Coherent Raman scattering (CRS) microscopy is a multi-photon microscopy technique based on Raman-active vibrational modes of molecules. The two major techniques in CRS microscopy are stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS). SRS and CARS were theoretically predicted and experimentally realized in the 1960s. [1] [2] [3] In 1982 the first CARS microscope was demonstrated. [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. [6] Since then, CRS's popularity in biomedical research started to grow. CRS is mainly used to image lipid, protein, and other bio-molecules in live or fixed cells or tissues without labeling or staining. [7] CRS can also be used to image samples labeled with Raman tags, [8] [9] [10] which can avoid interference from other molecules and normally allows for stronger CRS signals than would normally be obtained for common biomolecules. CRS also finds application in other fields, such as material science [11] and environmental science. [12]
Coherent Raman scattering is based on Raman scattering (or spontaneous Raman scattering). In spontaneous Raman, only one monochromatic excitation laser is used. 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. The laser with a higher photon energy is normally called the pump laser and the laser with a lower photon energy is called Stokes laser. In order to produce a signal their photon energy differences must match the energy of a vibrational mode:
,
where the .
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. Therefore, most CRS microscopy experiments are performed with pulsed lasers, where higher peak power improved the signal levels of CRS significantly. [13]
In CARS, anti-Stokes photons (higher in energy, shorter wavelength than the pump) are detected as signals.
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 , the number of molecules in the focus of the lasers and the third order Raman susceptibility of the molecule: [16]
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]
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:
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
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]
Confocal Raman microscopy normally uses continuous wave lasers to provide a spontaneous Raman spectrum over a broad wavenumber range for each point in an image. It takes a long time to scan the whole sample, since each pixel requires seconds for data acquisition. The whole imaging process is long and therefore, it is more suitable for samples that do not move. CRS on the other hand measures signals at single wavenumber but allows for fast scanning. 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. [43]
In CRS microscopy, we can regard SRS and CARS as two aspects of the same process. CARS signal is always mixed with non-resonant four-wave mixing background and has a quadratic dependence on concentration of chemicals being imaged. SRS has much smaller background and depends linearly on the concentration of the chemical being imaged. Therefore, SRS is more suitable for quantitative imaging than CARS. 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. Therefore, the instrumentation required is more complicated for SRS than CARS. [16]
On the sensitivity side, SRS and CARS normally provide similar sensitivities. [44] Their differences are mainly due to detection methods. In CARS microscopy, PMT, APD or CCDs are used as detectors to detect photons generated in the CARS process. PMTs are most commonly used due to their large detection area and high speed. In SRS microscopy, photodiodes are normally used to measure laser beam intensities. Because of such differences, the applications of CARS and SRS are also different. [16]
PMTs normally have relatively low quantum efficiency compared with photodiodes. This will negatively impact the SNR of CARS microscopy. PMTs also have reduced sensitivity for lasers with wavelengths longer than 650 nm. Therefore, with the commonly used laser system for CRS (Ti-sapphire laser), CARS is mainly used to image at high wavenumber region (2800–3400 cm−1). The SNR of CARS microscopy is normally poor for fingerprint imaging (400–1800 cm−1). [16]
SRS microscopy mainly uses silicon photodiode as detectors. 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. However, the sensitivity is still relatively high and allows for imaging in fingerprint region (400–1800 cm−1). [16]
Spectroscopy is the field of study that measures and interprets electromagnetic spectrum. In narrower contexts, spectroscopy is the precise study of color as generalized from visible light to all bands of the electromagnetic spectrum.
An optical amplifier is a device that amplifies an optical signal directly, without the need to first convert it to an electrical signal. An optical amplifier may be thought of as a laser without an optical cavity, or one in which feedback from the cavity is suppressed. Optical amplifiers are important in optical communication and laser physics. They are used as optical repeaters in the long distance fiber-optic cables which carry much of the world's telecommunication links.
Raman spectroscopy is a spectroscopic technique typically used to determine vibrational modes of molecules, although rotational and other low-frequency modes of systems may also be observed. Raman spectroscopy is commonly used in chemistry to provide a structural fingerprint by which molecules can be identified.
In chemistry and physics, Raman scattering or the Raman effect is the inelastic scattering of photons by matter, meaning that there is both an exchange of energy and a change in the light's direction. Typically this effect involves vibrational energy being gained by a molecule as incident photons from a visible laser are shifted to lower energy. This is called normal Stokes-Raman scattering.
