Holographic Microscopy Techniques for Multifocus Phase Imaging of Living Cells - Biomedical Optical Phase Microscopy and Nanoscopy - page 119

the image sharpness quantification process that is used for the determination of the axial
sample displacements. Furthermore, these disturbances affect the correct determination of
the lateral object position from the quantitative phase contrast images. This limits the
density of objects under investigations to an amount in which the specimens are imaged
laterally separated. Nevertheless, label-free 3D tracking with DHM prospects new
application areas of quantitative phase imaging for particle and cell tracking in fluidics and
3D cell migration monitoring in cancer research [54] .
6.8 Conclusions
The presented concepts and the experimental results demonstrate that DHM permits reliable
multifocus quantitative phase contrast imaging of adherent and suspended cells. DHM in
off-axis and self-interference configuration enables a vibration insensitive (hologram capture
time in or below the millisecond range) and noncontact full-field measurement of dynamic
cellular processes. The results further demonstrate that the presented DHM concepts can be
utilized for investigations on dynamic cell morphology changes and for the quantitative
analysis of cellular reactions on toxin treatment. The obtained information opens up new
quantitative ways for label-free dynamic cell monitoring, and may help to access new
parameters, for example, for a minimally invasive support in apoptosis and necrosis recognition
or for the label-free quantitative characterization of subcellular structures like nucleoli or
vacuoles. Future prospects include the use of DHM for multimodal imaging [87] ,3Dcell
tracking in a 3D environment, further analysis of the quantitative phase images for light
scattering (see Ref. [88] ), and new evaluation parameters [51] . A higher specificity of the
technology and an improvement in the lateral and axial resolutions may be expected by the
integration of nonlinear effects like second harmonic generation [89,90] . In addition, recently
developed novel algorithms for the deconvolution of the holographically retrieved wave fields
[91,92] and the corresponding quantitative phase images [93] prospect a further improvement
in the spatial resolution. In conclusion, the presented methods have the potential to form
versatile microscopy tools for label-free quantitative analysis in the field of live cell imaging.
Acknowledgments
Financial support by the German Federal Ministry for Education and Research (BMBF) as part of the
“Biophotonics” research program and the European Network of Excellence “Photonics for Life (P4L)” is
gratefully acknowledged.
References
[1] E.M. Goldys (Ed.), Fuorescence Applications in Biotechnology and the Life Siences, Wiley, New Jersey,
2009.
[2] V. Ntziachristos, Fluorescence molecular imaging, Annu. Rev. Biomed. Eng. 8 (2006) 1 33.