Neuronal Image Analysis - Fast and Easy
Leica MM AF Module "Neurite Outgrowth"
Dr. Oliver Schlicker, Leica Microsystems
For the fields of neurobiology and neurology, Leica Microsystems offers the application module “Neurite Outgrowth” for its software platform Leica MM AF powered by MetaMorph® for multidimensional image acquisition and analysis. Using Neurite Outgrowth the user can easily quantify neuronal branches, neurite lengths and also the number of neuron cell bodies. This module provides the user with the additional benefit that most of the steps needed for segmentation and structure counting are done in an automatic way even at images with uneven background.
Up until the 1990s, researchers thought that neurogenesis was finished in the adult human organism. But researchers also knew that in the brains of singing birds, for example, neuronal development occurs after sexual maturity. Neurogenesis plays a crucial role in learning and refining pairing songs. New investigations in neurogenesis show that in older humans and other mammals, a progeny of neuronal stem cells and formation of new neurons can take place. For many mammals (rats, mice) it is shown that the formation of new cells is dependent on intellectual and physical activity. It is assumed that adult neurogenesis is regulated in humans the same way. The field of neurogenesis describes the growth of neuronal cells from distinct stem or progenitor cells. One can distinguish between neurogenesis during embryonic development (prenatal neurogenesis) and after birth (postnatal neurogenesis). During embryogenesis the cortex is built up from the ventricular system. First, neurons develop, which then migrate towards the outer brain surface using processes of radial glia cells and then build up the cortex.
Various options for identifying neurites
The Neurite Outgrowth application module provides options that, depending on image content and acquired wavelengths, enable the researcher to isolate cell bodies and identify their attached neurites with different degrees of sensitivity. Image sets of two wavelengths, which include the stained nucleus in one image, will typically provide the greatest degree of accuracy for isolating closely grouped cell bodies. The use of a nuclear stain insures a one-to-one correlation between nuclei and cell bodies (Fig. 3).
Cell bodies and associated neurites can still be isolated and identified even if only a single wavelength of the cell body sample is available. Different options enable the researcher to customise the settings to obtain the best possible results. After the application module has processed the image, the researcher can use the “Cellular Results” table to view data belonging to an individual cell or group of cells interactively by clicking the cell(s) in the image (Figs. 4 + 5).
Easy-to-use dialog box
Neurite Outgrowth dialog box settings are used to define the measurement criteria needed to identify and isolate cell bodies, neurites, and the associated physical characteristics of each. This dialog box can optionally produce separate images for the neurite result or the nuclear result.
Innovative software solutions
Leica Microsystems offers a variety of software solutions for a multitude of applications in microscopic imaging. Besides Leica Application Suite (LAS), a modular-designed software for transmitted light and basic fluorescence applications, Leica Microsystems also offers LAS AF (AF = Advanced Fluorescence) imaging software optimised for multidimensional acquisition in real time. The key benefit of Leica MM AF is the huge number of image analysis tools, which complement LAS AF’s capabilities for microscope control and realtime image acquisition. The optimal solution for both real-time acquisition and professional data processing and analysis is the combination of LAS AF and Leica MM AF.
Additional modules available
The Neurite Outgrowth analysis module is only one of the various modules of Leica MM AF which help save the time to get a perfect result. The many steps, like thresholding, background, and shading correction, that are normally necessary, are reduced to entering a few values.
- Fig. 1: Growth cones of growing neurites. The outer extremes of a neuron show which neurons are particularly agile. This agility is due to a tremendous polymerisation and depolymerisation of actin (red). The neuronal extensions contain a high amount of tubulin (blue). The nuclei located in the cell bodies are stained green.
- Fig. 2: Primary neuronal culture (false colour). Cultured neurons are stained with a dye that stains whole cells including the finest extensions. Note that the signal-to-noise ratio is very high (bad) due to weak staining.
- Fig. 3: Neurite Outgrowth window. In this window the user can select either fluorescence or transmission illumination. Furthermore the user only has to enter the approximate width and area of the cell bodies and then define the minimum and maximum width of the extensions to detect.
- Fig. 4: Resulting window of detecting neurites. The resulting window contains the information the program has calculated. Here, each colour represents a single neuron with its neurites.
- Fig. 5: Quantitative results of the neurite outgrowth calculation. Quantitative results contain, for example, the number of cells processed, their mean length, and the number of branches of each cell. After clicking on one row in the table, the representing cell is shown in the branching window (see Fig. 4).