The Raman microscope is a laser-based microscopic device used to perform Raman spectroscopy. [1] The term MOLE (molecular optics laser examiner) is used to refer to the Raman-based microprobe. [1] The technique used is named after C. V. Raman, who discovered the scattering properties in liquids. [2]
The Raman microscope begins with a standard optical microscope, and adds an excitation laser, laser rejection filters, a spectrometer or monochromator, and an optical sensitive detector such as a charge-coupled device (CCD), or photomultiplier tube, (PMT). Traditionally Raman microscopy was used to measure the Raman spectrum of a point on a sample, more recently the technique has been extended to implement Raman spectroscopy for direct chemical imaging over the whole field of view on a 3D sample.
In direct imaging, the whole field of view is examined for scattering over a small range of wavenumbers (Raman shifts). For instance, a wavenumber characteristic for cholesterol could be used to record the distribution of cholesterol within a cell culture. The other approach is hyperspectral imaging or chemical imaging, in which thousands of Raman spectra are acquired from all over the field of view. The data can then be used to generate images showing the location and amount of different components. Taking the cell culture example, a hyperspectral image could show the distribution of cholesterol, [3] as well as proteins, nucleic acids, and fatty acids. [4] [5] [6] Sophisticated signal- and image-processing techniques can be used to ignore the presence of water, culture media, buffers, and other interference.
Raman microscopy, and in particular confocal microscopy, can reach down to sub-micrometer lateral spatial resolution. [7] Because a Raman microscope is a diffraction-limited system, its spatial resolution depends on the wavelength of light and the numerical aperture of the focusing element. In confocal Raman microscopy, the diameter of the confocal aperture is an additional factor. As a rule of thumb, the lateral spatial resolution can reach approximately the laser wavelength when using air objective lenses, while oil or water immersion objectives can provide lateral resolutions of around half the laser wavelength. This means that when operated in the visible to near-infrared range, a Raman microscope can achieve lateral resolutions of approx. 1 µm down to 250 nm, while the depth resolution (if not limited by the optical penetration depth of the sample) can range from 1-6 µm with the smallest confocal pinhole aperture to tens of micrometers when operated without a confocal pinhole. [8] [9] [10] Since the objective lenses of microscopes focus the laser beam down to the micrometer range, the resulting photon flux is much higher than achieved in conventional Raman setups. This has the added effect of increased photobleaching of molecules emitting interfering fluorescence. However, the high photon flux can also cause sample degradation, and thus, for each type of sample, the laser wavelength and laser power have to be carefully selected.
Another tool that is becoming more popular is global Raman imaging. This technique is being used for the characterization of large scale devices, mapping of different compounds and dynamics study. It has already been used for the characterization of graphene layers, [11] J-aggregated dyes inside carbon nanotubes and multiple other 2D materials such as MoS2 [12] and WSe2. Since the excitation beam is dispersed over the whole field of view, those measurements can be done without damaging the sample. By using Raman microspectroscopy, in vivo time- and space-resolved Raman spectra of microscopic regions of samples can be measured. As a result, the fluorescence of water, media, and buffers can be removed. Consequently, it is suitable to examine proteins, cells and organelles.
Raman microscopy for biological and medical specimens generally uses near-infrared (NIR) lasers (785 nm diodes and 1064 nm Nd:YAG are especially common). This reduces the risk of damaging the specimen by applying higher energy wavelengths. However, the intensity of NIR Raman scattering is low (owing to the ω4 dependence of Raman scattering intensity), and most detectors require very long collection times. Recently, more sensitive detectors have become available, making the technique better suited to general use. Raman microscopy of inorganic specimens, such as rocks, ceramics and polymers, [13] can use a broader range of excitation wavelengths.
A related technique, tip-enhanced Raman spectroscopy, can produce high-resolution hyperspectral images of single molecules [14] and DNA. [15]
Confocal Raman microscopy can be combined with numerous other microscopy techniques. By using different methods and correlating the data, the user attains a more comprehensive understanding of the sample. Common examples of correlative microscopy techniques are Raman-AFM, [16] [13] Raman-SNOM, [17] and Raman-SEM. [18]
Correlative SEM-Raman imaging is the integration of a confocal Raman microscope into an SEM chamber which allows correlative imaging of several techniques, such as SE, BSE, EDX, EBSD, EBIC, CL, AFM. [19] The sample is placed in the vacuum chamber of the electron microscope. Both analysis methods are then performed automatically at the same sample location. The obtained SEM and Raman images can then be superimposed. [20] [21] Moreover, adding a focused ion beam (FIB) on the chamber allows removal of the material and therefore 3D imaging of the sample. Low-vacuum mode allows analysis on biological and non-conductive samples.
