Biological Applications of Raman Spectroscope

Raman spectroscopy is widely used to detect the chemical composition of solid and liquid materials. It can use the spectral "fingerprint" information of substances to distinguish various material samples and detect cells with different physiological conditions and their biomolecules.

Raman spectroscopy has become a multifunctional tool for biomedical analysis. Single-cell Raman spectroscopy usually contains thousands of Raman bands, which can provide a wealth of cellular molecular information, such as nucleic acid, protein, lipid, etc., and can reflect the genotype, phenotype and physiological state of cells.

But there are two big obstacles to its development:

1. Low signal strength

2. Overlapped spectral bands

The traditional Raman spectrum is weak in intensity and has some fluorescence interference. With the development of science and technology, more new Raman techniques have been developed to address the above shortcomings and broaden the application range of Raman.

New developments

Surface enhanced Raman spectroscopy

Surface Enhanced Raman Scattering (SERS), the samples adsorbed on the Surface of glial metal particles such as silver, gold or copper, or the samples adsorbed on the rough Surface of these metal pieces are determined by ordinary Raman spectroscopy.

SERS is widely used in the medical field. On the cellular and molecular level, it is DNA, which provides a new method for protein detection. As a non-labeled technology, SERS can quickly monitor the substances with low concentration in biological matrix, making it an effective real-time detection tool for some drugs with narrow treatment window. SERS can not only detect the growth of bacteria on the wound surface, but also play a role of sterilization or bacteriostasis to a certain extent. SERS tag combined with laser Raman spectroscopy and microscope technology showed a unique potential in optical labeling and imaging.

Coherent Raman scattering

Coherent Raman Scattering (CRS) is an effect induced by a nonlinear optical process to produce coherent light, in which the specific vibration of the target molecule can be used as the contrast required for imaging. Therefore, a new optical microscopy method, namely coherent Raman Scattering microscopy, has been developed. Compared with spontaneous Raman scattering, coherent Raman scattering is at least 3 orders of magnitude stronger than spontaneous Raman scattering, and the imaging speed is 3~4 orders of magnitude faster.

Coherent Raman scattering can be divided into two kinds: coherent anti-Stokes Raman scattering (CRAS) and stimulated Raman scattering (SRS).

1. Coherent inverse Stokes Raman scattering

Because of the large number of C-H bonds in lipids, large scattering surface and relatively strong signal, CRAs are often used to detect lipid signals in the field of biomedical research to study cell activities. The detection of the characteristics of target molecules by CRAS can be used to image in vivo, in vitro and pathological tissue sections without labeling and assist in disease diagnosis, which also has a broad development prospect in clinical biopsy.

2. Stimulated Raman scattering

SRS imaging technology is characterized by:

(1) There will be no non-resonant background;

(2) The signal peak does not shift during imaging, and the component analysis can be carried out directly by using Raman spectral database;

(3) The signal intensity of SRS has a positive linear correlation with the molecular concentration, which makes the quantitative analysis more convenient.

SRS can be used for selective imaging of substances, studying cell lipid, protein and other signals, as well as the metabolism and distribution of specific substances in the cell. In order to improve the specificity of signal recognition, Raman tags have been widely used in SRS in recent years. The specific Raman signal characteristics of the Raman label can be used to change the original signal of the tested substance so as to achieve specific detection in the signal silent area (1800-2800cm-1) without interference of endogenous substances, and the cell metabolism will not be affected.

3. Resonance Raman spectroscopy

When the frequency of the excitation light is close to or equal to one of the electron absorption peaks of the molecule, the intensity of certain Raman bands will increase dramatically. The technique using this effect is called resonance Raman spectroscopy (RRS) technique.

RRS can enhance the Raman spectral signal by 4~6 orders of magnitude, improve the detection sensitivity and shorten the detection time. Compared with the conventional Raman spectrum, the fluorescence background of resonance Raman spectrum is more significant, the SNR is reduced, and the band is easily distorted. Resonant Raman spectroscopy selectively enhances the vibration of specific hair chromatic groups of biomolecules, so it can be used for non-destructive detection of pigment molecules, such as lycopene, carotenoids, chlorophyll, etc. Most of the proteins and other biomolecules are absorbed in the ultraviolet region, so UV resonance Raman spectroscopy has more advantages in biomedical research.

4. Spatial displacement Raman spectroscopy

Spatial Displaced Raman Spectroscopy (SORS) enables chemical analysis of materials within logarithmic millimeters and opaque packaging.

In addition to the inherent advantages of SORS Raman spectroscopy, the SORS technique has a number of unique advantages:

(1) Can effectively inhibit the fluorescence, improve the detection sensitivity;

(2) Within a certain range, the larger the offset distance, the larger the signal and the deeper the penetration depth of the Raman signal collected, which can realize the deep detection;

(3) In the testing process, the sample can be tested without damaging the packaging, so as to reduce the user's testing and production costs.

In recent years, Raman spectroscopy and its derivative development of other technologies because of its non-invasive, real-time, high repeatability, etc, in the biological medicine, especially in cancer diagnosis, treatment and prognosis of many aspects, such as a widely used with the continuous development of Raman technology, Raman spectra future will be more widely in scientific research in various fields of applications.