Log phase yeast culture cells were spotted on a glass slide and imaged with an Olympus BX-51 microscope equipped with Infinity software v.5.0.3 (Lumenera, Ottawa, Canada) for image acquisition. Alternatively, live cells were imaged using a laser scanning confocal microscope (Zeiss LSM410). MetaMorph v6.1 software (Universal Imaging Corporation; Downington, PA) was used to construct calibrated overlays of z-stacked images. Worms were mounted on 2% agarose pads spotted with 20 μl of 10 mM Na-azide. Confocal microscopy was performed using a Zeiss LSM410 confocal laser scanning microscope. Z-stacks were acquired and processed using the Zeiss LSM software package. Alternatively, imaging was performed using an Applied Precision DeltaVision microscope (GE). Image processing was performed using Softworx software (G.E./Applied Precision).
Applied precision deltavision microscope
Manufactured by GE Healthcare
The Applied Precision DeltaVision microscope is a high-performance imaging system designed for advanced biological research. It utilizes state-of-the-art optics and imaging technologies to enable high-resolution, three-dimensional microscopy of living and fixed samples.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 410, by Zeiss (1 mentions)
Bx 51 microscope, by Teledyne (1 mentions)
Infinity software v 5, by Teledyne (1 mentions)
Metamorph v6, by Universal Imaging (1 mentions)
Rapamycin, by Merck Group (1 mentions)
1.4 na oil immersion objective, by Olympus (1 mentions)
Anti ha, by Roche (1 mentions)
Softworx software, by GE Healthcare (1 mentions)
4 protocols using applied precision deltavision microscope
1
Imaging of Yeast and Worm Samples
2
Live-cell Imaging of Autophagy Dynamics
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For all live-cell imaging, mid log phase cells were gently pelleted and washed with SC media containing 2% D and immediately imaged directly on cover glass. All images were acquired on an Applied Precision DeltaVision microscope (GE Healthcare, Life Sciences) equipped with a 100×/1.4 NA oil immersion objective (Olympus), solid state illumination, CoolSNAP HQ2 charge-couple device (CCD; Photometrics) or electron-multiplying CCD (Photometrics) cameras. The microscope stage was maintained at 30°C within an environmental chamber.
Time-lapse imaging of GFP-Atg8 and Atg39-mCherry (DTCPL1683) was performed in microfluidic plates (Y04C; CellASIC) with the ONIX Microfluidic Platform (CellASIC; EMD Millipore). Prior to loading cells in the microfluidic chamber, Atg39-mCherry expression was induced for 2 h with YPG followed by the addition of D to arrest expression. SC with 2% D containing rapamycin (final concentration, 250 ng/ml; Sigma-Aldrich) was perfused through the chamber at 0.25 psi for 1 h before the start of imaging. Z-stacks (0.4-μm sections) were acquired at 2-min intervals for 2 h.
Time-lapse imaging of GFP-Atg8 and Atg39-mCherry (DTCPL1683) was performed in microfluidic plates (Y04C; CellASIC) with the ONIX Microfluidic Platform (CellASIC; EMD Millipore). Prior to loading cells in the microfluidic chamber, Atg39-mCherry expression was induced for 2 h with YPG followed by the addition of D to arrest expression. SC with 2% D containing rapamycin (final concentration, 250 ng/ml; Sigma-Aldrich) was perfused through the chamber at 0.25 psi for 1 h before the start of imaging. Z-stacks (0.4-μm sections) were acquired at 2-min intervals for 2 h.
3
Live-cell Imaging and Statistical Analysis
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Live-cell imaging was performed using an Applied Precision DeltaVision microscope (GE Healthcare, Issaquah, WA), and data were acquired and processed with the accompanying software. Fluorescence intensity was measured using ImageJ. All data are expressed as the mean ± standard deviation (SD). Data were analyzed using an unpaired t-test (Student's t-test) for two groups and a one-way ANOVA with Bonferroni post hoc tests for multiple comparisons, as noted in the figure legends (SPSS 13.0 software; SPSS Inc., Chicago, IL). Differences with p < 0.05 were deemed statistically significant. Error bars in the figures represent the SD.
4
Visualizing Toxoplasma Mitochondrial and Apicoplast Proteins
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Tachyzoites of T. gondii derived from strain RH were cultivated and genetically manipulated as described previously [66 ]. HCF101 (TGME49_318590) and Nbp35 (TGME49_280730) coding sequences were amplified from T. gondii complementary DNA (cDNA) and cloned in frame with a triple hemagglutinin (HA) epitope tag at the 3′ end into plasmid pDt7s4HA. The constructs were transiently transfected into the T. gondii Δku80/TATi strain [67 (link)] using a BTX ECM 630 electroporator (Harward Apparatus). Confluent human foreskin fibroblasts (HFF) were infected with transfected parasites and fixed after 24 h of infection with 4% formaldehyde and permeabilized with 0.2% Triton X-100. Immunofluorescence microscopy was performed using the primary antibodies anti-HA (Roche), mouse anti- T. gondii mitochondrial F1-adenosinetriphosphatase (ATPase) [68 (link)], and rabbit anti-apicoplast Cpn60 [69 (link)]. Secondary antibodies used were goat anti-rat Alexa Fluor 488, goat anti-mouse Alexa Fluor 546, and goat anti-rabbit Alexa Fluor 546. Images were obtained on an Applied Precision Delta Vision microscope and were deconvolved and adjusted using Softworx software (GE Healthcare).
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