Quantitative
Iatia's QPI calculates phase values for each point in an image based on the degree of phase shift induced in light traversing through the sample. The magnitude of phase shift (changes in optical thickness) is the product of cell thickness and refractive index. When one of these cellular properties is known, the other can be determined.
Case study - cell volume measurement
In the following example, researchers at the Department of Physiology and Pharmacology at the University of Melbourne, Australia, calculated the volume of rat red blood cells exposed to imidazole-buffered solutions of varied tonicity 4.
Blood was collected from rats and transferred to standard haematological imidazole-buffered solutions (22oC) of different osmolality and graded tonicity (170, 240, 400, 540 mosm/kg), allowing 10 minutes equilibration prior to imaging. Brightfield and QPI phase images were acquired using an inverted Zeiss Axiovert 100M fitted with a Zeiss LD-Achroplan (x63, 0.8NA). Analysis of QPI phase data and calculation of cell volume was performed using IDL software (v5.5). Steady-state cell volume was calculated by integration of the cell delineated QPI phase values, assuming an intracellular refractive index (RI) of 1.367 (phase = cell thickness x RI). To delineate cell boundaries standard nearest-neighbour gradient thresholding techniques for edge detection were applied to the QPI phase images.
The following images are extracts from the results of this study showing the brightfield and QPI phase images of an Isotonic, Hypotonic and Hypertonic red blood cell along with a corresponding line profile of the phase shift of the respective sample.
Isotonic
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Hypotonic
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Hypertonic
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In osmotic conditions approximating isotonicity (240 mosm/kg, isotonic), red blood cells exhibited biconcave morphology evident in the brightfield image and appeared annular in the QPI phase image. A line profile of phase shift values measured across the cell diameter indicated a dual peak phase consistent with biconcave morphology.
In solutions of low tonicity (170 mosm/kg, hypotonic), cell bi-concavity was not evident in bright field images. In these conditions the cell phase map exhibited a centralised region of maximum phase shift. A linear profile of phase shift values measured across the cell diameter indicated spherical morphology consistent with osmotically-induced cell volume expansion.
Red blood cells exposed to solutions of high tonicity (400 and 540 mosm/kg, hypertonic) exhibited a centrally thinned appearance characterised by a plateau in the line profile, consistent with crenated morphology.
The assumed spherical shape of the cell under hypotonic conditions provided a means by which cell thickness values could be computed from phase shift measurement for cells in all osmotic environments.
Mean red blood cell volume measured in buffer solutions of different tonicity
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As shown above, an inverse relationship between buffer tonicity and mean red blood cell volume was observed. Phase-computed volume was found to increase with decreasing solution osmolality as anticipated: 42.81 ± 2.37, 48.71 ± 2.29, 62.64 ± 2.33, 90.82 ± 7.7 (µm3) in solutions of 540, 400, 240 and 170 mosm/kg respectively.
Conclusion
Erythrocyte morphology in different osmotic environments can be quantitatively assessed using QPI phase images. QPI provides an optically simple, accurate and non-destructive approach for evaluating cell structure, shape and volume which has potentially broad application for a range of cell types.
References
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Quantitative Optical Phase Microscopy
A Barty, KA Nugent, D Paganin and A Roberts, Optics Letters 23, 1-3 (1998). -
Quantitative Phase Tomography
A Barty, KA Nugent, A Roberts and D Paganin, Optics Communications 175 (2000) pp 329-336. -
Refractive index profiling of optical fibers with axial symmetry by use of quantitative phase microscopy
A Roberts, E Ampem-Lassen, A Barty A, KA Nugent, Optics Letters 2002;27:2061–2063. -
Single Cell Volume Measurement by Quantitative Phase Microscopy (QPM): A Case Study of Erythrocyte Morphology
Claire L. Curl, , Catherine J. Bellair, Peter J. Harris, Brendan E. Allman, Ann Roberts, Keith A. Nugent, and Lea M.D. Delbridge, Cellular Physiology and Biochemistry, 17, (2006).