Coherent Raman scattering microscopy

[4] In 1999, CARS microscopy using a collinear geometry and high numerical aperture objective were developed in Xiaoliang Sunney Xie's lab at Harvard University.

[5] This advancement made the technique more compatible with modern laser scanning microscopes.

CRS is mainly used to image lipid, protein, and other bio-molecules in live or fixed cells or tissues without labeling or staining.

Spontaneous Raman scattering's signal intensity grows linearly with the average power of a continuous-wave pump laser.

In CRS,[7] two lasers are used to excite specific vibrational modes of molecules to be imaged.

CRS is a nonlinear optical process, where the signal level is normally a function of the product of the powers of the pump and Stokes lasers.

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

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.

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-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]

Those signals are normally called non-resonant (NR) four-wave-mixing (FWM) background in CARS microscopy.

The other is to measure the decrease of power in the pump laser, which is called stimulated Raman loss (SRL).

[22] The SRS signal depends on the pump and Stokes laser powers in the following way:

In the shot noise limited case, the signal-to-noise ratio (SNR) of SRS[16] is

One method is to detect the back-scattered light in front of the objective by a photodiode with a hole at the center.

In this case, the spectrum of the transmitted broadband laser can be spread by a grating and measured by an array of detectors.

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.

CRS is routinely used to image myelin in live or fixed tissues to study neurodegenerative diseases or other neural disorders.

[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.

Specially engineered molecules containing isotopes can be used as Raman tags to achieve super-multiplexing multi-color imaging with SRS.

It takes a long time to scan the whole sample, since each pixel requires seconds for data acquisition.

If more spectral information is needed, multi-color or hyperspectral CRS can be used and the scanning speed or data quality will be compromised accordingly.

On the instrument side, SRS requires modulation and demodulation (e.g. lock-in amplifier or resonant detector).

For multi-channel imaging, SRS requires multichannel demodulation while CARS only needs a PMT array or a CCD.

PMTs are most commonly used due to their large detection area and high speed.

Si photodiodes have much higher quantum efficiency than PMTs, which is one of the reasons that the SNR of SRS can be better than CARS in many cases.

Si photodiodes also suffer reduced sensitivity when the wavelength of laser is longer than 850 nm.