ix81 fluorescence microscope  (Thermo Fisher)

Journal: Scientific Reports

Article Title: Optimized laboratory techniques for assessing the quality of pre-stripped DMEK grafts

doi: 10.1038/s41598-025-91512-z

Figure Lengend Snippet: Influence of calcein AM concentration and diluent on staining and CECs. ( A ) Comparison of calcein staining in four different conditions: 2 µM or 4 µM calcein diluted in PBS or Opti-MEM (Opt). ( A1 ) The DMEK graft was separated from the cornea and then cut into four parts. Each part was incubated in one of the four solutions differentiated by calcein concentration (2 or 4 µM) and diluent (PBS or Opt). After incubation for 45 min at RT, the four different parts of the same DMEK graft were spread on the same glass slide and observed simultaneously under the same field using a fluorescence macroscope at a magnification of ×0.8. ( A2 ) The fluorescence intensity of calcein in each of the four parts of the endothelium was measured and then compared. Five different DMEK grafts were used ( n = 5). The condition PBS-2 µM was considered as the control condition. The fluorescence intensity of the four parts of each graft was standardized with their respective control. The standardized fluorescence intensity was expressed as Mean +/− SD (Min, Max). One-sample t-test was utilized for statistical analysis, resulting in p = 0.0460 (*) for Opt-2 µM, p = 0.0111 (*) for PBS-4 µM, and p = 0.0096 (**) for Opt-4 µM. ( B ) Side-effects of using PBS as a diluent: alteration of cell junctions. ( B1 ) Calcein staining of endothelium was performed using dilutions in PBS or Opti-MEM under a microscope with a 10× objective. ( B2) Cell junctions were highlighted using IF under a microscope with a 40× objective. The arrowheads indicate dead zones, and the arrows show the cracks that formed between cell islands consisting of several to several dozen CECs.

Article Snippet: For IF imaging, an Olympus IX81 fluorescence inverted microscope (Olympus, Tokyo, Japan), equipped with CellSens imaging systems software and a monochrome camera (ORCA-Flash 4.0, Hamamatsu), was employed.

Techniques: Concentration Assay, Staining, Comparison, Incubation, Fluorescence, Control, Microscopy

Journal: Dentistry Journal

Article Title: Extracellular Phosphate Modulation and Polyphosphate Accumulation by Corynebacterium matruchotii and Streptococcus mutans

doi: 10.3390/dj12110366

Figure Lengend Snippet: A schematic diagram of the experimental workflow. ( A ). Sterile BHI was inoculated with fresh seed culture, and growth was monitored hourly at 600 nm. Over time, the concentration of available soluble orthophosphate in the medium decreases as the bacteria take up phosphate and accumulate it as polyP. Cells were aliquoted at regular intervals during growth and one aliquot was centrifuged. The supernatant was used for an ascorbate assay to determine phosphate drawdown. The second aliquot was filtered, and the filtrate was used for the analysis of calcium drawdown from the medium using ICP-OES. ( B ). The two circles show bacterial cells at an early log phase and at an early stationary phase, respectively. PolyP accumulation is significantly increased as cells transition from the exponential log phase to the stationary phase (shown as yellow chains of polyP inside blue cells). ( C ). A typical bacterial growth curve; arrows indicate the growth phases (I = early log phase and II = early stationary phase) where cells were aliquoted for polyP study. ( D ). Cells aliquoted at both the early log and early stationary phases were stained with DAPI and imaged using an inverted fluorescence microscope for the qualitative identification of polyP. ( E ). Shows a typical epifluorescence image of C. matruchotii with accumulated polyP inclusions (yellowish green).

Article Snippet: A total of 40 µL DAPI (5 µg/mL) was added to each well and the plate was incubated in a hybridization chamber in the dark for 30 min. An Olympus IX81 fluorescence microscope fitted with an XM10 CCD camera and CellSens Dimensions Imaging Software (version 1.18 Build 16686) was used ( D).

Techniques: Sterility, Concentration Assay, Bacteria, Staining, Fluorescence, Microscopy