SPECTROSCOPY, IMAGING, OTHER TECHNIQUES |
Nonperturbative Chemical Imaging of Organelle Transport in Living Cells with Coherent Anti-Stokes Raman Scattering Microscopy
Xiaolin Nan 1, Eric Potma 1 and Sunney Xie 1*
1 Harvard University
* To whom correspondence should be addressed. E-mail: xie{at}chemistry.harvard.edu.
Submitted on September 14, 2005
Revised on November 12, 2005
Accepted on 5 April 2006
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Abstract |
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Nonperturbative monitoring of intracellular organelle transport in unstained living cells was achieved with coherent anti-Stokes Raman scattering (CARS) microscopy. To avoid possible interference with the organelle transport introduced by laser radiation, we first examined different illumination conditions. Using a new photodamage criterion based on morphological changes of the cells, we determined the threshold values of both pulse energy and average power at relevant wavelengths. Under excitation conditions much milder than the threshold levels, we were able to monitor the motions of lipid droplet (LD) organelles in steroidogenic mouse adrenal cortical (Y-1) cells with CARS microscopy in real time without perturbations to the cells. Particle tracking analyses revealed sub-diffusion as well as active transport of LDs along microtubules. Interestingly, LD active transport is only present in Y-1 cells that rounded up in culture, a morphological change associated with steroidogenesis, suggesting possible involvements of LD active transport in the latter. Simultaneous imaging of LDs and mitochondria with CARS and two-photon fluorescence microscopy clearly showed that interactions between the two organelles could be facilitated by high LD motility. These observations demonstrate CARS microscopy as a powerful noninvasive imaging tool for studying dynamic processes in living cells.
Key Words:
CARS Microscopy, Lipid Droplets, Organelle Transport, Steroidogenesis, Two Photon Fluorescence Microscopy, Y-1
