Uplan fl n objective lens

Cells were imaged on a DeltaVision microscope (Applied Precision) using either a 60× 1.3 NA Plan-Apochromat or a 40× 1.3NA UPlan-FL N objective lens (Olympus). z-stacks were acquired with a CoolSNAP HQ camera (Photometrics) and SoftWoRx acquisition software (Applied Precision). Images were deconvolved using Deltavision SoftWoRx software and objective specific point spread function. Projections of three z-stacks were made using FIJI.

For histological analysis, the fixed samples were decalcified in 10% ethylenediaminetetraacetic acid (EDTA) for 1 month and then embedded in paraffin. The paraffin-embedded tissues were cut from the center at 3-µm intervals using a Leica microtome (RM2245; Leica Microsystems, Bannockbrun, IL, USA), and the most central sections from each sample were subjected to hematoxylin-eosin (H&E) and Masson’s trichrome staining. Histological evaluations were performed by observing the stained sections under a microscope (Olympus BX53, Olympus, Tokyo, Japan) equipped with a 10 × 0.30 UPLANFL N objective lens (Olympus, Tokyo, Japan). Images were captured using a digital camera (Olympus, Tokyo, Japan) attached to the microscope. Histomorphometric analysis was performed using image analysis software (I-solution; iMTechnology, Daejeon, Korea) to calculate the amount of new bone ingrowth in the defect sites: (1) the percentage of new bone (%) = the area of new bone between the defect margins (mm²)/the total augmented area (mm²) × 100 (%) and (2) the percentage of residual bone material (%) = the area of residual bone material between the defect margins (mm²)/the total augmented area (mm²) × 100 (%).