Automated acquisition and processing of multidimensional image data in confocal in vivo microscopy

Kozubek, Michal; Matula, Petr; Matula, Pavel; Kozubek, Stanislav

doi:10.1002/jemt.20068

Cited by 26 publications

(16 citation statements)

References 19 publications

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“…A 100 W mercury lamp was used as a source of excitation light. The HRCM system was upgraded for living-cell experiments with a heated microscopic stage insert (Carl Zeiss), objective heating (Carl Zeiss), and an atmospheric chamber for the microscope stage containing humidified warm air enriched to 5% CO 2 [40]. The HRCM system was controlled by FISH 2.0 software developed in our laboratory [38][39][40].…”

Section: Plasmid Dna Preparation and Transfectionmentioning

confidence: 99%

Bioinformatic and image analyses of the cellular localization of the apoptotic proteins endonuclease G, AIF, and AMID during apoptosis in human cells

et al. 2007

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We studied the cellular localization of the apoptotic proteins endonuclease G, AIF, and AMID in silico using three prediction tools and in living cells using both single-cell colocalization image analysis and nuclear translocation analysis. We confirmed the mitochondrial localization of endonuclease G and AIF by prediction analysis and by single-cell colocalization image analysis. We found the AMID protein to be cytoplasmic, most probably incorporated into the cytoplasmic side of the membranes of various organelles. The highest concentration of AMID was observed associated with the Golgi. Colocalization of AMID with lysosomes was also indirectly confirmed by analysis of AMID-rich vesicle velocity using manual tracking analysis. Bioinformatic analysis also detected nuclear localization signals in endonuclease G and AIF, but not in AMID. A novel analysis of time-lapse fluorescence image data during staurosporine-induced apoptosis revealed nuclear translocation only for endonuclease G and AIF.

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Section: Plasmid Dna Preparation and Transfectionmentioning

confidence: 99%

Bioinformatic and image analyses of the cellular localization of the apoptotic proteins endonuclease G, AIF, and AMID during apoptosis in human cells

et al. 2007

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“…The images were obtained by means of a high-resolution confocal cytometer (Kozubek et al 2004). The cytometer is based on a completely automated Leica DM RXA fluorescence microscope equipped with a confocal unit CSU-10a (Yokogawa, Japan), a CoolSnap HQ CCD camera (Photometrix) or alternatively an iXon DV 887ECS-BV (Andor), and an Ar/Kr laser Inova 70C (Coherent).…”

Section: Living Cell Observation and Time-lapse Microscopymentioning

confidence: 99%

Directional motion of foreign plasmid DNA to nuclear HP1 foci

et al. 2006

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Movement of labelled plasmid DNA relative to heterochromatin foci in nuclei, visualized with HP1-GFP, was studied using live-cell imaging and object tracking. In addition to Brownian motion of plasmid DNA we found a pronounced, non-random movement of plasmid DNA towards the nearest HP1 focus, while time-lapse microscopy showed that HP1 foci are relatively immobile and positionally stable. The movement of plasmid DNA was much faster than that of the HP1 foci. Contact of transgene DNA with an HP1 focus usually resulted in cessation of the directional motion. Moreover, the motion of plasmid DNA inside the heterochromatin compartment was more restricted (limited to 0.25 microm) than when the plasmid DNA was outside heterochromatin (R = 0.7 microm). Three days after transfection most of the foreign labelled DNA colocalized with centromeric heterochromatin.

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“…Second, the method must be robust to various acquisition artifacts that are specific to confocal microscopy. [25][26][27][28] Last, the large amount of data that is involved imposes a subtle balance between memory and time constraints.…”

Section: Introductionmentioning

confidence: 99%

Four-dimensional cardiac imaging in living embryos via postacquisition synchronization of nongated slice sequences

et al. 2005

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Abstract. Being able to acquire, visualize, and analyze 3D time series ͑4D data͒ from living embryos makes it possible to understand complex dynamic movements at early stages of embryonic development. Despite recent technological breakthroughs in 2D dynamic imaging, confocal microscopes remain quite slow at capturing optical sections at successive depths. However, when the studied motion is periodicsuch as for a beating heart-a way to circumvent this problem is to acquire, successively, sets of 2D + time slice sequences at increasing depths over at least one time period and later rearrange them to recover a 3D + time sequence. In other imaging modalities at macroscopic scales, external gating signals, e.g., an electro-cardiogram, have been used to achieve proper synchronization. Since gating signals are either unavailable or cumbersome to acquire in microscopic organisms, we have developed a procedure to reconstruct volumes based solely on the information contained in the image sequences. The central part of the algorithm is a least-squares minimization of an objective criterion that depends on the similarity between the data from neighboring depths. Owing to a wavelet-based multiresolution approach, our method is robust to common confocal microscopy artifacts. We validate the procedure on both simulated data and in vivo measurements from living zebrafish embryos.

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Automated acquisition and processing of multidimensional image data in confocal in vivo microscopy

Cited by 26 publications

References 19 publications

Bioinformatic and image analyses of the cellular localization of the apoptotic proteins endonuclease G, AIF, and AMID during apoptosis in human cells

Bioinformatic and image analyses of the cellular localization of the apoptotic proteins endonuclease G, AIF, and AMID during apoptosis in human cells

Directional motion of foreign plasmid DNA to nuclear HP1 foci

Four-dimensional cardiac imaging in living embryos via postacquisition synchronization of nongated slice sequences

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