CARS Microscopy Made Simple
Abstract: Coherent Anti-Stokes Raman Scattering (CARS) Microscopy has emerged as an important tool for label-free yet chemical-specific imaging of live cells and tissue. The contrast mechanism in CARS derives from the natural vibrational Raman spectrum of the molecule of interest. Since Raman linewidths in the condensed phase are typically 10 cm-1 or so, for the past decade it was assumed that picosecond lasers, having bandwidths in the 10cm-1 range, will be required for CARS microscopy. Femtosecond pulses, being >100x broader, were thought to give poor contrast. In fact, this is not the case if one takes advantage of the control over optical phase that is implicit in femtosecond pulses. Recently, a much simplified approach was demonstrated wherein a single femtosecond oscillator, combined with a photonic crystal fibre, is used for high performance CARS microscopy. In this approach, control over the linear 'chirp' of the optical phase is used as a flexible tool to optimize performance. The method is simple, robust and flexible enough that a major microscope manufacturer recently used it to develop the first commercially available CARS microscope. An overview of the optimally chirped approach is presented, including recent developments and new opportunities, and illustrate these with applications to selected problems in biomedical imaging. Full Text available: “CARS Microscopy Made Simple,” Biophotonics, October 2009, Optics Express 17, p 2984, (2009)
Adrian F. Pegoraro, Albert Stolow, National Research Council Canada and Queen's University; Andrew Ridsdale, Douglas J. Moffatt, John Paul Pezacki, National Research Council Canada; and Yiwei Jia, Olympus America Inc.
All-fiber multimodal CARS microscopy of live cells
Abstract: Using an all-fiber laser system consisting of a femtosecond Er/Yb fiber oscillator as the pump and an ultra-highly nonlinear fiber for Stokes generation, we demonstrate multimodal (TPF+SHG+CARS) non-linear optical microscopy of both tissue samples and live cells. Multimodal imaging was successfully performed with pixel dwell times as short as 4 µs at low laser powers (< 40 mW total). Optics Express Vol. 17, Issue 23, pp. 20700-20706 (2009)
Adrian F. Pegoraro, Andrew Ridsdale, Douglas J. Moffatt, John P. Pezacki, Brian K. Thomas, Libin Fu, Liang Dong, Martin E. Fermann, and Albert Stolow
Multimodal nonlinear optical imaging of atherosclerotic plaque development in myocardial infarction-prone rabbits
Abstract: Label-free imaging of bulk arterial tissue is demonstrated using a multimodal nonlinear optical microscope based on a photonic crystal fiber and a single femtosecond oscillator operating at 800 nm. Colocalized imaging of extracellular elastin fibers, fibrillar collagen, and lipid-rich structures within aortic tissue obtained from atherosclerosis-prone myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits is demonstrated through two-photon excited fluorescence, second harmonic generation, and coherent anti-Stokes Raman scattering, respectively. These images are shown to differentiate healthy arterial wall, early atherosclerotic lesions, and advanced plaques. Clear pathological changes are observed in the extracellular matrix of the arterial wall and correlated with progression of atherosclerotic disease as represented by the age of the WHHLMI rabbits. Journal of Biomedical Optics Vol. 15, 020501 (2010)
Alex C. T. Ko, Andrew Ridsdale, Michael S. D. Smith, Leila B. Mostaço-Guidolin, Mark D. Hewko, Adrian F. Pegoraro, Elicia K. Kohlenberg, Bernie Schattka, Masashi Shiomi, Albert Stolow, and Michael G. Sowa
Direct imaging of the disruption of hepatitis C virus replication complexes by inhibitors of lipid metabolism
