Miss Claudia Di Napoli
Label-free multiphoton microscopy of intracellular lipids using Coherent Antistokes Raman Scattering (CARS) and differential CARS (D-CARS
The current first world “obesity epidemic” is a result of a reduction in physical activity and increase in calorie consumption as changes in modern lifestyle take place. Lipid storage mechanisms, which have evolved to allow animals to survive periods of nutrient deprivation, contribute to obesity resulting in clinical problems such as type 2 diabetes and atherosclerosis. The bulk of the excess energy derived from diet is stored as triacylglycerol, which can be efficiently packaged within mammalian cells as a cytosolic lipid droplet. However, still little is known about what controls the formation, trafficking and breakdown of lipid droplets in living cells.
Optical microscopy is still the only practical means of obtaining high spatial and temporal resolution within living cells and tissues. Coherent Antistokes Raman Scattering microscopy has recently emerged as a new multiphoton (i.e. nonlinear) microscopy technique which overcomes the need of fluorescent labelling and yet retains biomolecular specificity. CARS also features intrinsic spatial resolution in three dimensions (optical sectioning). This technique has proven to be very successful in imaging unstained lipid membranes and lipid vesicles via the Raman scattering of light with the stretching vibration of the dense carbon-hydrogen bonds in the aliphatic chain in lipids. CARS chemical specificity can be improved with differential CARS (D-CARS), a technique recently invented in my group that allows the elimination of non-resonant CARS background.
[See Rocha-Mendoza I et al., Optics Letters 34, 2258 (2009);
Langbein W. et al., Appl. Phys. Lett. 95, 081109 (2009)]
In collaboration with the School of Physics, my group has built a CARS/D-CARS microscope that incorporates two-photon fluorescence and second harmonic generation for comparative/complementary studies. In my project I am developing correlative CARS/fluorescence microscopy to visualise unstained lipid droplets with CARS/D-CARS and associated protein components with two-photon fluorescence, in order to determine the dynamics of lipid droplet formation and development in living cells.