High resolution imaging of 3d organoids at low magnification

Acquisition @ Scuola di Microscopia 2019
A) Organoids cultured in Matrigel on optical multi-well plates and generated from primary stem cells from colon cancer biopsies and stained for cytoskeleton by Phalloidin-568.

Organoids cultured in Matrigel

Widefield acquisition

The acquisitions obtained were transformed into a Z-projection (Average Intentiy)

Confocal Spinning Disk acquisition

B) Organoids cultured in Matrigel on optical multi-well plates and generated from primary stem cells from colon cancer biopsies and stained for cytoskeleton by Phalloidin-568

Organoids cultured in Matrigel

Confocal Spinning Disk acquisition

The acquisitions obtained were transformed into a Z-projection (Maximum Intensity

Video Confocal Super-resolution (VCS) acquisition

Acquisition parameters:
Images acquisition was performed through an Olympus IX83 inverted microscope equipped with X-Light V2 spinning disk combined with a VCS (Video Confocal Super resolution) module (CrestOptics), Prime BSI Scientific CMOS Camera (Photometrics) and LDI laser source (89 North), integrated by Crisel Instruments.
The images were acquired by using Metamorph software version 7.10.2. (Molecular Devices) with a 10x air objective (0,4 NA) and sectioning the slice in Z with a step size of 3 um for widefield, confocal spinning disk and VCS to obtain a total Z-stack of about 63 um (Fig A) and 45 um (Fig B).

Only for B (picture on the right):
In order to achieve super-resolution, raw data obtained by the VCS module have been processed with a modified version of the joint Richardson151 Lucy (jRL) algorithm (Strolh et al. 2015; Ingaramo et al., 2014; Chakrova et al. 2016), where the out of focus contribution of the signal has been explicitly added in the image formation model used in the jRL algorithm, and evaluated as a pixel-wise linear “scaled subtraction” (Heintzmann et al., 2006) of the raw signal.

Sample courtesy of Chiara Cordiglieri
INGM Imaging Facility
Istituto Nazionale Genetica Molecolare (Milan)
http://www.ingm.org/en/imaging/

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