CONICET | Buscador de Institutos y Recursos Humanos

The use of tissueculture as an experimental model requires high-quality microscopic images. Thecommon 2-D cell culture systems are being complemented by 3-D images to mimic in vivo conditions for drug screeningand regenerative medicine. This supports the current concept that themicroscope is a tool not only for magnifying biological specimens but also forimaging, measuring and recording fine structural details, as reported in theeditorial by Le Bacon (1). For our routineresearch activities, microscopy is a crucial tool. Along with creative imaging, it is alsoessential to clearly show results in cells, axon or chromosomes. Thus,laboratories involved in this research are continuously developing originalstrategies for this purpose. Stem cells, chromosome damageor, and retinalaxogenesis are always analyzed by 2-D optical, laser confocal and scanningelectron microscopy. Imaging of z levels requires several accessories and more powerfulsoftware than routine procedures. In both confocal and normal planeillumination, subsequent deconvolution would improve the imaging of deepness ofthe image but it has not yet been applied for live cultures topography.This report describesa specific adaptation of a soft procedure to generate 3-D images. Thisprocedure adds a technical tool to reconstitute the topographic view of culturestem cells, a complete group of chromosomes, or embryonic retinal axons. Thenreconstruction can also be seen as stereo red-cyan anaglyphs or videos. Thiscommunication is strictly a short description of the steps for simple imagingto recreate different virtual z levels in the reconstitution of finalstereography or video. Photos, frequentlytaken as good quality microscopic views, are processed as a series of zsequenced images by different atomized procedures to minute particles in the AdobePhotoshop software. Images are layered by modification with contraction andfiltration. Each new layer is a new photo that is reconstructed to formulate anew virtual 3-D image. Normal microscopicphotos are taken with a fixed illumination and a discrete focus on the z axis(routinely adjusted to the cells or chromosome details -disregarding upperfocus or lower edges-). It resembles a focus where everything is seen well, butpicture detail is never so high as to reconstruct the topography. For this reasonresearchers make great effort taking a sequential series of z photos for thereconstruction. The use of fluorescentantibodies gives very important in situinformation of surface topography but has serious limitations in the resolutionof the anaglyph and video reconstruction. The specimen at the microscope alwaysenables the operator to visualize the fine details of the epifluorescence, butthere are great difficulties in transforming this into a good photo, with theadequate focus of all the positive parts of the surface without becomingovercolored. This kind of photos, anaglyphs or videos always have overlapping colours,which means that fine subcellular details of the immunolabel are lost in a multicolourfluorescent universe. This rapid communicationdescribes our personal experience imaging the z stack to reconstruct with goodresolution a 3-D image, a red-cyan anaglyph, or a video using the Helicon®program. This procedure is ideal and unique for reconstructing delicatechromosomes, retinal axons or stem cells.It is well known thatthese enhancement protocols of 3D figures of microscope specimens can be usedfor constructing cells in the new 4D printers to make lab models. Our modestcontribution -Carri et al., (2)- describing part of the fundamentals of thistechnique results quite interesting and the method is valuable for severalgroups. 1 P. Le Baccon . Biology and microscopy: The friendshipstrengthens. Bioessays 34: 329 (2012). 2 Carri N.G. S. NoBermudez, J. Di Napoli, L. Fiore and G. Scicolone. Anaglyph of Retinal StemCells and Developing Axons: Selective Volume Enhancement in Microscopy Images,Anat Rec 297,770 (2014)