Phase Contrast Microscopy
Phase contrast microscopy (PCM) was pioneered by Fritz Zernike to enable the study and visualisation of thin transparent biological samples, such as cells on a coverslip, for which he was awarded the Nobel Prize in Physics in 1953. PCM is usually implemented using transmitted light that undergoes phase changes as it propagates through a sample, e.g. due to variations in local thickness and/or refractive index. Initially established as a qualitative imaging technique, PCM typically exploits interference between scattered and unscattered transmitted light (e.g., Zernike phase contrast microscopy1) and reports differences in optical pathlength, or exploits interference between light rays that have passed through adjacent parts of the sample and so is sensitive to phase gradients (e.g., Nomarski differential interference contrast microscopy2 or Hoffman modulation contrast microscopy3).
Today phase contrast microscopy is attracting significant interest with the development of quantitative or semi-quantitative techniques that can provide label-free means to image cell dynamics and/or to provide readouts for diverse potential applications4 such as cell sizing, sorting and/or tracking, microbiology, neuroscience, and pathology. We are particularly interested to explore the potential for label-free segmentation of cells to enhance fluorescence microscopy. There are many approaches to realize quantitative phase microscopy (QPM) including (a) interferometric measurement techniques, such as phase stepping interferometry, off-axis (digital) holography and shearing interferometry, (b) techniques that reconstruct the phase of the electric field scattered by the sample from intensity measurements, such as transport of intensity5,6, and (Fourier) ptychography7 , and (c) non-interferometric wavefront sensing approaches including the use of Shack Hartman sensors8 or other phase gradient measurement techniques9 , including differential phase contrast microscopy10,11.
Most PCM techniques require significant modification of the microscope configuration (e.g. for interferometry) or specialist optical components (e.g. objective lenses with phase rings, Nomarski prisms) and/or the acquisition of multiple images to calculate quantitative phase contrast images. We have recently developed a single-shot semi-quantitative PCM technique, which we describe as polarisation differential phase contrast (pDPC) microscopy. Since pDPC is wavelength agnostic, it can be readily implemented on existing fluorescence microscopes.
1. F. Zernike, Physica 9 (1942) 686-674
2. Patent: US2924142, Georges Nomarski, "INTERFERENTIAL POLARIZING DEVICE FOR STUDY OF PHASE OBJECTS"
3. Robert Hoffman and Leo Gross, "Modulation Contrast Microscope," Appl. Opt. 14 (1975) 1169-1176
4. Y.K. Park, C. Depeursinge and G. Popescu, Nature Photonics 2018, 12, 578
5. M. Shribak, J. Opt. Soc. Am. A, 2013, 30, 769
6. C. Zuo, J. Sun, J. Li, J. Zhang, A. Asundi and Q. Chen, Sci. Rep., 2017, 7, 7654
7. G. Zheng, R. Horstmeyer and C. Yang, Nature Photonics 2013, 7, 739
8. Hai Gong, Temitope E. Agbana, Paolo Pozzi, Oleg Soloviev, Michel Verhaegen, and Gleb Vdovin, Opt. Lett., 2017, 42, 2122
9. I. Iglesias, Pyramid phase microscopy”, Opt. Lett., 2011, 36, 3636
10. S. B. Mehta and C.J.R. Sheppard, Opt Lett., 2009, 34, 1924
11. L. Tian and L. Waller, Opt. Expr., 2015, 23, 11394