Structured Illumination Microscopy Imaging

Images were captured with a commercial inverted SIM microscope (Zeiss ELYRA, Oberkochen, Germany) using an oil-immersion objective (Plan-Apochromat 63 × /1.4 Oil Dic M27) (Gustafsson, 2000 (link); Wegel et al., 2016 (link)). Excitation of the fluorophores was performed by laser illumination at 642 nm (Alexa Fluor 647), 561 nm (Alexa Fluor 555), 488 nm (Alexa Fluor 488), and 405 nm (DAPI) and fluorescence light was filtered by appropriate detection filters: LP 655 (Alexa Fluor 647), BP 570–620 + LP 750 (Alexa Fluor 555), BP 495–550 + LP 750 (Alexa Fluor 488), and BP 420–480 + LP 750 (DAPI). Images were recorded with five rotations and five phase steps of the illumination pattern. Recorded data were processed with the ZEN imaging software (Zeiss). They were processed under standard ELYRA settings of the manual mode, selecting the Raw Scale option to keep the original dynamic range and therefore ensure a reliable comparison of the actual sample and the control samples. Following the structured illumination processing, the four channels were aligned (ZEN imaging software).

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Forero A., Rivero O., Wäldchen S., Ku H.P., Kiser D.P., Gärtner Y., Pennington L.S., Waider J., Gaspar P., Jansch C., Edenhofer F., Resink T.J., Blum R., Sauer M, & Lesch K.P. (2017). Cadherin-13 Deficiency Increases Dorsal Raphe 5-HT Neuron Density and Prefrontal Cortex Innervation in the Mouse Brain. Frontiers in Cellular Neuroscience, 11, 307.

Publication 2017

ELYRA Plan-Apochromat 63 × /1.4 Oil Dic M27 ZEN imaging software

Alexa fluor 488 Alexa fluor 555 Alexa fluor 647 Dapi Fluorescence Illumination Immersion Microscope

Corresponding Organization : Sechenov University

Other organizations : Institut du Fer à Moulin, Inserm, Universität Innsbruck, University of Basel, University Hospital of Basel

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Variable analysis

independent variables

Type of microscope (commercial inverted SIM microscope (Zeiss ELYRA))
Objective (oil-immersion Plan-Apochromat 63 × /1.4 Oil Dic M27 objective)
Excitation wavelengths (642 nm, 561 nm, 488 nm, 405 nm)
Fluorescence detection filters (LP 655, BP 570–620 + LP 750, BP 495–550 + LP 750, BP 420–480 + LP 750)
Number of rotations and phase steps of the illumination pattern (five rotations and five phase steps)

dependent variables

Fluorescence images of the samples

control variables

Image processing settings (standard ELYRA settings, Raw Scale option to keep original dynamic range)

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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