Dynamic phase differences based on quantitative phase imaging for the objective evaluation of cell behavior.

Quantitative phase imaging (QPI) brought innovation to noninvasive observation of live cell dynamics seen as cell behavior. Unlike the Zernike phase contrast or differential interference contrast, QPI provides quantitative information about cell dry mass distribution. We used such data for objective evaluation of live cell behavioral dynamics by the advanced method of dynamic phase differences (DPDs). The DPDs method is considered a rational instrument offered by QPI. By subtracting the antecedent from the subsequent image in a time-lapse series, only the changes in mass distribution in the cell are detected. The result is either visualized as a two dimensional color-coded projection of these two states of the cell or as a time dependence of changes quantified in picograms. Then in a series of time-lapse recordings, the chain of cell mass distribution changes that would otherwise escape attention is revealed. Consequently, new salient features of live cell behavior should emerge. Construction of the DPDs method and results exhibiting the approach are presented. Advantage of the DPDs application is demonstrated on cells exposed to an osmotic challenge. For time-lapse acquisition of quantitative phase images, the recently developed coherence-controlled holographic microscope was employed.

Automated alignment method for coherence-controlled holographic microscope

A coherence-controlled holographic microscope (CCHM) was developed particularly for quantitative phase imaging and measurement of live cell dynamics, which is the proper subject of digital holographic microscopy (DHM). CCHM in low-coherence mode extends DHM in the study of living cells. However, this advantage is compensated by sensitivity of the system to easily become misaligned, which is a serious hindrance to wanted performance. Therefore, it became clear that introduction of a self-correcting system is inevitable. Accordingly, we had to devise a theory of a suitable control and design an automated alignment system for CCHM. The modulus of the reconstructed holographic signal was identified as a significant variable for guiding the alignment procedures. From this, we derived the original basic realignment three-dimensional algorithm, which encompasses a unique set of procedures for automated alignment that contains processes for initial and advanced alignment as well as long-term maintenance of microscope tuning. All of these procedures were applied to a functioning microscope and the tested processes were successfully validated. Finally, in such a way, CCHM is enabled to substantially contribute to study of biology, particularly of cancer cells in vitro.

Numerical refocusing in digital holographic microscopy with extended-sources illumination.

Numerical refocusing can be seen as a method of compensating the defocus aberration based on deconvolution by inverse filtering [1] in digital holographic microscopy (DHM). It is well-understood in cases when a coherent (ie point and monochromatic) light source such as a collimated laser beam is used [2]. This paper extends the theory to the case of illumination by a quasi-monochromatic extended (spatially incoherent) source. Refocusing methods for spatially incoherent illumination are derived and benefits of this type of illumination are demonstrated. We have proved both theoretically and experimentally that coherent-based refocusing gives incorrect results for extended-source illumination, while results obtained using the newly derived method are correct.