A Raman laser is a specific type of laser in which the fundamental light-amplification mechanism is stimulated Raman scattering. In contrast, most "conventional" lasers rely on stimulated electronic transitions to amplify light.
Medical optical imaging is the use of light as an investigational imaging technique for medical applications, pioneered by American Physical Chemist Britton Chance. Examples include optical microscopy, spectroscopy, endoscopy, scanning laser ophthalmoscopy, laser Doppler imaging, and optical coherence tomography. Because light is an electromagnetic wave, similar phenomena occur in X-rays, microwaves, and radio waves.
Two-photon excitation microscopy is a fluorescence imaging technique that is particularly well-suited to image scattering living tissue of up to about one millimeter in thickness. Unlike traditional fluorescence microscopy, where the excitation wavelength is shorter than the emission wavelength, two-photon excitation requires simultaneous excitation by two photons with longer wavelength than the emitted light. The laser is focused onto a specific location in the tissue and scanned across the sample to sequentially produce the image. Due to the non-linearity of two-photon excitation, mainly fluorophores in the micrometer-sized focus of the laser beam are excited, which results in the spatial resolution of the image. This contrasts with confocal microscopy, where the spatial resolution is produced by the interaction of excitation focus and the confined detection with a pinhole.
Coherent anti-Stokes Raman spectroscopy, also called Coherent anti-Stokes Raman scattering spectroscopy (CARS), is a form of spectroscopy used primarily in chemistry, physics and related fields. It is sensitive to the same vibrational signatures of molecules as seen in Raman spectroscopy, typically the nuclear vibrations of chemical bonds. Unlike Raman spectroscopy, CARS employs multiple photons to address the molecular vibrations, and produces a coherent signal. As a result, CARS is orders of magnitude stronger than spontaneous Raman emission. CARS is a third-order nonlinear optical process involving three laser beams: a pump beam of frequency ωp, a Stokes beam of frequency ωS and a probe beam at frequency ωpr. These beams interact with the sample and generate a coherent optical signal at the anti-Stokes frequency (ωpr+ωp-ωS). The latter is resonantly enhanced when the frequency difference between the pump and the Stokes beams (ωp-ωS) coincides with the frequency of a Raman resonance, which is the basis of the technique's intrinsic vibrational contrast mechanism.
Raman amplification is based on the stimulated Raman scattering (SRS) phenomenon, when a lower frequency 'signal' photon induces the inelastic scattering of a higher-frequency 'pump' photon in an optical medium in the nonlinear regime. As a result of this, another 'signal' photon is produced, with the surplus energy resonantly passed to the vibrational states of the medium. This process, as with other stimulated emission processes, allows all-optical amplification. Optical fiber is today most used as the nonlinear medium for SRS for telecom purposes; in this case it is characterized by a resonance frequency downshift of ~11 THz. The SRS amplification process can be readily cascaded, thus accessing essentially any wavelength in the fiber low-loss guiding windows. In addition to applications in nonlinear and ultrafast optics, Raman amplification is used in optical telecommunications, allowing all-band wavelength coverage and in-line distributed signal amplification.
Chemical imaging is the analytical capability to create a visual image of components distribution from simultaneous measurement of spectra and spatial, time information. Hyperspectral imaging measures contiguous spectral bands, as opposed to multispectral imaging which measures spaced spectral bands.
In optics, a supercontinuum is formed when a collection of nonlinear processes act together upon a pump beam in order to cause severe spectral broadening of the original pump beam, for example using a microstructured optical fiber. The result is a smooth spectral continuum. There is no consensus on how much broadening constitutes a supercontinuum; however researchers have published work claiming as little as 60 nm of broadening as a supercontinuum. There is also no agreement on the spectral flatness required to define the bandwidth of the source, with authors using anything from 5 dB to 40 dB or more. In addition the term supercontinuum itself did not gain widespread acceptance until this century, with many authors using alternative phrases to describe their continua during the 1970s, 1980s and 1990s.
Harmonic generation is a nonlinear optical process in which photons with the same frequency interact with a nonlinear material, are "combined", and generate a new photon with times the energy of the initial photons.
Xiaoliang Sunney Xie is a Chinese biophysicist well known for his contributions to the fields of single-molecule biophysical chemistry, coherent Raman Imaging and single-molecule genomics. In 2023, Xie renounced his U.S. citizenship in order to reclaim his Chinese citizenship.
The Raman microscope is a laser-based microscopic device used to perform Raman spectroscopy. The term MOLE is used to refer to the Raman-based microprobe. The technique used is named after C. V. Raman, who discovered the scattering properties in liquids.
