Published: July 17th, 2016
Coherent anti-Stokes Raman scattering (CARS) microscopy based on inherent vibration of molecule bonds permits label-free chemically selective live cell imaging. This work presents the implementation of a complementary microscopy technique on a standard multiphoton laser scanning microscope based on a femtosecond Ti:sapphire laser and an OPO laser.
Laser scanning microscopes combining a femtosecond Ti:sapphire laser and an optical parametric oscillator (OPO) to duplicate the laser line have become available for biologists. These systems are primarily designed for multi-channel two-photon fluorescence microscopy. However, without any modification, complementary non-linear optical microscopy such as second-harmonic generation (SHG) or third harmonic generation (THG) can also be performed with this set-up, allowing label-free imaging of structured molecules or aqueous medium-lipid interfaces. These techniques are well suited for in-vivo observation, but are limited in chemical specificity. Chemically selective imaging can be obtained from inherent vibration signals based on Raman scattering. Confocal Raman microscopy provides 3D spatial resolution, but it requires high average power and long acquisition time. To overcome these difficulties, recent advances in laser technology have permitted the development of nonlinear optical vibrational microscopy, in particular coherent anti-Stokes Raman scattering (CARS). CARS microscopy has therefore emerged as a powerful tool for biological and live cell imaging, by chemically mapping lipids (via C-H stretch vibration), water (via O-H stretch vibrations), proteins or DNA. In this work, we describe the implementation of the CARS technique on a standard OPO-coupled multiphoton laser scanning microscope. It is based on the in-time synchronization of the two laser lines by adjusting the length of one of the laser beam path. We present a step-by-step implementation of this technique on an existing multiphoton system. A basic background in experimental optics is helpful and the presented system does not require expensive supplementary equipment. We also illustrate CARS imaging obtained on myelin sheaths of sciatic nerve of rodent, and we show that this imaging can be performed simultaneously with other nonlinear optical imaging, such as standard two-photon fluorescence technique and second-harmonic generation.
Optical microscopy has become a major technique for nondestructive visualization of dynamic processes in living biological systems with a subcellular resolution. Fluorescence microscopy is currently the most popular imaging contrast used in live cells due to its high specificity and sensitivity1. A large palette of fluorescent probes has emerged (exogenous dyes, genetically encoded proteins, semiconductor nanoparticles). Various sample illumination fluorescent-based techniques have flourished (such as confocal or two-photon microscopy) to perform 3D imaging and to reduce a main drawback of this technique which is photobleaching2. Other limitation....
Figure 1. Schematic view of the general set-up. It includes the Ti:sapphire (680 - 1,080 nm) and the OPO (1,050 - 1,300 nm) lasers, the delay line with the 4 mirrors (M1 to M4), the fast oscilloscope, the photodiode and two fixed iris diaphragms I1 and I2. Mirrors M2 and M3 are fixed on a linear translation stage enabling to change the.......
The pulse train frequency of standard Ti:sapphire laser is typically around 80 MHz. The OPO has the same frequency since it is pumped by the Ti:sapphire laser. A fast oscilloscope of at least 200 MHz is therefore required. A fast photodiode in the range 600 to 1,100 nm is also required. The maximal temporal shift occurs when the Ti:sapphire and the OPO signals are shifted of 1/(2×80×106) = 6.2 nanoseconds. It corresponds to a maximum beam path shift of 1.9 m. .......
The most challenging part of the work is the temporal synchronization of the laser beams. It requires a fast photodiode combined with a fast oscilloscope, but only a rough overlapping in time can be performed at first. Then a further adjustment of few cm is required. Finally, micrometer moves by a linear translation stage allows performing the final fine adjustment of the delay line length in order to trigger the CARS signal. This signal is maintained in a narrow range of around 20 micrometers, as observed by tuning the .......
The authors want to thank Dr. Philippe Combette (IES, UM, Montpellier, France) for the loan of the fast oscilloscope and acknowledge financial supports from Montpellier RIO Imaging (MRI). HR acknowledges ANR grants France Bio Imaging (ANR-10-INSB-04-01) and France Life Imaging (ANR-11-INSB-0006) infrastructure networks for coherent Raman imaging developments. This work was mainly supported by an European Research Council grant (FP7-IDEAS-ERC 311610) and an INSERM - AVENIR grant to NT.....
|5 GHz InGaAs, 800-1700 nm
|Ti:Sapphire laser Chameleon Ultra Family II
|Optical parametric oscillator OPO Compact Family
|Axio Examiner microscope LSM 7 MP
|Objective W Plan-Apochromat 20x/1.0
|OPO power attenuator
|LSM BiG 1935-176
|400-480 nm ; 500-550 nm ; 465-610 nm
|Cutoff wavelength 760 nm
|λ/10, 480-20,000 nm , Quantity 4
|Single-axis translation stage with standard micrometer
|Mirror holder for Ø1" Optics
|TB4, XE25L900/M, T205-1.0, RM1S
|TR40/M, PH50/M, PH75/M, BA2/M
|Quantity 8 (lengths depending on the set-up)
|661-690 nm bandpass filter
|676/29 nm BrightLine® single-band bandpass filter
|TetraSpeck™ Fluorescent Microspheres Size Kit
|Laser viewing card
|IR laser viewing card
|Laser safety glass
|FluoroMyelin™ Red Fluorescent Myelin Stain
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