scholarly journals Fluorescence excitation analysis by two-photon confocal laser scanning microscopy: a new method to identify fluorescent nanoparticles on histological tissue sections

2012 ◽  
pp. 5545 ◽  
Author(s):  
Edmond Kahn ◽  
Tissot ◽  
Frere ◽  
Dauphin ◽  
Boumhras ◽  
...  
Keyword(s):  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
New Method ◽  
Confocal Laser ◽  
Fluorescent Nanoparticles ◽  
Fluorescence Excitation ◽  
Two Photon ◽  
Tissue Sections

scholarly journals Implementation of Accurate and Fast DNA Cytometry by Confocal Microscopy in 3D

2005 ◽  
Vol 27 (4) ◽  
pp. 225-230
Author(s):  
Lennert S. Ploeger ◽  
André Huisman ◽  
Jurryt van der Gugten ◽  
Dionne M. van der Giezen ◽  
Jeroen A. M. Beliën ◽  
...  
Keyword(s):  
Genomic Instability ◽  
Confocal Laser Scanning Microscopy ◽  
Dna Content ◽  
Laser Scanning ◽  
Clinical Applications ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Dna Cytometry ◽  
Tissue Sections

Background: DNA cytometry is a powerful method for measuring genomic instability. Standard approaches that measure DNA content of isolated cells may induce selection bias and do not allow interpretation of genomic instability in the context of the tissue. Confocal Laser Scanning Microscopy (CLSM) provides the opportunity to perform 3D DNA content measurements on intact cells in thick histological sections. Because the technique is technically challenging and time consuming, only a small number of usually manually selected nuclei were analyzed in different studies, not allowing wide clinical evaluation. The aim of this study was to describe the conditions for accurate and fast 3D CLSM cytometry with a minimum of user interaction to arrive at sufficient throughput for pilot clinical applications. Methods: Nuclear DNA was stained in 14 μm thick tissue sections of normal liver and adrenal stained with either YOYO-1 iodide or TO-PRO-3 iodide. Different pre-treatment strategies were evaluated: boiling in citrate buffer (pH 6.0) followed by RNase application for 1 or 18 hours, or hydrolysis. The image stacks obtained with CLSM at microscope magnifications of ×40 or ×100 were analyzed off-line using in-house developed software for semi-automated 3D fluorescence quantitation. To avoid sectioned nuclei, the top and bottom of the stacks were identified from ZX and YZ projections. As a measure of histogram quality, the coefficient of variation (CV) of the diploid peak was assessed. Results: The lowest CV (10.3%) was achieved with a protocol without boiling, with 1 hour RNase treatment and TO-PRO-3 iodide staining, and a final image recording at ×60 or ×100 magnifications. A sample size of 300 nuclei was generally achievable. By filtering the set of automatically segmented nuclei based on volume, size and shape, followed by interactive removal of the few remaining faulty objects, a single measurement was completely analyzed in approximately 3 hours. Conclusions: The described methodology allows to obtain a largely unbiased sample of nuclei in thick tissue sections using 3D DNA cytometry by confocal laser scanning microscopy within an acceptable time frame for pilot clinical applications, and with a CV small enough to resolve smaller near diploid stemlines. This provides a suitable method for 3D DNA ploidy assessment of selected rare cells based on morphologic characteristics and of clinical samples that are too small to prepare adequate cell suspensions.

scholarly journals Confocal Laser Scanning Microscopy and Two Photon Excitation Microscopy as Tools to Study Testate Amoebae

2010 ◽  
Vol 16 (S2) ◽  
pp. 1142-1143
Author(s):  
Z Burdíková ◽  
M Čapek ◽  
P Ostasou ◽  
EAD Mitchell ◽  
J Machač ◽  
...  
Keyword(s):  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Testate Amoebae ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Laser ◽  
Two Photon ◽  
Photon Excitation ◽  
Two Photon Excitation

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.

scholarly journals QUANTITATIVE CONFOCAL LASER SCANNING MICROSCOPY

2011 ◽  
Vol 25 (3) ◽  
pp. 111 ◽  
Author(s):  
Merete Krog Raarup ◽  
Jens Randel Nyengaard
Keyword(s):  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
3D Structure ◽  
Correlation Spectroscopy ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Laser ◽  
Two Photon ◽  
Photon Excitation

This paper discusses recent advances in confocal laser scanning microscopy (CLSM) for imaging of 3D structure as well as quantitative characterization of biomolecular interactions and diffusion behaviour by means of one- and two-photon excitation. The use of CLSM for improved stereological length estimation in thick (up to 0.5 mm) tissue is proposed. The techniques of FRET (Fluorescence Resonance Energy Transfer), FLIM (Fluorescence Lifetime Imaging Microscopy), FCS (Fluorescence Correlation Spectroscopy) and FRAP (Fluorescence Recovery After Photobleaching) are introduced and their applicability for quantitative imaging of biomolecular (co-)localization and trafficking in live cells described. The advantage of two-photon versus one-photon excitation in relation to these techniques is discussed.

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