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Quantitative phase imaging - A new way to 'see' cells
Abstract
A new technique for quantitative phase imaging offers a solution to the problem of microscopic visualisation of virtuality transparent viable cells. Using this technique the refractive and absorptive structures of a cell can be seen independently and analogue image types can be generated from simple bright field views.
... A difference in cytoplasmic and nuclear RI values has been reported for Chinese hamster ovary cells in studies using light scattering techniques. We also previously observed a marked increase in the phase value corresponding to the nuclei of epithelial cells, which is suggestive of increased RI (22). A higher nuclear RI may be associated with more active cell cycling and growth. ...
... QPM is a newly developed method of visualizing unstained, live cellular specimens, which may be simply implemented, and requires only brightfield imaging conditions (13,19,22,24). With QPM the growth of cell cultures can be tracked and cell functional responses monitored in situ by repeated imaging without compromising the cell viability. ...
The refractive index (RI) of cellular material provides fundamental biophysical information about the composition and organizational structure of cells. Efforts to describe the refractive properties of cells have been significantly impeded by the experimental difficulties encountered in measuring viable cell RI. In this report we describe a procedure for the application of quantitative phase microscopy in conjunction with confocal microscopy to measure the RI of a cultured muscle cell specimen.
The experimental strategy involved calculation of cell thickness by using confocal optical sectioning procedures, construction of a phase map of the same cell using quantitative phase microscopy, and selection of cellular regions of interest to solve for the cell RI.
Mean cell thickness and phase values for six cell regions (five cytoplasmic and one nuclear) were determined. The average refractive index calculated for cytoplasmic and nuclear regions was 1.360 +/- 0.004. The uncertainty in the final RI value represents the technique measurement error.
The methodology we describe for viable cell RI measurement with this prototype cell has broad generic application in the study of cell growth and functional responses. The RI value we report may be used in optical analyses of cultured cell structure and morphology.
... The QPM methodology described has general application potential for cell volume measurement in a variety of cell types including epithelial, muscle, neural and stem cells [34][35][36]. QPM may be applied to cell suspensions, cultures and sections with appropriate specification of optical and imaging conditions. Consideration and validation of the QPM methodology in relation to magnification selected, depth of field of objective and specimen dimensional aspects has been previously documented [20]. ...
The measurement of the volume of intact, viable cells presents challenging problems in many areas of experimental and diagnostic science involved in the evaluation of cellular morphology, growth and function. This investigation details the implementation of a recently developed quantitative phase microscopy (QPM) method to measure the volume of erythrocytes under a range of osmotic conditions. QPM is a computational approach which utilizes simple bright field optics to generate cell phase maps which, together with knowledge of the cellular refractive index, may be used to measure cellular volume. Rat erythrocytes incubated in imidazole-buffered solutions (22 degrees C) of graded tonicity were analysed using QPM (n=10 cells/group, x63, 0.8 NA objective). Erythrocyte refractive index (1.367) was measured using a combination of phase and morphological data obtained from cells adopting spherical geometry under hypotonic conditions. Phase-computed volume increased with decreasing solution osmolality: 42.8 +/- 2.4, 48.7 +/- 2.3, 62.6 +/- 2.3, 90.8 +/- 7.7 microm3 in solutions of 540, 400, 240, and 170 mosmol/kg respectively. These volume changes were associated with crenated, bi-concave and spherical morphological states associated with increasing tonicity. This investigation demonstrates that QPM is a valid, simple and non-destructive approach for measuring cellular phase properties and volume. QPM cell volume analysis represents a significant advance in viable cell experimental capability and provides for acquisition of 'real-time' data - an option not previously available using other approaches.
The quantitative phase imaging (QPI), a patented algorithm, developed by the University of Melbourne, that quantifies the relationship between phase and intensity while propagating, was described. The phase information was used to visualize and measure the refractive (phase) properties of the sample from a simple through-focal series of brightfield images. The images were produced using QPm™ software, the commercial implementation of the QPI technique. QPI is not limited to the common microscopies, and has also been applied in exotic imaging environments such as X-ray microscopy, X-ray and neutron radiography and in production of three-dimensional phase tomograms.
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.
Quantitative phase microscopy (QPM) is a recently developed computational approach that provides quantitative phase measurements of specimen images obtained under bright-field conditions without phase- or interference-contrast optics. To perform QPM, an in-focus bright-field image is acquired, together with one positive and one negative de-focus image. An algorithm is then applied to produce a specimen phase map. In this investigation we demonstrate that manipulation of the phase map intensity histogram using novel, non-subjective thresholding and segmentation methods provides enhanced delineation of cells in culture. QPM was utilised to measure the growth behaviour of cultured airway smooth muscle cells over a 92-h period. There was a high degree of correlation between parallel QPM-derived confluency measurements and haemocytometry-derived counts of airway smooth muscle cells over this time period. Using QPM, translucent cells can be visualised with improved cell boundary definition allowing precise and reproducible measurements of cell culture confluency. Quantitative phase imaging provides a rapid, optically simple and non-destructive approach for measurement of cellular morphology. Further development of the QPM-based analysis methodology has the potential to provide even more refined measures of cellular growth.
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