Three‐dimensional intracellular transport in neuron bodies and neurites investigated by label‐free dispersion‐relation phase spectroscopy

Due to the limitations of fluorescence imaging techniques, the study of intracellular cargo is typically restricted to two‐dimensional analyses. To overcome low light levels and the risk of phototoxicity, we employ quantitative phase imaging, a family of full‐field imaging techniques that measure the optical path length shift introduced by the specimen. Specifically, we use spatial light interference microscopy (SLIM) to study the transport of mass in whole tomographic volumes and show that a time‐correlation technique, dispersion‐relation phase spectroscopy (DPS), can be used to simultaneously assay the horizontal and vertical traffic of mass through a cell. To validate our method, we compare the traffic inside cell bodies and neuronal extensions, showing that the vertical transport of mass may prove a more sensitive and interesting metric than similar measurements limited to a 2D, horizontal plane. © 2017 International Society for Advancement of Cytometry

[1]  K. Pearson VII. Note on regression and inheritance in the case of two parents , 1895, Proceedings of the Royal Society of London.

[2]  Pavel Tomancak,et al.  Guide to light-sheet microscopy for adventurous biologists , 2014, Nature Methods.

[3]  G. Popescu Quantitative Phase Imaging of Cells and Tissues , 2011 .

[4]  Gabriel Popescu,et al.  Label-Free Imaging of Single Microtubule Dynamics Using Spatial Light Interference Microscopy. , 2017, ACS nano.

[5]  Gabriel Popescu,et al.  Optical Assay of Erythrocyte Function in Banked Blood , 2014, Scientific Reports.

[6]  Nils O. Petersen,et al.  Image cross-correlation spectroscopy: A new experimental biophysical approach to measurement of slow diffusion of fluorescent molecules , 1996 .

[7]  Gabriel Popescu,et al.  Nuclear dynamics in metastatic cells studied by quantitative phase imaging , 2015, Photonics West - Biomedical Optics.

[8]  Jaeduck Jang,et al.  Dynamic spectroscopic phase microscopy for quantifying hemoglobin concentration and dynamic membrane fluctuation in red blood cells. , 2012, Optics express.

[9]  Sen Han,et al.  Live cell interferometry reveals cellular dynamism during force propagation. , 2008, ACS nano.

[10]  Molly J. Rossow,et al.  Raster image correlation spectroscopy in live cells , 2010, Nature Protocols.

[11]  Paul R. Selvin,et al.  Kinesin and Dynein Move a Peroxisome in Vivo: A Tug-of-War or Coordinated Movement? , 2005, Science.

[12]  Santiago Costantino,et al.  Spatiotemporal image correlation spectroscopy (STICS) theory, verification, and application to protein velocity mapping in living CHO cells. , 2005, Biophysical journal.

[13]  S. B. Kater,et al.  Isolated hippocampal neurons in cryopreserved long-term cultures: Development of neuroarchitecture and sensitivity to NMDA , 1988, International Journal of Developmental Neuroscience.

[14]  W. Webb,et al.  Thermodynamic Fluctuations in a Reacting System-Measurement by Fluorescence Correlation Spectroscopy , 1972 .

[15]  Gabriel Popescu,et al.  Active intracellular transport in metastatic cells studied by spatial light interference microscopy , 2015, Journal of biomedical optics.

[16]  H. B. Mann,et al.  On a Test of Whether one of Two Random Variables is Stochastically Larger than the Other , 1947 .

[17]  Jyothi Arikkath,et al.  Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex , 2012, Nature Protocols.

[18]  T. Meyvis,et al.  Fluorescence Recovery After Photobleaching: A Versatile Tool for Mobility and Interaction Measurements in Pharmaceutical Research , 1999, Pharmaceutical Research.

[19]  J. Rogers,et al.  Spatial light interference microscopy (SLIM) , 2010, IEEE Photonic Society 24th Annual Meeting.

[20]  H. Yokoyama,et al.  Fluorescence correlation spectroscopy with traveling interference fringe excitation , 1996 .

[21]  Marcin Wozniak,et al.  Trafficking Inside Cells: Pathways, Mechanisms and Regulation , 2009 .

[22]  Radim Chmelik,et al.  Dynamic phase differences based on quantitative phase imaging for the objective evaluation of cell behavior , 2015, Journal of biomedical optics.

[23]  Zhuo Wang,et al.  Dispersion-relation phase spectroscopy of intracellular transport , 2011, Optics express.

[24]  Michael T. Heath,et al.  Scientific Computing: An Introductory Survey , 1996 .