluorescence images were captured by an EMCCD camera (iXon Ultra 888, Andor, Ltd.). A water immersion objective (0.3 NA, UMPLFLN, Olympus, Corp.) was used for GCaMP5G-expressing fly brain imaging. A objective (0.14 NA, XLFluor2X/340, Olympus, Corp.) was used for GCaMP6f-expressing mouse brain slice imaging.
3
Imaging Neural Activity in Embedded Fish
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For the imaging of neural activity in embedded fish in experiments 2, 4, and 5, a 5-d-old larva was embedded at the bottom of the central glass compartment in 2% low-gelling-temperature agarose (Merck). The agarose was carefully removed around the eyes and mouth upon hardening and the arena was filled with 3 mL of water. Calcium fluorescence was recorded with a custom-made epifluorescence microscope equipped with a ×10 water-immersion objective (UMPLFLN, Olympus), a set of green fluorescent protein emission-excitation filters (FGL400, MD498, and MF525-9, Thorlabs) and a mounted blue light-emitting diode (LED) controlled by a driver (MWWHL4, LEDD1B, Thorlabs). Images were collected at 5 Hz via a custom-written Python script using the Pymba wrapper for interfacing with the camera (Mako G319B, Allied Vision).
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