TITLE: Preparation of human Red Blood Cells for confocal imaging
AUTHORS: Marie Anne Balanant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used to label red blood cells (RBC) for confocal imaging. Fluorescent beads are used to calibrate voxel size on the confocal microscope. The imaging protocol was optimised to obtain high quality data, by mounting the cells in a high refractive index and optimising acquisition parameters.</div><div class = "text-block">Spherical aberration results in a decrease in image intensity when the imaging plan gets further away from the coverslip. It is caused by difference in refractive indices between the different components present over the beam pathway. In order to reduce spherical aberration, the cells are fixed in glutaraldehyde after staining so they can be resuspended in a medium with a high glycerol content and maintain their shape. Glycerol increases the reflective index of the medium, bringing it closer to the reflective indices of the coverslip and the immersion oil.</div><div class = "text-block">Acquisition parameters are first chosen using the system optimisation function, then adjusted. The z-axis step size is chosen as close as possible to the lateral resolution to avoid under- or over-sampling. The pinhole is reduced to 50 μm to increase resolution and reduce out-of-focus light. A 2-step line averaging is selected during scanning to remove noise.</div><div class = "text-block">Data is analysed and quantitative volume and surface area values were extracted using a home-written matlab code (not included in this protocol).</div></div>

[STEPS]
?. [Imaging chamber]
In the center of the chamber, add  of 4µm calibration beads, and let the slide dry in the dark
2 µl
?. [Imaging chamber]
Prepare imaging chamber by using double-sided tape as a spacer between two coverslips : cut a square within the tape such as to form a frame. Without removing the non-adhesive cover, place this pre-cut frame on a coverslip. The chamber will be closed at the end of the protocol, by removing the non-adhesive cover and placing a second coverslip on top of the double-sided tape.
?. [RBC isolation]
Centrifuge fresh whole blood in a citrate tube for
[at 1000g]
Until screening for diseases is completed, consider all blood samples as potentially contagious and use the appropriate PPE
Ethical clearance must be obtained before any exeriment using human blood samples.
?. [RBC isolation]
Remove supernantant and buffy coat, keeping the RBC fraction
?. [Staining]
Wash of RBC in PBS, centrifuge for , and remove the supernantant
5 µl
500 µl
[at 1000g]
?. [Staining]
Resuspend in PBS
500 µl
?. [Staining]
Add of DiI and incubate for at
5 µl
[in the dark]
?. [Staining]
Wash 3 times in PBS () and remove supernanant
500 µl
[at 1000g]
?. [Fixation]
Mix of the pellet containing the RBC with of glutaraldehyde in PBS
10 µl
20 µl
3 Volume Percent
Prepare glutaraldehyde solution under a fume hood
?. [Fixation]
Incubate
[in the dark]
?. [Fixation]
Wash 3 times in PBS () and remove supernanant
500 µl
[at 1000g]
?. [Imaging]
Resuspend in glycerol to obtain a glycerol solution, deposit on the prepared slide containing the calibration beads, then close the imaging chamber by placing a second coverslip on top of the double-sided tape spacer
50 Volume Percent
?. [Imaging]
Image with x63 oil immersion objective