Abstract: Here we have simultaneously characterized the influence of inhibitors of peroxisome proliferator-activated receptor alpha (PPARalpha) and the mevalonate pathway on hepatocyte lipid metabolism and the subcellular localization of hepatitis C virus (HCV) RNA using two-photon fluorescence (TPF) and coherent anti-Stokes Raman scattering (CARS) microscopy. Using this approach, we demonstrate that modulators of PPARalpha signaling rapidly cause the dispersion of HCV RNA from replication sites and simultaneously induce lipid storage and increases in lipid droplet size. We demonstrate that reductions in the levels of cholesterol resulting from inhibition of the mevalonate pathway upregulates triglyceride levels. We also show that the rate of dispersion of HCV RNA is very rapid when using a PPARalpha antagonist. This occurs with a faster rate to that of direct inhibition of 3-hydroxy-3-methyglutaryl CoA reductase (HMG-CoA reductase) using lovastatin in living cells, demonstrating the potential therapeutic value of modulating host cell pathways as part of a strategy to eliminate chronic HCV infection. DOI: (Virology, Volume 394, Issue 1, 10 November 2009, Pages 130-142)
Rodney K. Lyn, David C. Kennedy, Selena M. Sagan, David R. Blais, Yanouchka Rouleau, Adrian F. Pegoraro, X. Sunney Xie, Albert Stolow, John Paul Pezacki
All-fiber multimodal CARS microscopy of live cells
Abstract: Coherent anti-Stokes Raman scattering (CARS) microscopy is a promising tool for live cell imaging and has been applied to the study of diseases such as multiple sclerosis, atherosclerosis and hepatitis. Combining PCFs with optimally chirped fs pulses, high performance multimodal (i.e. simultaneous two-photon fluorescence (TPF), second harmonic generation (SHG) and CARS) imaging of live cells and tissues is possible using only a single laser. Here the paper demonstrates a fully operational multimodal live cell and tissue imaging system based on a compact fs Er fiber oscillator system. A key component is a newly developed ultra-highly nonlinear silica fiber (UHNLF) which greatly reduces the peak power requirement for Stokes pulse generation. A commercial fully integrated frequency doubled fs Er fiber laser (IMRA F100) was the pump for TPF and SHG imaging. Here, in this paper a multimodal image of a fixed rabbit atherosclerotic arterial sample where TPF (elastin), SHG (collagen) and CARS (lipid) signals were collected simultaneously. This demonstrates for the first time the feasibility of a compact all-fiber source for multimodal CARS microscopy of live cells and tissue. ( Lasers and Electro-Optics 2009 and the European Quantum Electronics Conference. CLEO Europe - EQEC, 14-19 June 2009.)
Pegoraro, A.F.; Ridsdale, A.; Lausten, R.; Moffatt, D.J.; Stolow, A.; Thomas, B.K.; Libin Fu; Liang Dong; Fermann, M.E.
Intracellular imaging of HCV RNA and cellular lipids by using simultaneous two-photon fluorescence and coherent anti-Stokes Raman scattering microscopies
Abstract: Hepatitis C virus (HCV) infection is associated with changes in host-cell lipid metabolism. Here we describe a new approach for detecting HCV RNA using two-photon fluorescence (TPF), and HCV-associated changes in cellular lipids using coherent anti-Stokes Raman scattering (CARS) microscopy. By combining the two types of microscopy with a common laser source, we visualized both phenomena simultaneously and profiled cellular lipids and subcellular localization of RNA in real time. (DOI: ChemBioChem 7, 1895 (2006)
X-L. Nan, A.M. Tonary, A. Stolow, X.S. Xie, J.P. Pezacki
Optimally chirped multimodal CARS microscopy based on a single Ti:sapphire oscillator
Astract: We demonstrate high performance coherent anti-Stokes Raman scattering (CARS) microscopy of live cells and tissues with user-variable spectral resolution and broad Raman tunability (2500 - 4100 cm(-1)), using a femtosecond Ti:Sapphire pump and photonic crystal fiber output for the broadband synchronized Stokes pulse. Spectral chirp of the fs laser pulses was a user-variable parameter for optimization in a spectral focusing implementation of multimodal CARS microscopy. High signal-to-noise, high contrast multimodal imaging of live cells and tissues was achieved with pixel dwell times of 2-8 micros and low laser powers (< 30 mW total). (Opt Express. 2009 Feb 16;17(4):2984-96)
Pegoraro AF, Ridsdale A, Moffatt DJ, Jia Y, Pezacki JP, Stolow A.
Introduction to Laser Scanning Microscopy NRC CARSLab Course
Workshop Presentation by Andrew Millar, Olympus Canada, Laser Scanning Microscopy Basics