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Publication Detail
Pushing the limits - using Imaris Filament Tracer to analyze dendritic spines
  • Publication Type:
  • Authors:
  • Pagination:
    819.25, ?
  • Published proceedings:
    Neuroscience Meeting Planner Online
  • Name of conference:
    Society for Neuroscience
  • Conference place:
    San Diego, California, USA
  • Conference start date:
  • Conference finish date:
  • Keywords:
    imaging, dendritic spines, Imaris
Hippocampal neurones in rodent brain slices are filled with fluorescent dye via patch electrodes and imaged using confocal microscopy. Our projects require data about dendritic spine densities, classification of spines (thin, stubby, mushroom etc) and changes in spine sizes. Dendritic spine heads (diameter 0.2 - 1.0 µm) are close to the resolution limit of light microscopy (~0.2 µm), challenging accurate measurement. We have used 3 main approaches: 1) Classification. Z-stacks are visualised with ImageJ (NIH, Bethesda) and each spine is classified against set criteria (De Simoni & Edwards 2006). In spite of objective criteria, results vary between investigators and it uses a 2D approach to analyze a 3D object. 2) Intensity profile. The z-stack is explored in ImageJ and the line tool is used to produce an intensity profile across each spine head. A Gaussian curve is fitted to the profile and spine width is estimated as a multiple of the SD based on calibration with beads. This approach is 2D and slow but is easy to understand and relatively objective. 3) Imaris Filament Tracer. Imaris is a powerful 3D imaging package produced by Bitplane (Zurich). Filament Tracer is a module of Imaris which can model neuronal dendrites and spines and produce a wealth of statistical data. To our knowledge, Imaris is the leading software package in the field of 3D analysis of dendrites/spines and promises more objectivity than manual methods. (See www.bitplane.com for publications.) It is excellent for collecting spine counts and densities but some issues have arisen when using Imaris to measure individual spines in our experimental set up: a) Pixelation of data. Outputs such as spine head diameter are clustered rather than smoothly distributed because Imaris measures in whole voxels. b) Possible distribution artefacts. Our data from Imaris show a double frequency distribution for spine head diameter. While this may reflect a true biological division in the distribution, various tests suggest that it is an artefact, probably due to the clustering of values (a). This is important to resolve because, if real, it may reflect spine classes such as thin and mushroom which would be very valuable in analysing plasticity mechanisms. c) A remaining element of subjectivity. Although considerably more objective than manual methods Filament Tracer wizard requests user inputs that have some impact on the measurements produced. Bitplane are working with us to resolve these issues that stem from our use of Imaris on objects close to or even at the resolution limit of light microscopy. Once resolved this software will prove an even more powerful tool in our investigations of synaptic function and plasticity.
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