A simple approach to the virtual reconstruction of difficult z axis of 2-D images

CARRI, N.G.; NOO BERMÚDEZ, S.; TORRES RUIZ, N.M.; BOLZAN, A.D.; SCICOLONE, G.

La Plata

Workshop; Imaging Techniques for Biotechnology and Biomedical Applications; 2016

CONICET-UNLP

The use of tissue culture as an experimental model requires high-quality microscopic images. The common 2-D cell culture systems are being complemented by 3-D images to mimic in vivo conditions for drug screening and regenerative medicine. This supports the current concept that the microscope is a tool not only for magnifying biological specimens but also for imaging, measuring and recording fine structural details, as reported in the editorial by Le Bacon (1). For our routine research activities, microscopy is a crucial tool. Along with creative imaging, it is also essential to clearly show results in cells, axon or chromosomes. Thus, laboratories involved in this research are continuously developing original strategies for this purpose. Stem cells, chromosome damageor, and retinal axogenesis are always analyzed by 2-D optical, laser confocal and scanning electron microscopy. Imaging of z levels requires several accessories and more powerful software than routine procedures. In both confocal and normal plane illumination, subsequent deconvolution would improve the imaging of deepness of the image but it has not yet been applied for live cultures topography. This report describes a specific adaptation of a soft procedure to generate 3-D images. This procedure adds a technical tool to reconstitute the topographic view of culture stem cells, a complete group of chromosomes, or embryonic retinal axons. Then reconstruction can also be seen as stereo red-cyan anaglyphs or videos. This communication is strictly a short description of the steps for simple imaging to recreate different virtual z levels in the reconstitution of final stereography or video. Photos, frequently taken as good quality microscopic views, are processed as a series of z sequenced images by different atomized procedures to minute particles in the Adobe Photoshop software. Images are layered by modification with contraction and filtration. Each new layer is a new photo that is reconstructed to formulate a new virtual 3-D image. Normal microscopic photos are taken with a fixed illumination and a discrete focus on the z axis (routinely adjusted to the cells or chromosome details -disregarding upper focus or lower edges-). It resembles a focus where everything is seen well, but picture detail is never so high as to reconstruct the topography. For this reason researchers make great effort taking a sequential series of z photos for the reconstruction. The use of fluorescent antibodies gives very important in situ information of surface topography but has serious limitations in the resolution of the anaglyph and video reconstruction. The specimen at the microscope always enables the operator to visualize the fine details of the epifluorescence, but there are great difficulties in transforming this into a good photo, with the adequate focus of all the positive parts of the surface without becoming overcolored. This kind of photos, anaglyphs or videos always have overlapping colours, which means that fine subcellular details of the immunolabel are lost in a multicolour fluorescent universe. This rapid communication describes our personal experience imaging the z stack to reconstruct with good resolution a 3-D image, a red-cyan anaglyph, or a video using the Helicon® program. This procedure is ideal and unique for reconstructing delicate chromosomes, retinal axons or stem cells. It is well known that these enhancement protocols of 3D figures of microscope specimens can be used for constructing cells in the new 4D printers to make lab models. Our modest contribution -Carri et al., (2)- describing part of the fundamentals of this technique results quite interesting and the method is valuable for several groups. References 1- P. Le Baccon . Biology and microscopy: The friendship strengthens. Bioessays 34: 329 (2012). 2- Carri N.G. S. No Bermudez, J. Di Napoli, L. Fiore and G. Scicolone. Anaglyph of Retinal Stem Cells and Developing Axons: Selective Volume Enhancement in Microscopy Images, Anat Rec 297,770 (2014)