Abstract
We develop a coherent anti-Stokes Raman scattering (CARS) microscopy system equipped with a tunable picosecond laser for high-speed wavelength scanning. An acousto-optic tunable filter (AOTF) is integrated in the laser cavity to enable wavelength scanning by varying the radio frequency waves applied to the AOTF crystal. An end mirror attached on a piezoelectric actuator and a pair of parallel plates driven by galvanometer motors are also introduced into the cavity to compensate for changes in the cavity length during wavelength scanning to allow synchronization with another picosecond laser. We demonstrate fast spectral imaging of 3T3-L1 adipocytes every 5 cm-1 in the Raman spectral region around 2850 cm-1 with an image acquisition time of 120 ms. We also demonstrate fast switching of Raman shifts between 2100 and 2850 cm-1, corresponding to CD2 symmetric stretching and CH2 symmetric stretching vibrations, respectively. The fast-switching CARS images reveal different locations of recrystallized deuterated and nondeuterated stearic acid.
Highlights
Raman microscopy is a powerful tool for biochemical observation and analysis that enables nonstaining, noninvasive imaging with high selectivity of specific chemical structures and properties by directly detecting their molecular vibrations.[1,2] Some biological studies demand observation times on the order of several seconds to minutes, such as observation of lipid droplet dynamics,[3,4] fatty acid uptake into lipid droplets for energy storage,[5] and fatty acid oxidation for providing bioenergy in muscle and heart tissue.[6,7] biological processes may occur faster, and an imaging system capable of acquiring images at video rates or faster would be advantageous
Spectral coherent anti-Stokes Raman scattering (CARS) images of 3T3-L1 adipocytes were observed by scanning the wavelength of the acousto-optic tunable filter (AOTF) laser every 0.5 nm from 875.0 to 905.0 nm, which corresponded to a Raman shift of 2663.21 to 3049.41 cm−1
Fast imaging was achieved by use of a microlens array, and good spectral imaging was demonstrated by using the AOTF laser
Summary
Raman microscopy is a powerful tool for biochemical observation and analysis that enables nonstaining, noninvasive imaging with high selectivity of specific chemical structures and properties by directly detecting their molecular vibrations.[1,2] Some biological studies demand observation times on the order of several seconds to minutes, such as observation of lipid droplet dynamics (e.g., exchange and transfer of lipid droplet contents),[3,4] fatty acid uptake into lipid droplets for energy storage,[5] and fatty acid oxidation for providing bioenergy in muscle and heart tissue.[6,7] biological processes may occur faster, and an imaging system capable of acquiring images at video rates or faster would be advantageous. A picosecond mode-locked laser has been used as a narrowband tunable laser source to achieve high spectral resolution.[12,15,16,17] In biological applications, Raman spectra are usually observed in fingerprint and CH vibrational regions, typically around 1000 and 300 cm−1, respectively.[10,11,14] Further enhancement can be achieved by extending the observation range to the silent region at 1800 to 2800 cm−1 where some Raman-tag molecules generate signals without any interference from biological specimens.[18,19,20]. The AOTF laser was tunable from 800 to 940 nm with a typical pulse duration of 10 ps and a resynchronization duration of 2~0 ms∕wavelength change.[30] An average laser power >300 mW was high enough for obtaining CARS images with multifocus scanning.[30] For demonstration purposes, fast spectral images of lipid droplets in 3T3-L1 adipocytes were obtained every 5 cm−1 within a Raman spectral region around 2850 cm−1 at 120 ms∕image. Those wavelengths correspond to Raman shifts of 2100 cm−1 (CD2 stretching in deuterated stearic acid) and 2845 cm−1 (CH2 stretching in nondeuterated stearic acid)
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