Orca er camera

Manufactured by Olympus

Sourced in Japan

The Orca-ER camera is a high-performance scientific imaging camera designed for a variety of laboratory applications. It features a large sensor, high resolution, and low noise, making it suitable for a range of imaging tasks.

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9 protocols using orca er camera

Visualizing Mitochondria and Nuclei

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Fluorescent probes, MitoTracker^TM Red CMXRos and NucBlue^TM Live ReadyProbes Reagent (both Molecular Probes, Eugene, OR, USA) were used to visualize the mitochondria and nuclei of cells. MitoTracker^TM passively passes into mitochondria and accumulates there. NucBlue^TM Reagent contains Hoechst 33342 (2′-[4-ethoxyphenyl]-5-[4-methyl-1-piperazinyl]-2,5′-bi-1H-benzimidazole) which emits a blue fluorescence when bound to DNA (Figure 4).
The cells were first stripped of differentiation medium, which was replaced with special medium for live cell imaging—Live Cell Imaging Solution (Molecular Probes, Eugene, OR, USA). Subsequently, Mitotracker^TM was added to the medium (final concentration 100 nM) and two drops per millilitre of NucBlue^TM were added too. Cells were incubated in the dark for 30 min and then visualized by the Hamamatsu Orca-ER camera mounted on the Olympus IX 81 inverted microscope at 200× magnification (Olympus, Tokyo, Japan).

Kladnicka I., Cedikova M., Jedlicka J., Kohoutova M., Muller L., Plavinova I., Kripnerova M., Bludovska M., Kuncova J, & Mullerova D. (2021). Chronic DDE Exposure Modifies Mitochondrial Respiration during Differentiation of Human Adipose-Derived Mesenchymal Stem Cells into Mature Adipocytes. Biomolecules, 11(8), 1068.

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Immunofluorescent Labeling of Transfected COS-7 Cells

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Transfected COS-7 cells were washed with 1x PBS and then fixed in 4% paraformaldehyde for 20 minutes at room temperature. For immunolabeling, cells were washed in PBS then blocked for 1 hour in blocking buffer (10% donkey serum, 1% fish gelatin, 5% bovine serum albumin, and 0.2% Triton X-100 in PBS). Cells were incubated in primary antibodies (see Table 1) in blocking buffer overnight at 4°C. Cells were then washed in PBS, incubated with appropriate AlexaFluor-conjugated secondary antibodies (Thermo Fisher), rinsed again, and mounted using ProlongGold mounting medium containing 4',6-diamidino-2-phenylindole (DAPI; Thermo Fisher). Images were captured on a BX-62 spinning disk confocal microscope equipped with an ORCA-ER camera (Olympus, Japan) and analyzed with Slidebook 5 software (Intelligent Imaging Innovations, Denver, CO, USA). Images were captured with a 100x/1.40 oil objective, and exposure times and display settings (brightness and contrast) for all images were normalized to a control section where primary antibody was omitted during processing. No gamma adjustments were made to immunofluorescent images.

Conley S.M., McClard C.K., Mwoyosvi M.L., Alkadhem N., Radojevic B., Klein M., Birch D., Ellis A., Icks S.W., Guddanti T, & Bennett L.D. (2022). Delineating the Clinical Phenotype of Patients With the c.629C>G, p.Pro210Arg Mutation in Peripherin-2. Investigative Ophthalmology & Visual Science, 63(8), 19.

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Quantifying Lignin Deposition in Cacao Leaves

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Six E− and six E+ cacao plants used for the first microarray analysis were preserved in RNAlater. Three to four approximately 1 cm² sections were cut from each leaf. The sections from each treatment (E+ and E−) were stained with 2% Phloroglucinol-HCL for 20 min. After the staining, 14 E+ and 18 E− sections were immediately assayed for the development of the purple color under an Olympus BX61Epi-Fluorescence Microscope (Olympus America Inc., Melville, NY) using a 10× objective. Images were acquired for with a Hamamatsu Orca-ER camera and processed with Olympus SlideBook 4.1 software. The purple color intensity (total pixel intensity) in the vascular tissue of each sample was measured using ImageJ software (http://rsbweb.nih.gov/ij/). Average intensity of each section was calculated and randomization tests indicated significant differences between the E+ and E− treatments (p < 0.001).