The technique of vibrational analysis with scanning probe microscopy allows probing vibrational properties of materials at the submicrometer scale, and even of individual molecules. This is accomplished by integrating scanning probe microscopy (SPM) and vibrational spectroscopy. This combination allows for much higher spatial resolution than can be achieved with conventional Raman/FTIR instrumentation. The technique is also nondestructive, requires non-extensive sample preparation, and provides more contrast such as intensity contrast, polarization contrast and wavelength contrast, as well as providing specific chemical information and topography images simultaneously.
Rotating-polarization coherent anti-Stokes Raman spectroscopy, (RP-CARS) is a particular implementation of the coherent anti-Stokes Raman spectroscopy (CARS). RP-CARS takes advantage of polarization-dependent selection rules in order to gain information about molecule orientation anisotropy and direction within the optical point spread function.
Optical rogue waves are rare pulses of light analogous to rogue or freak ocean waves. The term optical rogue waves was coined to describe rare pulses of broadband light arising during the process of supercontinuum generation—a noise-sensitive nonlinear process in which extremely broadband radiation is generated from a narrowband input waveform—in nonlinear optical fiber. In this context, optical rogue waves are characterized by an anomalous surplus in energy at particular wavelengths or an unexpected peak power. These anomalous events have been shown to follow heavy-tailed statistics, also known as L-shaped statistics, fat-tailed statistics, or extreme-value statistics. These probability distributions are characterized by long tails: large outliers occur rarely, yet much more frequently than expected from Gaussian statistics and intuition. Such distributions also describe the probabilities of freak ocean waves and various phenomena in both the man-made and natural worlds. Despite their infrequency, rare events wield significant influence in many systems. Aside from the statistical similarities, light waves traveling in optical fibers are known to obey the similar mathematics as water waves traveling in the open ocean, supporting the analogy between oceanic rogue waves and their optical counterparts. More generally, research has exposed a number of different analogies between extreme events in optics and hydrodynamic systems. A key practical difference is that most optical experiments can be done with a table-top apparatus, offer a high degree of experimental control, and allow data to be acquired extremely rapidly. Consequently, optical rogue waves are attractive for experimental and theoretical research and have become a highly studied phenomenon. The particulars of the analogy between extreme waves in optics and hydrodynamics may vary depending on the context, but the existence of rare events and extreme statistics in wave-related phenomena are common ground.
Stimulated Raman spectroscopy, also referred to as stimulated Raman scattering (SRS), is a form of spectroscopy employed in physics, chemistry, biology, and other fields. The basic mechanism resembles that of spontaneous Raman spectroscopy: a pump photon, of the angular frequency , which is scattered by a molecule has some small probability of inducing some vibrational transition, as opposed to inducing a simple Rayleigh transition. This makes the molecule emit a photon at a shifted frequency. However, SRS, as opposed to spontaneous Raman spectroscopy, is a third-order non-linear phenomenon involving a second photon—the Stokes photon of angular frequency —which stimulates a specific transition. When the difference in frequency between both photons resembles that of a specific vibrational transition the occurrence of this transition is resonantly enhanced. In SRS, the signal is equivalent to changes in the intensity of the pump and Stokes beams. The signals are typically rather low, of the order of a part in 10^5, thus calling for modulation-transfer techniques: one beam is modulated in amplitude, and the signal is detected on the other beam via a lock-in amplifier. Employing a pump laser beam of a constant frequency and a Stokes laser beam of a scanned frequency allows for unraveling the molecule's spectral fingerprint. This spectral fingerprint differs from those obtained by other spectroscopy methods, such as Rayleigh scattering, as the Raman transitions confer different exclusion rules than those that apply to Rayleigh transitions.
Pump–probe microscopy is a non-linear optical imaging modality used in femtochemistry to study chemical reactions. It generates high-contrast images from endogenous non-fluorescent targets. It has numerous applications, including materials science, medicine, and art restoration.
Ji-Xin Cheng is an academic, inventor, and entrepreneur. He holds the Moustakas Chair Professorship in Optoelectronics and Photonics at Boston University. His inventions span optical imaging, cancer diagnosis, neuromodulation, and phototherapy of infectious diseases. He holds positions of co-founder of Vibronic and of Pulsethera. He is also the scientific advisor of Photothermal Spectroscopy and Axorus.
{{cite book}}
: CS1 maint: location missing publisher (link) CS1 maint: others (link){{cite book}}
: CS1 maint: multiple names: authors list (link) CS1 maint: numeric names: authors list (link)