AFM/CLSM data visualization and comparison using an open‐source toolkit

Abstract

There is a vast difference in the traditional presentation of AFM data and confocal data. AFM data are presented as surface contours while confocal data are usually visualized using either surface- or volume-rendering techniques. Finding a common meaningful visualization platform is not an easy task. AFM and CLSM technologies are complementary and are more frequently being used to image common biological systems. In order to provide a presentation method that would assist us in evaluating cellular morphology, we propose a simple visualization strategy that is comparative, intuitive, and operates within an open-source environment of ImageJ, SurfaceJ, and VolumeJ applications. In order to find some common ground for AFM-CLSM image comparison, we have developed a plug-in for ImageJ, which allows us to import proprietary image data sets into this application. We propose to represent both AFM and CLSM image data sets as shaded elevation maps with color-coded height. This simple technique utilizes the open source VolumeJ and SurfaceJ plug-ins. To provide an example of this visualization technique, we evaluated the three-dimensional architecture of living chick dorsal root ganglia and sympathetic ganglia measured independently with AFM and CLSM.

Healthcare Cloud Integration using Distributed Cloud Storage and Hybrid Image Compression

“…Custom acquisition, analysis and processing plugins can be developed using ImageJ's built-in editor and a Java compiler. Userwritten plugins make it possible to solve many image processing and analysis problems, from three-dimensional live-cell imaging [21], to radiological image processing [22], multiple imaging system data comparisons [23] and automated hematology systems [24]. ImageJ can read many image formats including TIFF, PNG, GIF, JPEG, BMP, DICOM, FITS, as well as raw formats.…”

Integration of confocal and atomic force microscopy images

“…The 200 nm resolution limit of the CLSM is restricted by the diffraction limit of the microscope objective and does not permit accurate evaluation of the sample height. The z-resolution of CLSM for any given wavelength is always at least 2 times less than the corresponding xy-resolution (Rajwa et al, 2004). Rajwa et al (2004) and McNally et al (2005) have emphasized that difference in image collection modes between AFM and CLSM make any image comparison a very difficult task and have pointed out the need for an effective integration of these techniques.…”

“…CLSM using fluorescence mode can collect emissions originating from the interior of a biological sample and therefore CLSM has been widely used to map intracellular mechanisms. When antibodies are used to stain specific biological structures, fluorescent confocal microscopy provides a powerful way to simultaneously map the distribution of different cellular components (Dailey et al, 1999;Rajwa et al, 2004). The 200 nm resolution limit of the CLSM is restricted by the diffraction limit of the microscope objective and does not permit accurate evaluation of the sample height.…”

Characterization of Embryonic Stem (ES) Neuronal Differentiation Combining Atomic Force, Confocal and DIC Microscopy Imaging

“…Confocal laser scanning microscopy (CLSM) utilizes the optical pathway of a regular optical microscope and in combination with immunofluorescence histochemistry has been widely used to simultaneously map the distribution and localization of different cellular components (extracellular and intracellular macro-molecules, including proteins, nucleic acids and lipids, as well as intracellular ions such as calcium) helping to understand intracellular mechanisms (Dailey et al, 1999;Rajwa et al, 2004). Moreover, it is possible to monitor the process and dynamics of living cells with high temporal resolution (Hell, 2007;Park et al, 2010).…”

“…The 200 nm resolution limit of the CLSM is restricted by the diffraction limits of the microscope objective and does not permit accurate evaluation of the sample height. The zresolution of CLSM for any given wavelength is always at least 2 times less than the corresponding xy-resolution thus restricting a detailed structural image that can be achieved when exploring biological samples (Doak et al, 2008;Rajwa et al, 2004). Great advances in optical microscopy such as near field scanning optical microscopy (NSOM), stimulated emission depletion microscopy (STED), photoactivated localization microscopy (PALM), and stochastic optical reconstruction microscopy (STORM) have been achieved to overcome the optical diffraction limit (Hell, 2007 and2009;Schermelleh et al, 2010), but there are still constraints for realizing time-resolved dynamics or three dimensional imaging (Park et al, 2010).…”