Phase contrast microscopy
Phase contrast microscopy (PCM) is generally composed of a bright-field microscope with the addition of a condenser annulus and a phase plate. Traditional bright-field imaging with transmitted light illumination solely relies on the intensity of light to form contrast based on the visible light absorption from the sample. However, due to the requirement of relative transparency of the imaging subject (cell culture in most cases), the contrast formed by the absorption is typically very limited within the cell, thus resulting in cellular structures that are hardly visible and therefore significantly restricting its usefulness for cell observation. Although exogenous staining can be introduced for bright-field microscopy to increase the imaging contrast, staining methods are often toxic to cells and reduce experimental efficiency due to the extra procedures (Smith et al., 2010).
Utilizing the phase of light, PCM employs coherent light interference to transform the phase difference of light into an amplitude difference that can be directly picked up by the eye or detected by the camera. Here, the interference is formed between the light diffracted from the sample and the light passed through or around the sample. Since the optical phase retardation is related to the optical path length from the sample, differences of both the refractive index and the thickness of the structure contribute to the imaging contrast of PCM. Because of the ultra-high sensitivity of the optical phase to the change of path length of light, as well as the great transforming efficiency and amplifying effect from phase to amplitude by using light interference, PCM possesses a significant improvement of contrast for live cell imaging without a large sacrifice of the resolution compared with bright-field microscopy (Zernike, 1955). This has positioned PCM as an efficient and convenient imaging tool for screening a thin, transparent layer of cultured cells without staining or labeling (Yang et al., 2005; Schmedlen et al., 2002; Fukuda, 2001).
The lateral resolution of PCM is mainly determined by the objective lens and condenser as well as the reduction of aperture introduced by the condenser annulus and phase plate. Under optimal conditions, PCM can reach a spatial resolution of ~0.3 pm (Snyder et al., 1987). Due to the lack of depthwise sectioning capability, PCM is primarily used for two-dimensional (2-D) visualizations. PCM has been widely utilized for the morphological imaging of cells in culture, and various image processing and analyzing methods have been developed for PCM to achieve cell detection (Pan et al., 2009) and segmentation (Su et al., 2013; Ambuhl et al., 2012). With time-lapse PCM imaging, these methods have successfully enabled the large-scale tracking of cell migration and proliferation (Kang et al., 2006), the automatic mitosis identification of stem cell populations (Huh et al., 2011), and the dynamic monitoring of cell apoptosis (Huh and Kanade, 2013).