All fluorescent microscopy imaging experiments were performed on an inverted Olympus IX81 microscope equipped with a Hamamatsu C8484 camera, 60X oil immersion Plan S-Apo objective and CFP, TxRed, and Cy5.5 filter cubes from Semrock. Images were taken at 250 ms exposure for CFP fluorescence, 500 ms exposure for mCh fluorescence, and 1000 ms exposure for Alexa 647 fluorescence. An ROI surrounding the entire cell was defined for cells that contain both the PKA-Reporter and Cry2-mCh. The minimum background fluorescence for each image was subtracted from each cell and then the ratio of the phosphoPKA-Reporter fluorescence/PKA-Reporter fluorescence (Alexa 647/CFP) was calculated. Values were normalized to the ratio from cells that were never exposed to light. All imaging analysis was done using Image J software and reported as the mean ± standard error of 10 – 25 cells per experiment.
Ix81 microscope
Manufactured by Hamamatsu Photonics
Sourced in Japan, Canada
The IX81 is an inverted research microscope manufactured by Hamamatsu Photonics. It features a motorized nosepiece and stage, as well as compatibility with a variety of optical accessories and imaging techniques.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Orca er camera, by Hamamatsu Photonics (3 mentions)
Ccd camera, by Hamamatsu Photonics (3 mentions)
Orca 2, by Hamamatsu Photonics (2 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
In situ cell death detection kit, by Merck Group (2 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Triton x, by Merck Group (2 mentions)
C4742 95 camera, by Hamamatsu Photonics (2 mentions)
Polyclonal rabbit anti active caspase 3, by Abcam (2 mentions)
Monoclonal mouse anti cardiac troponin t, by Developmental Studies Hybridoma Bank (2 mentions)
Alexa fluor 594 conjugated wheat germ agglutinin, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 594 goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Dmi6000 microscope, by Leica (2 mentions)
M165 fc stereomicroscope, by Leica (2 mentions)
Bx41 microscope, by Olympus (2 mentions)
Dfc290 color digital camera, by Leica (2 mentions)
C8484 camera, by Hamamatsu Photonics (1 mentions)
28 protocols using ix81 microscope
1
Quantifying Light-Induced PKA Activation
2
Confocal Microscopy Fluorescence Analysis
3
3D Spheroid Assay for Mammary Cancer
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MCF7-ras cells ([68 (link)] kindly provided by Dr. Ula Polanska) were expanded in DMEM/F12 supplemented with 10% FBS, 100 U/ml of penicillin, and 100 μg/ml of streptomycin and incubated in non-adherent PolyHEMA-coated dish overnight to form spheroids. Next day, the spheroids were embedded either alone (200 spheroids in 45 μl of Matrigel per well) or with 5 × 104 mammary fibroblasts per well and plated in domes in 24-well plates. After setting the gel for 45 to 60 min at 37°C, the cultures were overlaid with basal organoid medium, supplemented with growth factors [2.5 nM FGF2 (Enantis) or EGF (Peprotech)] small molecule inhibitors (S1 Table ) according to the experiment. The (co-)cultures were incubated in a humidified atmosphere of 5% CO2 at 37°C on Olympus IX81 microscope equipped with Hamamatsu camera and CellR system for time-lapse imaging and photographed every 60 min for 5 days with manual refocusing every day (high-detail imaging) or photographed only once per day for 5 days (low-detail imaging). The images were exported and analyzed using Image J. Spheroid budding was evaluated from the videos and it was defined as formation of a new bud from the spheroid. Spheroids that fused with other spheroids were excluded from the quantification.
4
Visualizing Actin Filament Assembly
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Unlabeled Mg-ATP-actin was assembled as per standard spontaneous assembly reactions. Actin filaments were then incubated with 1 μM TRITC-Phalloidin (Fluka Biochemika, Switzerland) for 5 min. Reactions were terminated by diluting assembled filaments in fluorescence buffer (50 mM KCl, 1 mM MgCl2, 100 mM DTT, 20 μg/ml catalase, 100 μg/ml glucose oxidase, 3 mg/ml glucose, 0.5% methylcellulose, and 10 mM imidazole, pH 7.0) and were absorbed to coverslips coated with 0.05 μg/μl poly-l -lysine. Fluorescence microscopy images were collected on an Olympus IX-81 microscope and cooled CCD camera (Orca-ER, Hamamatsu).
5
Quantitative Cellular Analysis Protocols
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In the case of black 96-well plates, the signal was measured using the Infinite 200 Pro Plate Reader (Tecan).
