Biological imaging with coherent Raman scattering microscopy: a tutorial

Abstract

Coherent Raman scattering (CRS) microscopy is gaining acceptance as a valuable addition to the imaging toolset of biological researchers. Optimal use of this label-free imaging technique benefits from a basic understanding of the physical principles and technical merits of the CRS microscope. This tutorial offers qualitative explanations of the principles behind CRS microscopy and provides information about the applicability of this nonlinear optical imaging approach for biological research.

Rapid spontaneous Raman light sheet microscopy using cw-lasers and tunable filters

“…We choose working with 532 and 636 nm sources in our experiments to take advantage of the CMOS quantum efficiency which is >30% for the excited Raman wavelengths. Notice that the spectral resolution obtained with green and red lasers is good enough to resolve vibrational bands in the C-H region (2700-3200 cm −1 ) and that such region has been successfully exploited for biological studies on lipids and tissue using CARS and/or SRS nonlinear Raman imaging [4][5][6].…”

“…Additionally, the potential use of the technique for studying biological living samples is also demonstrated using spontaneous Raman imaging in the C-H (2600-3200 cm −1 ) specific region as widely used in CARS and SRS microscopy [3][4][5][6]15]. …”

“…In this sense, cost-effective SRS imaging has been implemented using a pair of continuous wave (cw) lasers [7,8] which are more stable, easy to operate and can be used in combination with fluorescence imaging. However, the SRS signal is induced via a Raman gain (or loss) process at one of the available wavelengths of the two sources [4,[6][7][8]; therefore, a fast modulation and lock-in based detection should be implemented to detect the SRS signal. As this is based on a point detection scheme, a laser scanning system is normally used.…”

“…Confocal micro-Raman based systems are able of obtaining optical sectioning using the label-free contrast mechanism intrinsically induced by the molecules contained in the sample [1]; however, due to the very low efficiency of the spontaneously emitted Raman signal (1 in 10 8 incident photons is Raman-shifted [2]), the long image acquisition time (normally from a few seconds to several hours) makes it unsuitable for studying most of the dynamic processes of living microorganisms. To exploit the benefits of the chemical contrast of the Raman effect, novel microscopic techniques based on nonlinear coherent anti-Stokes Raman scattering (CARS) or Stimulated Raman Scattering (SRS) processes have emerged providing high detection sensitivity, fast imaging speeds and intrinsic confocal capabilities [3][4][5][6]. Despite that, the optical systems involved are usually complex to be implemented and the richness of the full Raman spectral information is commonly limited by the wavelength tuning range (or bandwidth) of the ultrashort pulse laser sources used; which can be expensive.…”

Multimodal fiber source for nonlinear microscopy based on a dissipative soliton laser

“…Secondand third-harmonic generation imaging share some benefits with multiphoton imaging, while offering complementary capabilities. Imaging techniques based on Raman processes, including coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) microscopy, also continue to proliferate [1]. Raman microscopies typically require two synchronized picosecond pulse trains, with the frequency difference between the two colors tunable to match the vibrational resonances of interest.…”