Using scale bar on the screen for diameter, and count the layers that come into focus for the thickness, the lymphocyte nuclei in formalin-fixed, paraffin section showed almost spherical shape. This result assures that we can use our virtual slide in semi-morphometrical analysis. Then we measured lymphocyte nuclei in thick cytology slide, and found that they also keep spherical shape still after embedding in the medium. Nuclear size was about 4 to 5μm, smaller than the value shown in histology textbook (6 to 15μm). This result is possibly due to preparation procedure. Histological slide suffered formalin fixation and heat coagulation in melted paraffin, and cytological slide suffers alcoholic fixation. In the months-old slides, the nuclei diameter was nearly the same, but the thickness got flat to about 2 to 3μm. This result is probably due to drying of mounting medium. Lymphocytes in air-dried Giemsa slide and improperly dried cells in Papanicolaou slides were highly flattened.
Small-sized cells like myoepithelial cells of breast duct, surface squamous epithelial cells, and shrunk cells in urine showed around 2μm thickness. If these cell mass is cut into 2μm layers, some of the nuclei may not come into focus on the virtual slide. Less than 2μm layer thickness is required for focus simulation. Thinner layer results fine pictures, but the thinner the layer set, the more the total number of layers increase. This causes longer scan time, more data size, and slower response to observe.
Thinking about total thickness to scan, number of stacked nuclei and the nuclear size should be considered. Papillary cell mass of urothelial carcinoma, aspiration cytology specimen of breast or thyroid, and uterine cervical smear showed different optimal thickness. Using our virtual slide, nuclei stacked more than 5 to 6 layers was difficult to distinguish. Stacked 5 nuclei of 2μm thickness each results in 10μm to scan. But usually carcinoma cells have big nuclei, as big as 50μm diameter and 10μm thickness. If these big cells stack together, we need to scan to 50μm or more. Usually, slides with more than 5 stacked cancer cell nuclei are exceptional and focal.
The “focus fusion” seems to contain all the cells in one plane, and easy for screening. But when trying to obtain detailed pictures like existence of myoepithelial cells, very important benign sign of breast, or to clarify the 3-dimensional tissue structure, multilayer image was better.
The efficiency of multilayer focus simulation depends on how easy we can change focus. The method to change focus differs from company to company. Too many layer causes slow response. From these findings, we conclude 10 to 15 layers with 1.5μm thick each provide sufficient information in most specimens. In case of thick slide, 30μm thick scan may require.