A simple and stable autofocusing protocol for long multidimensional live cell microscopy
ABSTRACT Focus maintenance is a challenging problem in multidimensional wide-field microscopy. Most automated microscopes use software algorithms, which are applied to z-sections of the object, to select for the plane with the best signal to noise ratio. When applied automatically in multidimensional imaging applications, autofocus routines significantly increase light exposure and can become cytotoxic if applied too frequently. In addition, automated focusing procedures can readily focus on unwanted high contrast objects. By labelling a defined position with a fluorescent marker, we were able to separate the focusing procedure from the actual image acquisition positions and therefore overcome some of the major drawbacks of routine autofocus procedures. To implement this method in a multidimensional acquisition experiment, we created a visual basic-based program, which is run prior to each image acquisition. This technique allows tight control of focus whilst keeping light toxicity in live cell imaging experiments to a minimum.
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ABSTRACT: Cytotoxic CD8(+) T cells are considered important effector cells contributing to neuronal damage in inflammatory and degenerative CNS disorders. Using time-lapse video microscopy and two-photon imaging in combination with whole-cell patch-clamp recordings, we here show that major histocompatibility class I (MHC I)-restricted neuronal antigen presentation and T cell receptor specificity determine CD8(+) T-cell locomotion and neuronal damage in culture and hippocampal brain slices. Two separate functional consequences result from a direct cell-cell contact between antigen-presenting neurons and antigen-specific CD8(+) T cells. (1) An immediate impairment of electrical signaling in single neurons and neuronal networks occurs as a result of massive shunting of the membrane capacitance after insertion of channel-forming perforin (and probably activation of other transmembrane conductances), which is paralleled by an increase of intracellular Ca(2+) levels (within <10 min). (2) Antigen-dependent neuronal apoptosis may occur independently of perforin and members of the granzyme B cluster (within approximately 1 h), suggesting that extracellular effects can substitute for intracellular delivery of granzymes by perforin. Thus, electrical silencing is an immediate consequence of MHC I-restricted interaction of CD8(+) T cells with neurons. This mechanism is clearly perforin-dependent and precedes, but is not causally linked, to neuronal cell death.The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 12/2009; 29(49):15397-409. DOI:10.1523/JNEUROSCI.4339-09.2009 · 6.75 Impact Factor
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ABSTRACT: Digital-holographic metrology enables quantitative phase contrast microscopy of reflective and (partially) transparent samples. In this way, new application fields are opened up for nondestructive investigations of technical samples as well as for marker-free and time-resolved analysis of cell biological processes. Studies on long-term biological processes require permanent focus position readjustment to maintain an optimum image quality. Digital holographic microscopy permits subsequent numerical focusing by variation of the propagation distance. Here, the determination of the optimal propagation distance for a sharply focused image is of particular importance. At the Laboratory of Biophysics image definition quantification algorithms were adapted to the requirements of digital holographic microscopy. In order to obtain robust and reliable algorithms, the object-dependent optical absorption properties were taken into consideration. Automatic focus tracking is demonstrated on investigations with digital holographic microscopy on both technical amplitude objects and cytological pure phase objects.Proceedings of SPIE - The International Society for Optical Engineering 06/2007; DOI:10.1117/12.727784 · 0.20 Impact Factor
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ABSTRACT: Digital holographic microscopy (DHM) is utilized for quantitative phase contrast microscopy in optical testing of reflective or transparent specimens and allows altering the focus numerically by propagating the complex wave. Especially for compensation of deformations or displacements and for long-term investigations of living cells, a reliable region selective numerical readjustment of the focus is of particular interest in digital holographic microscopy. Since this method is time consuming, a Halton point set with low discrepancy has been chosen. By this, the effective axial resolution can be enhanced numerically by post processing of complex wave fronts without narrowing the field of view leading to a loss of information around the focus plane by blurring. The concept of numerical parametric lenses is another key feature in DHM and used to correct aberrations in the reconstructed wave front caused by the setup. To reduce the number of parameters for parametric lenses, the polynomial basis by Forbes is applied for the needs of DHM. Both numerical approaches have been characterized and adapted to the requirements of DHM. The applicability is demonstrated by results of investigations of engineered surfaces and biological cells.Proceedings of SPIE - The International Society for Optical Engineering 08/2010; DOI:10.1117/12.860695 · 0.20 Impact Factor