Mejía L.C., Herre E.A., Sparks J.P., Winter K., García M.N., Van Bael S.A., Stitt J., Shi Z., Zhang Y., Guiltinan M.J, & Maximova S.N. (2014). Pervasive effects of a dominant foliar endophytic fungus on host genetic and phenotypic expression in a tropical tree. Frontiers in Microbiology, 5, 479.

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Cell Size Analysis by Microscopy

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Basic morphological evaluation was carried out by routine phase contrast microscopic observation. The cell size analysis was described by a published procedure [26 (link)]. Briefly, phase contrast photographs of growing cells were taken at a 10× magnification by the Hamamatsu Orca-ER camera mounted on the Olympus IX 70 inverted microscope (Olympus, Tokyo, Japan). The QuickPHOTO Industrial 2.3 (Promicra Ltd., Prague, Czech Republic) software was used for the photo evaluations. The area of the cells in each clone (n = 15) was calculated based on the polygon surface created by tracing the contour of cells.

Kripnerová M., Parmar H.S., Šána J., Kopková A., Radová L., Sopper S., Biernacki K., Jedlička J., Kohoutová M., Kuncová J., Peychl J., Rudolf E., Červinka M., Houdek Z., Dvořák P., Houfková K., Pešta M., Tůma Z., Dolejšová M., Tichánek F., Babuška V., Leba M., Slabý O, & Hatina J. (2021). Complex Interplay of Genes Underlies Invasiveness in Fibrosarcoma Progression Model. Journal of Clinical Medicine, 10(11), 2297.

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Quantification of Adipocyte Differentiation

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Fatty acid-binding protein (FABP4) is highly expressed in adipocytes and consists of about 1% of all soluble proteins in adipose tissue [28 (link)]. On day 21, the adipogenic marker FABP4 was measured to confirm the presence of adipocytes.
Cells were washed in phosphate buffered saline (PBS), fixed for 60 min in 4% formaldehyde with PBS at room temperature, and permeabilized in PBS containing 0.3% Triton X-100 for 15 min followed by blocking in PBS with 1% bovine serum albumin (BSA) and 10% normal donkey serum at room temperature for 60 min. After blocking, cells were incubated with anti-mFABP4 antibody (anti-mouse Fatty Acids Binding Protein 4, R&D Systems, Inc., Minneapolis, MN, USA) working solution (PBS containing 0.03% Triton X-100, 1% BSA, 10% normal donkey serum and anti-mFABP4 in final concentration 10 µg/mL) overnight at 2–8 °C. After three 5-min rinses in PBS with 1% BSA, cells were incubated for 1 h in NL557-conjugated anti-goat secondary antibody (R&D Systems, Inc., Minneapolis, MN, USA) diluted 1:200 in 1% BSA in PBS in the dark for 60 min at room temperature. The coverslips were washed, placed on microscope slides with a mounting medium (ProLong Gold Antifade Mountant with DAPI, Molecular Probes, Eugene, OR, USA) and visualized using the Hamamatsu Orca-ER camera mounted on the Olympus IX 81 inverted microscope at 200× magnification (Olympus, Tokyo, Japan).

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Immunofluorescence Microscopy of GFP-Positive Cells

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Images of GFP-positive live cells were acquired with the AMG-Evos inverted microscope. Immunofluorescence microscopy was performed as described^{30 (link)}, with antibody dilutions: HA (1/500, Sigma), γH2AX (1/500, Millipore), GFP (1/500, Roche). Samples were examined either with a microscope (Zeiss) equipped with a 10X, a 40X dry objective and a 100X oil immersion objective and a Hamamatsu Orca ER camera, or a confocal microscope (Olympus IX71) equipped with a 40X, 60X and 100X oil immersion objective and a CoolSNAP HQ2 camera. Pictures were analysed with ImageJ software.

Brustel J., Kozik Z., Gromak N., Savic V, & Sweet S.M. (2018). Large XPF-dependent deletions following misrepair of a DNA double strand break are prevented by the RNA:DNA helicase Senataxin. Scientific Reports, 8, 3850.

