Coherent Raman scattering microscopy

Forward and epi-detected CARS

In CARS, anti-Stokes photons (higher in energy, shorter wavelength than the pump) are detected as signals.

${\displaystyle E_{CARS}=E_{pump}+\Omega =2\times E_{pump}-E_{Stokes}}$

In CARS microscopy, there are normally two ways to detect the newly generated photons. One is called forward-detected CARS, the other called epi-detected CARS.[14][15] In forward-detected CARS, the generated CARS photons together with pump and Stokes lasers go through the sample. The pump and Stokes lasers are completely blocked by a high optical density (OD) notch filter. The CARS photons are then detected by a photomultiplier tube (PMT) or a CCD camera. In epi-detected CARS, back-scattered CARS photons are redirected by a dichroic mirror or polarizing beam splitter. After high OD filters are used to block back-scattered pump and Stokes lasers, the newly generated photons are detected by a PMT. The signal intensity of CARS has the following relationship with the pump and Stokes laser intensities ${\displaystyle I_{pump},I_{Stokes}}$, the number of molecules ${\displaystyle N}$ in the focus of the lasers and the third order Raman susceptibility ${\displaystyle \chi _{Raman}^{(3)}}$ of the molecule:[16]

${\displaystyle I_{CARS}\propto (N\chi _{Raman}^{(3)})^{2}I_{pump}^{2}I_{Stokes}}$

The signal-to-noise ratio (SNR), which is a more important characteristic in imaging experiments depends on the square root of the number of CARS photons generated, which is given below:[16]

${\displaystyle SNR_{CARS}\propto N\chi _{Raman}^{(3)}I_{pump}{\sqrt {I_{Stokes}}}}$

There are other non-linear optical processes that also generate photons at the anti-Stokes wavelength. Those signals are normally called non-resonant (NR) four-wave-mixing (FWM) background in CARS microscopy. These background can interfere with the CARS signal either constructively or destructively.[17] However, the problem can be partially circumvented by subtracting the on- and off-resonance images[18][19] or using mathematical methods to retrieve the background free images.[20]

In SRS, the intensity of the energy transfer from the pump wavelength to the Stokes laser wavelength is measured as a signal. There are two ways to measure SRS signals, one is to measure the increase of power in Stokes laser, which is called stimulated Raman gain (SRG). The other is to measure the decrease of power in the pump laser, which is called stimulated Raman loss (SRL). Since the change of power is on the order of 10⁻³ to 10⁻⁶ compared with the original power of pump and Stokes lasers, a modulation transfer scheme[21] is normally employed to extract the SRS signals.[22] The SRS signal depends on the pump and Stokes laser powers in the following way:

${\displaystyle I_{SRS}\propto N\chi _{Raman}^{(3)}I_{pump}I_{Stokes}}$

Shot noise limited detection can be achieved if electronic noise from detectors are reduced well below optical noise and the lasers are shot noise limited at the detection frequency (modulation frequency). In the shot noise limited case, the signal-to-noise ratio (SNR) of SRS[16] is

${\displaystyle SNR_{SRL}\propto N\chi _{Raman}^{(3)}{\sqrt {I_{pump}}}I_{Stokes}}$

${\displaystyle SNR_{SRG}\propto N\chi _{Raman}^{(3)}I_{pump}{\sqrt {I_{Stokes}}}}$

The signal of SRS is free from the non-resonant background which plagues CARS microscopy, although a much smaller non-resonant background from other optical process (e.g. cross-phase modulation, multi-color multi-photon absorption) may exist.

SRS can be detected in either the forward direction and epi directions. In forward-detected SRS, the modulated laser is blocked by a high OD notch filter and the other laser is measured by a photodiode. Modulation transferred from the modulated laser to the originally unmodulated laser is normally extracted by a lock-in amplifier from the output of photodiode. In epi-detected SRS, there are normally two methods to detect the SRS signal. One method is to detect the back-scattered light in front of the objective by a photodiode with a hole at the center. The other method is similar to the epi-detected CARS microscopy, where the back-scattered light goes through the objective and is deflected to the side of the light path, normally with the combination of a polarizing beam splitter and a quarter wave-plate. The Stokes (or pump) laser is then detected after filtering out the pump (or Stokes laser).

One pair of laser wavelengths only gives access to a single vibrational frequency. Imaging samples at different wavenumbers can provide a more specific and quantitative chemical mapping of the sample.[23][24][25][26][27][28] This can be achieved by imaging at different wavenumbers one after another. This operation always involves some type of tuning: tuning of one of the lasers' wavelengths, tuning of a spectral filtering device, or tuning of the time delay between the pump and Stokes lasers in the case of spectral-focusing CRS. Another way of performing multi-color CRS is to use one picosecond laser with a narrow spectral bandwidth (<1 nm) as pump or Stokes and the other laser with broad spectral bandwidth. In this case, the spectrum of the transmitted broadband laser can be spread by a grating and measured by an array of detectors.

CRS normally use lasers with narrow bandwidth lasers, whose bandwidth < 1 nm, to maintain good spectral resolution ~ 15 cm⁻¹. Lasers with sub 1 nm bandwidth are picosecond lasers. In spectral-focusing CRS, femtosecond pump and Stokes lasers are equally linearly chirped into picosecond lasers.[29][30][31] The effective bandwidth become smaller and therefore, high spectral resolution can be achieved this way with femtosecond lasers which normally have a broad bandwidth. The wavenumber tuning of spectral-focusing CRS can be achieved both by changing the center wavelength of lasers and by changing the delay between pump and Stokes lasers.

One of the major applications for CRS is label-free histology, which is also called coherent Raman histology, or sometimes stimulated Raman histology.[32][33][34][35] In CRH, CRS images are obtained at lipid and protein images and after some image processing, an image similar to H&E staining can be obtained. Different from H&E staining, CRH can be done on live and fresh tissue and doesn't need fixation or staining.

The metabolism of small molecules like glucose,[36] cholesterol,[37] and drugs[38] are studied with CRS in live cells. CRS provide a way to measure molecular distribution and quantities with relatively high throughput.

Myelin is rich in lipid. CRS is routinely used to image myelin in live or fixed tissues to study neurodegenerative diseases or other neural disorders.[39][40][41]

The functions of drugs can be studied by CRS too. For example, an anti-leukemia drug imatinib are studied with SRS in leukemia cell lines.[38] The study revealed the possible mechanism of its metabolism in cells and provided insight about ways to improve drug effectiveness.

Even though CRS allows label-free imaging, Raman tags can also be used to boost signal for specific targets.[42][9][8] For example, deuterated molecules are used to shift Raman signal to a band where the interference from other molecules is absent. Specially engineered molecules containing isotopes can be used as Raman tags to achieve super-multiplexing multi-color imaging with SRS.[10]