If the image cytometry or manual counting was used for the sample evaluation, the images were obtained by an Olympus IX81 microscope (objective: UPLFLN 10 × NA 0.3) equipped with a Hamamatsu ORCA II camera (a resolution of 1344 × 1024 pixels) using Cell ∧ R acquisition software.
The data were analysed using CellProfiler and Microsoft Excel and the final graphs were made in GraphPad Prism 6. The graphs were constructed using the following functions:
The Fig.1 was done using Rhinoceros 5 and Adobe Photoshop CS4 software.
If the image cytometry or manual counting was used for the sample evaluation, the images were obtained by an Olympus IX81 microscope (objective: UPLFLN 10 × NA 0.3) equipped with a Hamamatsu ORCA II camera (a resolution of 1344 × 1024 pixels) using Cell ∧ R acquisition software.
The data were analysed using CellProfiler and Microsoft Excel and the final graphs were made in GraphPad Prism 6. The graphs were constructed using the following functions:
Standard four parameter logistic nonlinear regression was used in the case of the analysis of the standard cytotoxic test by MTT assay or by the developed approach.
One-phase association was used for the analysis of the signal dependence on the number of HeLa cells in the case of MTT assay.
Linear regression or second order polynomial (quadratic) regression was used for the analysis of the signal dependence on the concentration of the DNA dyes diluted in 2% SDS, the signal dependence on the number of HeLa cells, IMR-90 cells and MRC-5 in the case of Hoechst 33342, DAPI and MTT assay
The Fig.
6
Bacterial Membrane Visualization Protocol
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To visualize the membrane structures, 1 ml of exponentially growing bacterial culture was stained by Nile Red (10 μg/ml) or by FM4–64 (1 μg/ml), washed twice with 1× PBS and re-suspended in 1× PBS containing 1 μM SYTOX green. The sample was then immediately either spotted onto a poly-l -lysine glass slide and covered with a poly-l -lysine coverslip (GLG method or P-GLG method when pressure ~80 kPa was applied for 10 s) or spotted on a 1× PBS agarose pad covered with a non-coated coverslip (GAG method). Pictures were obtained at the indicated time points (t = 0 is the start of microscopy, typically 30 s after coverslip addition) using a Olympus BX63 fluorescence microscope equipped with a Andor Zyla 5.5 sCMOS camera (alternatively, an Olympus IX81 microscope equipped with Hamamatsu Orca/ER camera was also used). Olympus CellP imaging software or Olympus Image-Pro Plus 6.0 software was used for image acquisition and analysis.
7
Fluorescent Peptide Binding to C. albicans
8
Immunohistochemical Analysis of Neonatal Intestine
9
Microfluidic Assay of Platelet-Collagen Binding
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Whole blood was assessed for its ability to bind collagen under physiologic shear stress using a microfluidic assay as previously described.20 (link) Briefly, blood was collected into a vacutube containing sodium heparin. Type I collagen (500 μg/mL) was patterned onto a microfluidic device and platelets in whole blood were labeled with fluorescent dye 3,3′-dihexyloxacarbocyanine iodide (1 μg/mL) and then perfused through the vacuum-sealed microfluidic device at a wall shear rate of 650 s−1 for 5 minutes using a PHD 2000 Syringe Pump (Harvard Apparatus, Holliston, MA). Platelet accretion was captured in real time in relief contrast and fluorescence with an Olympus IX81 microscope (Center Valley, PA) using an ORCA Flash4 camera (Hamamatsu, Japan). Images were captured using cellSens software (Center Valley, PA). Platelet surface area covered with time was analyzed using FIJI/ImageJ 1.51n (National Institutes of Health, Bethesda, MD) and data were processed with GraphPad (Dotmatics, Boston, MA).
10
Time-lapse Imaging and Cell Migration Analysis
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Cells were sparsely seeded in wells of a culture plate 24 h prior to filming. Time-lapse imaging was conducted using an Olympus IX81 microscope attached to a Hamamatsu Photonics Orca-R2 cooled CCD camera (Hamamatsu Photonics., Welwyn Garden City, UK). Phase contrast images were captured at varying positions using a motorised stage (LUDL Electronic Products, Hawthorne, NY, USA) every 5 min for 16 h. Individual cells were manually tracked throughout consecutive frames using an ImageJ software plugin (ImageJ1, National Institutes of Health (NIH), Bethesda, MD, USA). Using these x-y coordinates, migrational velocity and persistence were determined using Mathematica 6.0 (Wolfram Research Ltd., Witney, UK) custom-written notebooks kindly provided from Professor Graham Dunn and Daniel Soong, King’s College London.
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