Dp72 ccd camera

Cell morphology was monitored using an Olympus BX71 inverted microscope equipped with a DP72 CCD camera and a computer image analysis system CellB. Intracellular localization of AgNPs after 48-h treatment and after AgNP removal was evaluated using Nomarski microscopy. To analyze AgNP-mediated changes in nucleus, HT22 cells were fixed [32 (link)] and DNA was visualized using Hoechst 33342 staining. F-actin was stained using Alexa Fluor® 488 Phalloidin (a high-affinity filamentous actin, F-actin, probe conjugated to green fluorescent Alexa Fluor® 488 dye) according to manufacturer’s instructions (Life Technologies). Additionally, lamin B1 was immunodetected using lamin B1 antibody (1:100, Life Technologies) and secondary antibody conjugated with Texas red (1:1000, Life Technologies). Digital cell images were captured with an Olympus BX61 fluorescence microscope equipped with a DP72 CCD camera and Olympus CellF software. To analyze nuclear lamin B1 content, ImageJ software http://rsbweb.nih.gov/ij/ was used. We evaluated the integrated fluorescence density (red channel), which is the sum of all pixel values within the marked area of each nucleus analyzed and equivalent to the product of area and mean gray value. The integrated fluorescence density is presented in relative fluorescence units (RFUs). A total of 2000 cells were analyzed.

A denaturation mixture in volume 25 µL containing 12.5 µL 100% formamide, 5 µL 50% dextran sulphate, 2.5 µL 20× SSC, 3 µL water, and with 1 µL of each probe (26S, 5S) in a final concentration of 100 ng per used volume was denatured at 96 °C for 10 min, then rapidly cooled for 2 min. The mixture was applied onto a chosen slide and co-denatured on a hot plate at 80 °C for 2 min, then incubated overnight at 37 °C in a humid chamber box. Post-hybridization washing was carried out at 42 °C with the following steps: 2× SSC twice for 5 min, 10% formamide in 0.1× SSC twice for 5 min, 2× SSC for 5 min, and 4× SSC with 0.05% Tween-20 for 5 min. Biotin and digoxigenin-labelled probes were immunodetected using streptavidin-Cy3 (GE Healthcare, Buckinghamshire, United Kingdom; 1:750 dilution) and anti-DIG-FITC (Roche; 1:250 dilution) antibodies, respectively. Chromosomes were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) in Vectashield (Vector Laboratories, Burlingame, CA, USA).
Images were captured using an Olympus BX 51, Olympus, Tokyo, Japan fluorescence microscope equipped with an Olympus DP72 CCD camera. Three greyscale images of each mitosis event were taken, and images were pseudocoloured using Adobe Photoshop CS6 software (chromosomes—blue, 26S rDNA signals—green, 5S rDNA signals—red).

Digital images of Candida cells were captured using an Olympus BX61 fluorescence microscope equipped with a DP72 CCD camera and Olympus CellF software (Olympus,Warsaw, Poland). ImageJ software (http://rsbweb.nih.gov/ij/) was used to analyze the cell size. Cell size was expressed as arbitrary units [a.u.].