Download the appropriate version for your system.You can then see these emissions and take pictures of them using the confocal. Once excited, the fluorophores absorb some and release some energy, causing them to emit waves of a different length (emitted waves are longer than the waves used to excite them, due to energy loss). Fluorescent technique involves the tagging of desired targets with fluorophores and the excitation of those fluorophores using light. (*) BRIEF BACKGROUND ON FLUORESCENT TECHNIQUEĬonfocal microscopy is a type of fluorescent microscopy. Because these fluorophores are excited by different wavelengths, the image files contain 3 different channels: one for each laser used: 405nm, 647nm, and 488nm, respectively. The sample images I’m using in this mini tracing tutorial were of tissue sectioned into 100um-thick slices, (mounted, and tagged) in which DNA was labelled with DAPI (405nm), and we had stained for the immediate early gene (IEG) zif268 (Alexa 647), and retrovirally GFP-labelled cells (Alexa 488). DETAILS ON THE SAMPLE NEURON IMAGES USED IN THIS TUTORIAL
With a 2D view, this could just seem like a shorter branch, but looking at the 3D view, if the branch ends at the edge of the z plane, it’s possible that the branch is actually longer but was just included in a different section of tissue. For example, sometimes branches that are part of the neuron are cut off the slice. When tracing neurons imaged using a confocal, it helps to keep the z plane and the overall 3D shape in mind as it could interfere with some measurements like overall dendritic length.
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With this, the series of 2D photos can be combined, allowing for reconstruction and 3D visualization and analyses of the specimen in question. If the 2-dimensional slices are made of the x and y planes, the thickness that is available as a third-dimension is the z plane. A series of clear 2D images can then be taken across the z plane. Using fluorescent imaging technique (*) and an extra spatial filter, this confocal can selectively collect light at a specific z-plane, filtering out any out-of-focus illumination. The image files used in this tutorial were taken using a Leica confocal microscope (model SP8). (2012), " Fiji: an open-source platform for biological-image analysis", Nature methods 9(7): 676-682, PMID 22743772, doi: 10.1038/nmeth.2019, ( on Google Scholar).This post will show you how you can get one of these (dendrite tracing) using FIJI: a how-to FIJI on neuron tracing CONFOCAL IMAGING If you'd like to share an idea or project, please share them with the community. Individuals at many other institutions worldwide.įiji is an open source project, so everybody is welcome to contribute with plugins, patches, bug reports, tutorials, documentation, and artwork.The Saalfeld Lab at Janelia Research Campus.The Tomancak and Jug labs at MPI-CBG and the CSBD.The Eliceiri/LOCI lab at UW-Madison, home of ImageJ2.Plugins and other components have their own licenses.įiji is supported by several laboratories and institutions: See the Fiji Downloads page for Life-Line versions, etc.įiji is released as open source under the GNU General Public License.įiji builds on top of the ImageJ2 core, which is licensed under the permissive BSD 2-Clause license. Error creating thumbnail: Unable to save thumbnail to destination