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Mitochondrial and Nuclear Labeling Protocol

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Fluorescent probes, MitoTracker TM Red CMXRos and NucBlue® Live ReadyProbes® Reagent (both Molecular Probes, Eugene, OR, USA) were used to visualize mitochondria and nucleus. To label mitochondria, cells were incubated with MitoTracker TM , which passively diffuses across the plasma membrane and accumulates in active mitochondria. Cell-permeant nuclear counterstain NucBlue® Live ReadyProbes® Reagent containing Hoechst® 33342 dye (2'-[4ethoxyphenyl]-5-[4-methyl-1-piperazinyl]-2,5'-bi-1Hbenzimidazole) emits blue fluorescence when bound to DNA with an emission maximum at 460 nm. It is detected through a blue/cyan filter.
Culture medium was replaced with Live Cell Imaging Solution (Molecular Probes, Eugene, OR, USA). Two drops of NucBlue® Live ReadyProbes® Reagent were added per milliliter of medium and MitoTracker TM was added in the final concentration 100 nM. Cells were incubated in the dark for 30 min and then visualized by the Hamamatsu Orca-ER camera mounted on the Olympus IX 81 inverted microscope at 200× magnification (Olympus, Tokyo, Japan).

Kladnická I., Čedíková M., Kripnerová M., Dvořáková J., Kohoutová M., Tůma Z., Müllerová D, & Kuncová J. (2019). Mitochondrial respiration of adipocytes differentiating from human mesenchymal stem cells derived from adipose tissue. Physiological research, 68(Suppl 3).

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Morphological Evaluation of Cells

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Basic morphological evaluation was carried out by routine phase contrast microscopic observation by two independent observers in 59 HBO and 55 control cells. Briefly, phase contrast photos of growing cells were taken at a 60× magnification by the Hamamatsu Orca-ER camera mounted on the Olympus IX 81 inverted microscope (Olympus, Tokyo, Japan). The QuickPHOTO Industrial 2.3 software (Promicra Ltd., Prague, CZ) was used for the photo evaluations. The area and perimeter of HFL1 cells were calculated based on the polygon surface created by tracing the contours of cells (Holubova et al. 2012) . The circularity index was calculated as 4π × (area/perimeter squared).

Dejmek J., Kohoutová M., Kripnerová M., Čedíková M., Tůma Z., Babuška V., Bolek L, & Kuncová J. (2018). Repeated exposure to hyperbaric hyperoxia affects mitochondrial functions of the lung fibroblasts. Physiological research, 67(Suppl 4).

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Immunofluorescence Analysis of Cellular Cytoskeleton

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HFL1 cells were seeded in count 40,000 per well on the bottom of 12-well plate and treated as described above.
For further processing, Image-iT® Fixation/Permeabilization Kit (Life Technologies, Prague, CZ) was used. Samples were fixed with 4 % formaldehyde for 15 min, permeabilized in Permeabilization solution for 15 min and blocked in 3 % BSA for 60 min. Cells were then sequentially incubated overnight with the primary monoclonal Anti-Vinculin (V9131) and monoclonal Anti-Vimentin (V5255) antibodies. Secondary Anti-Mouse IgG-Atto 488 goat antibody (62197) and Anti-Mouse IgM-FITC goat antibody (F9259) were applied for 60 min at room temperature in dark. For actin staining, Phalloidin-Atto 488 (49409) was used. Samples were mounted in ProLong Gold antifade reagent with DAPI (Invitrogen, Life Technologies, Prague, CZ) and analyzed using the Olympus IX81 fluorescent microscope (Olympus; Tokyo, Japan) equipped with a Cell-R system at 40×, 100×, and 400× magnification.
Cellular mitochondrial network was visualized with MitoTracker™ Red CMXRos (No. M7512; Thermo Fisher Scientific, Prague, CZ) using 0.5 µl of 1 mmol/l stain for each well containing 1 ml of the cell culture medium. Basic evaluation was followed by fluorescent microscopy at a 100× magnification by the Hamamatsu Orca-ER camera mounted on the Olympus IX 81 inverted microscope (Olympus; Tokyo, Japan).

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