Noninvasive time-dependent cytometry monitoring by digital holography

Abstract

Using a digital holographic microscope setup, it is possible to measure dynamic volume changes in living cells. The cells were investigated time-dependently in transmission mode for different kinds of stimuli affecting their morphology. The measured phase shift was correlated to the cellular optical thickness, and then of the cell volume as well as the refractive index were calculated and interpreted. For the characterization of the digital holographic microscope setup, we have developed a transparent three-dimensional (3-D) reference chart that can be used as a lateral resolution chart and step-height resolution chart included in one substrate. For the monitoring of living cells, a biocompatible and autoclavable flow chamber was designed, which allows us to add, exchange, or dilute the fluid within the flow chamber. An integrated changeable coverslip enables inverse microscopic applications. Trypsinization, cell swelling and shrinking induced by osmolarity changes, and apoptosis served as model processes to elucidate the potential of the digital holographic microscopy (DHM).

Quantitative Phase Imaging for Label-Free Analysis of Cancer Cells—Focus on Digital Holographic Microscopy

“…The ability of QPI to differentiate between viable and non-viable cells without the use of staining has been extensively studied [21,83]. The first DH microscopy images of tumor spheroids were published in 2003 [84], and the first images of single cells one year after that [85].…”

“…The first DH microscopy images of tumor spheroids were published in 2003 [84], and the first images of single cells one year after that [85]. Indeed, DH microscopy has turned out to be a very useful technique for cell death studies [21,83]. Cells that go into programmed cell death, or apoptosis, show a decreased cell phase shift as the first indication of early apoptosis, but several morphological alterations take place, such as cell shrinkage, formation of small blebs, nuclear fragmentation, chromatin condensation, chromosomal DNA fragmentation, and breakdown into several apoptotic bodies [86].…”

Digital Holography and Cell Studies

“…Among others, Kemper and coworkers have shown that erythrocyte shape can be clearly visualized by DHM . Kemmler and colleagues studied the morphological changes during trypsinization using oligodendrocytes from rat (Kemmler et al, 2007) and Emery and colleagues detected cell swelling and shrinking in primary mouse cortical neurons (Emery et al, 2007). Schnekenburger et al (2007) have applied DHM to study the dynamics of cytoskeleton changes in the human pancreatic tumor cell lines PaTu 8988S and PaTu 8988T.…”

“…Using the refractive index to measure thickness, the study showed that cells treated with toxins or cell-death inducing drugs were thinner than control cells. DHM has also been used to follow the process of staurosporine-induced apoptosis in oligodendrocytes (Kemmler et al, 2007). More details of the apoptotic process have been shown by Colomb et al (2008), where the apoptotic blebbing in prostate cells was clearly visualized.…”

“…Movement in both 2-D and 3-D has been studied Persson et al, in press). In addition, cell viability status can be determined using DHM (Kemper et al, 2006;Kemmler et al, 2007). Interestingly, it is possible to study both single cells and entire populations simultaneously, allowing for very detailed studies.…”