University of Muenster, Department of Medicine B, Gastroenterological Molecular Cell Biology, Muenster, Germany.
J Biomed Opt. 2010 Jul-Aug;15(4):041509. doi: 10.1117/1.3449567.
The understanding of biological reactions and evaluation of the significance for living cells strongly depends on the ability to visualize and quantify these processes. Digital holographic microscopy (DHM) enables quantitative phase contrast imaging for high resolution and minimal invasive live cell analysis without the need of labeling or complex sample preparation. However, due to the rather homogeneous intracellular refractive index, the phase contrast of subcellular structures is limited and often low. We analyze the impact of the specific manipulation of the intracellular refractive index by microinjection on the DHM phase contrast. Glycerol is chosen as osmolyte, which combines high solubility in aqueous solutions and biological compatibility. We show that the intracellular injection of glycerol causes a contrast enhancement that can be explained by a decrease of the cytosolic refractive index due to a water influx. The underlying principle is proven by experiments inducing cell shrinkage and with fixated cells. The integrity of the cell membrane is considered as a prerequisite and allows a reversible cell swelling and shrinking within a certain limit. The presented approach to control the intracellular phase contrast demonstrated for the example of DHM opens prospects for applications with other quantitative phase contrast imaging methods.
对生物反应的理解和对活细胞意义的评估在很大程度上取决于可视化和量化这些过程的能力。数字全息显微镜（DHM）能够进行定量相位对比成像，具有高分辨率和最小侵入性，无需标记或复杂的样品制备。然而，由于细胞内折射率相当均匀，亚细胞结构的相位对比受到限制，通常较低。我们分析了通过微注射对细胞内折射率进行特定操作对 DHM 相位对比的影响。甘油被选为渗透剂，它在水溶液中的溶解度高且具有生物相容性。我们表明，甘油的细胞内注射会引起对比度增强，这可以通过水流入导致细胞质折射率降低来解释。通过诱导细胞收缩的实验和固定细胞的实验证明了这一原理。细胞膜的完整性被认为是前提条件，并允许在一定限度内进行可逆的细胞膨胀和收缩。所提出的控制细胞内相位对比的方法已通过 DHM 示例进行了演示，为其他定量相位对比成像方法的应用开辟了前景。
