CARS microscopy is a label-free imaging method that is capable of real-time, non-contact and nondestructive examination of various biological samples.
Biology Imaging, Medical Diagnosis of Disease, Material Science
In the CARS technique, a pump beam at frequency ωp and a Stokes beam at frequency ωs interact with a sample. When the beat frequency ωp - ωs matches one of the vibrational modes in the sample, a strong anti-Stokes signal at ωas = 2ωp – ωs is generated in the phase matching direction (Fig.1). Chemically selective information can be provided by tuning into characteristic vibrational resonances in the samples.
The advantages of CARS technique are summarized as follows:
• CARS signals are 3-4 orders of magnitude stronger than those of spontaneous Raman process,
• video-rate vibrational imaging at reasonable excitation powers,
• sample photodamage is minimized, because picosecond lasers are used,
• the CARS signals are blue-shifted from the pump and Stokes frequencies, and they can be easily detected in the presence of any fluorescence background,
• an enhanced spatial resolution, because the CARS signal is generated only from the focal volume, where two beams are overlapped (Stokes and pump beams),
• the capability of three-dimensional sectioning without the need of confocal geometry,
• it does not require exogenous dyes or markers (any labeling may strongly affect the specimen properties).
The commercial CARS imaging microscope, RAMOS CARS system (Ostec), is shown below.
The RAMOS CARS imaging microscope allows a complete multimodal investigation of tissues, in particular:
• Coherent Anti Stokes Raman Microscopy (CARS), including F-CARS, E-CARS and P-CARS
• Stimulated Raman Scattering (SRS) Microscopy
• Raman confocal imaging,
• Fluorescence Life Time Imaging (FLIM) Microscopy, including FRET and FRAP,
• Two-Photon Excited Fluorescence (TPFE) Microscopy,
• Second-harmonic Imaging (SHG) Microscopy,
• Laser reflection & transmission imaging,
• Upconversion Luminescence,
• SONICC imaging (SONICC is an emerging technique for crystal imaging based on second harmonic generation effect found in chiral crystals).
The following examples are provided to demonstrate the quality of results.
1. CARS/TPEF imaging of cancer HeLa cells (Fig.2).
Figure 2. HeLa cells imaging. Proteins (blue color) and lipids (grey color) have been observed in the CARS mode at their characteristic vibrations of 2930 cm-1 and 2840 cm-1, respectively. Nucleic acids, strained by acridine orange, have been acquired in the red (Ribonucleic acid, RNA) and green (DNA) fluorescence channels in TPEF mode.
(Courtesy of A.Kachynski, A. Kuzmin, and P. N. Prasad, The State University of New York at Buffalo)
2. Imaging of bacteriorhodopsin (BR) crystals (Fig.3).
Figure 3. BR crystals imaging
(Arzumanyan, Grigory M. J. Am. Chem. Soc., 2016, 138 (41), p. 13457)
RAMOS CARS system represents a novel and sensitive tool for biology and materials sciences. The experimental data demonstrate that the CARS imaging approach is a fast and nondestructive technique.