Mitotic spindle at high resolution

Acquisition @ Scuola di Microscopia 2019
Hematopoietic cells CD34+ isolated from chronic myeloid leukemia patient and stained for centrosomal and mitotic spindle markers (pericentrin is shown in red and gamma-tubulin green).

Mitotic cell

Confocal Spinning Disk acquisition

(Z-projection: Maximum Intensity)
Gamma-tubulin intensity profile (yellow arrow) is shown on the right

Mitotic cell

Video Confocal Super-resolution (VCS) acquisition

(Z-projection: Maximum Intensity)
Gamma-tubulin intensity profile (yellow arrow) is shown on the right

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 60x oil objective (1,35 NA) and sectioning the slice in Z with a step size of 0,25 um for confocal spinning disk and 0,2 VCS to obtain a total Z-stack of about 3 um.

Only for VCS:
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 Martina Ghetti
IRCCS, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (I.R.S.T.) S.r.l.
http://www.irst.emr.it

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