Rebenku István, Bartha Ferenc A, Katona Tamás, Zsebik Barbara, Antalffy Géza, Takács Lili, Molnár Béla, Vereb György
Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary.
ELKH-DE Cell Biology and Signaling Research Group, Faculty of Medicine, University of Debrecen, Debrecen, Hungary.
Cytometry A. 2023 Mar;103(3):198-207. doi: 10.1002/cyto.a.24675. Epub 2022 Aug 5.
The emergence and fast advance of digital pathology allows the acquisition, digital storage, interactive recall and analysis of morphology at the tissue level. When applying immunohistochemistry, it also affords the correlation of morphology with the expression of one or two specific molecule of interest. The rise of fluorescence pathology scanners expands the number of detected molecules based on multiplex labeling. The Pannoramic Confocal (created by 3DHistech, Hungary) is a first-of-the-kind digital pathology scanner that affords not only multiplexed fluorescent detection on top of conventional transmission imaging, but also confocality. We have benchmarked this scanner in terms of stability, precision, light efficiency, linearity and sensitivity. X-Y stability and relocalisation precision were well below resolution limit (≤50 nm). Light throughput in confocal mode was 4-5 times higher than that of a point scanning confocal microscope, yielding similar calculated confocal intensities but with the potential for improving signal to noise ratio or scan speed. Response was linear with R ≥ 0.9996. Calibrated measurements showed that using indirect labeling ≥2000 molecules per cell could be well detected and imaged on the cell surface. Both standard-based and statistical post-acquisition flatfield corrections are implemented. We have also measured the point spread function (PSF) of the instrument. The dimensions of the PSF are somewhat larger and less symmetric than of the theoretical PSF of a conventional CLSM, however, the spatial homogeneity of these parameters allows for obtaining a specific system PSF for each optical path and using it for optional on-the-fly deconvolution. In conclusion, the Pannoramic Confocal provides sensitive, quantitative widefield and confocal detection of multiplexed fluorescence signals, with optical sectioning and 3D reconstruction, in addition to brightfield transmission imaging. High speed scanning of large samples, analysis of tissue heterogeneity, and detection of rare events open up new ways for quantitatively analyzing tissue sections, organoid cultures or large numbers of adherent cells.
数字病理学的出现和快速发展使得在组织水平上能够获取、数字存储、交互式检索和分析形态学信息。在应用免疫组织化学时，它还能将形态学与一两种特定感兴趣分子的表达相关联。荧光病理扫描仪的兴起基于多重标记增加了可检测分子的数量。全景共聚焦扫描仪（由匈牙利的3DHistech公司制造）是首款数字病理扫描仪，它不仅能在传统透射成像的基础上进行多重荧光检测，还具备共聚焦功能。我们已在稳定性、精度、光效率、线性度和灵敏度方面对该扫描仪进行了基准测试。X-Y稳定性和重新定位精度远低于分辨率极限（≤50 nm）。共聚焦模式下的光通量比点扫描共聚焦显微镜高4至5倍，产生的计算共聚焦强度相似，但有提高信噪比或扫描速度的潜力。响应呈线性，R≥0.9996。校准测量表明，使用间接标记时，每个细胞表面≥2000个分子能够被良好地检测和成像。同时实施了基于标准和统计的采集后平场校正。我们还测量了该仪器的点扩散函数（PSF）。该PSF的尺寸比传统共聚焦激光扫描显微镜（CLSM）的理论PSF稍大且对称性稍差，然而，这些参数的空间均匀性允许为每个光路获得特定的系统PSF，并将其用于可选的实时去卷积。总之，全景共聚焦扫描仪除了能进行明场透射成像外，还能对多重荧光信号进行灵敏、定量的宽场和共聚焦检测，并具备光学切片和三维重建功能。对大样本的高速扫描、组织异质性分析以及罕见事件检测为定量分析组织切片、类器官培养物或大量贴壁细胞开辟了新途径。
