Quantitative phase microscopy: a new tool for investigating the structure and function of unstained live cells.
1. The optical transparency of unstained live cell specimens limits the extent to which information can be recovered from bright-field microscopic images because these specimens generally lack visible amplitude-modulating components. However, visualization of the phase modulation that occurs when light traverses these specimens can provide additional information. 2. Optical phase microscopy and derivatives of this technique, such as differential interference contrast (DIC) and Hoffman modulation contrast (HMC), have been used widely in the study of cellular materials. With these techniques, enhanced contrast is achieved, which is useful in viewing specimens, but does not allow quantitative information to be extracted from the phase content available in the images. 3. An innovative computational approach to phase microscopy, which provides mathematically derived information about specimen phase-modulating characteristics, has been described recently. Known as quantitative phase microscopy (QPM), this method derives quantitative phase measurements from images captured using a bright-field microscope without phase- or interference-contrast optics. 4. The phase map generated from the bright-field images by the QPM method can be used to emulate other contrast image modes (including DIC and HMC) for qualitative viewing. Quantitative phase microscopy achieves improved discrimination of cellular detail, which permits more rigorous image analysis procedures to be undertaken compared with conventional optical methods. 5. The phase map contains information about cell thickness and refractive index and can allow quantification of cellular morphology under experimental conditions. As an example, the proliferative properties of smooth muscle cells have been evaluated using QPM to track growth and confluency of cell cultures. Quantitative phase microscopy has also been used to investigate erythrocyte cell volume and morphology in different osmotic environments. 6. Quantitative phase microscopy is a valuable, new, non-destructive, non-interventional experimental tool for structural and functional cellular investigations.
Curl
Claire L
CL
Department of Physiology, University of Melbourne, Parkville, Victoria 3010, Australia.
Bellair
Catherine J
CJ
Harris
Peter J
PJ
Allman
Brendan E
BE
Roberts
Ann
A
Nugent
Keith A
KA
Delbridge
Lea M D
LM
D000818:Animals; D048429:Cell Size; D004847:Epithelial Cells / Q000166:cytology* / Q000502:physiology*; D005069:Evaluation Studies as Topic; D006801:Humans; D008857:Microscopy, Interference / Q000379:methods* / Q000592:standards; D008858:Microscopy, Phase-Contrast / Q000379:methods / Q000592:standards