Costa Clayton M, Yang Suann
Department of Biology, 208 Mueller Laboratory, Pennsylvania State University, University Park, PA 16802, USA.
Ann Bot. 2009 Oct;104(5):1005-10. doi: 10.1093/aob/mcp186. Epub 2009 Jul 29.
Although many methods exist for quantifying the number of pollen grains in a sample, there are few standard methods that are user-friendly, inexpensive and reliable. The present contribution describes a new method of counting pollen using readily available, free image processing and analysis software.
Pollen was collected from anthers of two species, Carduus acanthoides and C. nutans (Asteraceae), then illuminated on slides and digitally photographed through a stereomicroscope. Using ImageJ (NIH), these digital images were processed to remove noise and sharpen individual pollen grains, then analysed to obtain a reliable total count of the number of grains present in the image. A macro was developed to analyse multiple images together. To assess the accuracy and consistency of pollen counting by ImageJ analysis, counts were compared with those made by the human eye.
Image analysis produced pollen counts in 60 s or less per image, considerably faster than counting with the human eye (5-68 min). In addition, counts produced with the ImageJ procedure were similar to those obtained by eye. Because count parameters are adjustable, this image analysis protocol may be used for many other plant species. Thus, the method provides a quick, inexpensive and reliable solution to counting pollen from digital images, not only reducing the chance of error but also substantially lowering labour requirements.
尽管存在多种对样本中花粉粒数量进行量化的方法，但很少有用户友好、价格低廉且可靠的标准方法。本文介绍了一种使用易于获取的免费图像处理与分析软件来计数花粉的新方法。
从两种植物（刺苞飞廉和毛飞廉，菊科）的花药中采集花粉，然后将其置于载玻片上进行光照，并通过体视显微镜进行数码拍照。使用ImageJ（美国国立卫生研究院）对这些数码图像进行处理，以去除噪声并锐化单个花粉粒，然后进行分析，以获得图像中花粉粒数量的可靠总数。开发了一个宏来一起分析多个图像。为了评估通过ImageJ分析进行花粉计数的准确性和一致性，将计数结果与人眼计数结果进行了比较。
图像分析每张图像在60秒或更短时间内得出花粉计数，比人眼计数（5 - 68分钟）快得多。此外，通过ImageJ程序得出的计数结果与人眼计数结果相似。由于计数参数是可调节的，该图像分析方案可用于许多其他植物物种。因此，该方法为从数码图像中计数花粉提供了一种快速、廉价且可靠的解决方案，不仅减少了出错的机会，还大幅降低了人工需求。
