With the development of direct-detection camera and better algorisms dealing with sample movements and image processing, TRPV1 channel structure in the closed state was determined by cryo-EM at an astonishing resolution of 3.4 Å (Liao et al., 2013).
Colour imaging may be considered as a channel-by-channel intensity image processing scheme.
Decolorization is widely used in monotonic displaying, black-and-white printing, single-channel image processing, and many computer vision applications.
A number of single-channel image processing applications, including edge detection, image segmentation, color augmentation, and image stylization, are demonstrated to verify the feasibility of the proposed decolorization method.
Separate images for DAPI, Cy3, and FITC staining were captured digitally from double-stained specimens into colour-separated components using an AxioCam digital camera (Carl Zeiss, Göttingen, Germany) and AxioVision 2.05 multi-channel image processing (Carl Zeiss Vision GmbH, Hallbergmoos, Germany).
Image processing, including overlay of different fluorescence channels and generation of maximum intensity projections (MIP) of z-stacks were done with Zeiss AxioVision software (version 4.8.1).
To do so: Open all channels after CLAHE application with image processing software and identify the first and last z -slide that has no distortions in any channel.
Open all channels after CLAHE application with image processing software and identify the first and last z-slide that has no distortions in any channel.
(because eGFP signal bleeds into the Venus channel, overlap was confirmed via image processing; see Figure 1 figure supplement 1 ).
Given the substantially improved image quality, the increased post-acquisition image processing time is largely acceptable, though a dual illumination channel setup will be required to achieve the gains in frame rate made possible by flood illumination.
