There are two light microscopic methods for cell volume measurement based on volume exclusion. One method, sometimes referred to as FLEX, utilises negative staining by an external fluorescent dye, and cell volume is found from attenuation of fluorescence under a wide‐field microscope. The other method (TTD) is based on exclusion of an external absorbing dye, resulting in an increased intensity of transmission image. In this work, we compared these two methods. TTD measurements were consistent, reproducible and identical to those obtained by confocal scanning. In our hands, FLEX based on either sodium fluorescein of fluorescent dextran, usually resulted in underestimation of cell volume, which were insignificant in shallow chambers but became more severe with increased chamber depth. We have not been able to exactly pinpoint the source of the problem; it may have been undetected accumulation of dye in the cells or, more likely, some unappreciated aspects of image formation under epi‐illumination. We also discuss applicability of both methods to in‐flow volume measurements.

中文翻译：

---

荧光和吸收排除显微镜测量细胞体积的比较


基于体积排除的细胞体积测量有两种光学显微镜方法。一种方法有时称为 FLEX，利用外部荧光染料的负染色，并通过宽视野显微镜下的荧光衰减来确定细胞体积。另一种方法 (TTD) 基于排除外部吸收染料，从而提高透射图像的强度。在这项工作中，我们比较了这两种方法。 TTD 测量结果一致、可重复且与共焦扫描获得的测量结果相同。在我们手中，基于荧光素钠或荧光葡聚糖的 FLEX 通常会导致细胞体积的低估，这在浅室中微不足道，但随着室深度的增加而变得更加严重。我们无法准确查明问题的根源；这可能是细胞中未被检测到的染料积累，或更可能是落射照明下图像形成的一些未被意识到的方面。我们还讨论了两种方法对流入体积测量的适用性。