By using Raman microspectroscopy, in vivo time- and space-resolved Raman spectra of microscopic regions of samples can be measured. Sampling is non-destructive and water, media, and buffers typically do not interfere with the analysis. Consequently, in vivo time- and space-resolved Raman spectroscopy is suitable to examine proteins, cells and organs. In the field of microbiology, confocal Raman microspectroscopy has been used to map intracellular distributions of macromolecules, such as proteins, polysaccharides, and nucleic acids and polymeric inclusions, such as poly-β-hydroxybutyric acid and polyphosphates in bacteria and sterols in microalgae. Combining stable isotopic probing (SIP) experiments with confocal Raman microspectroscopy has permitted determination of assimilation rates of 13C and 15N-substrates as well as D2O by individual bacterial cells. [22]
An electron microscope is a microscope that uses a beam of electrons as a source of illumination. They use electron optics that are analogous to the glass lenses of an optical light microscope. As the wavelength of an electron can be up to 100,000 times shorter than that of visible light, electron microscopes have a higher resolution of about 0.1 nm, which compares to about 200 nm for light microscopes. Electron microscope may refer to:
Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye. There are three well-known branches of microscopy: optical, electron, and scanning probe microscopy, along with the emerging field of X-ray microscopy.
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.
The optical microscope, also referred to as a light microscope, is a type of microscope that commonly uses visible light and a system of lenses to generate magnified images of small objects. Optical microscopes are the oldest design of microscope and were possibly invented in their present compound form in the 17th century. Basic optical microscopes can be very simple, although many complex designs aim to improve resolution and sample contrast.
A fluorescence microscope is an optical microscope that uses fluorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances. "Fluorescence microscope" refers to any microscope that uses fluorescence to generate an image, whether it is a simple set up like an epifluorescence microscope or a more complicated design such as a confocal microscope, which uses optical sectioning to get better resolution of the fluorescence image.
Confocal microscopy, most frequently confocal laser scanning microscopy (CLSM) or laser scanning confocal microscopy (LSCM), is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a spatial pinhole to block out-of-focus light in image formation. Capturing multiple two-dimensional images at different depths in a sample enables the reconstruction of three-dimensional structures within an object. This technique is used extensively in the scientific and industrial communities and typical applications are in life sciences, semiconductor inspection and materials science.
Resonance Raman spectroscopy is a variant of Raman spectroscopy in which the incident photon energy is close in energy to an electronic transition of a compound or material under examination. This similarity in energy (resonance) leads to greatly increased intensity of the Raman scattering of certain vibrational modes, compared to ordinary Raman spectroscopy.
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.
Near-field scanning optical microscopy (NSOM) or scanning near-field optical microscopy (SNOM) is a microscopy technique for nanostructure investigation that breaks the far field resolution limit by exploiting the properties of evanescent waves. In SNOM, the excitation laser light is focused through an aperture with a diameter smaller than the excitation wavelength, resulting in an evanescent field on the far side of the aperture. When the sample is scanned at a small distance below the aperture, the optical resolution of transmitted or reflected light is limited only by the diameter of the aperture. In particular, lateral resolution of 6 nm and vertical resolution of 2–5 nm have been demonstrated.
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.
Photothermal microspectroscopy (PTMS), alternatively known as photothermal temperature fluctuation (PTTF), is derived from two parent instrumental techniques: infrared spectroscopy and atomic force microscopy (AFM). In one particular type of AFM, known as scanning thermal microscopy (SThM), the imaging probe is a sub-miniature temperature sensor, which may be a thermocouple or a resistance thermometer. This same type of detector is employed in a PTMS instrument, enabling it to provide AFM/SThM images: However, the chief additional use of PTMS is to yield infrared spectra from sample regions below a micrometer, as outlined below.
Super-resolution microscopy is a series of techniques in optical microscopy that allow such images to have resolutions higher than those imposed by the diffraction limit, which is due to the diffraction of light. Super-resolution imaging techniques rely on the near-field or on the far-field. Among techniques that rely on the latter are those that improve the resolution only modestly beyond the diffraction-limit, such as confocal microscopy with closed pinhole or aided by computational methods such as deconvolution or detector-based pixel reassignment, the 4Pi microscope, and structured-illumination microscopy technologies such as SIM and SMI.
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.
Light sheet fluorescence microscopy (LSFM) is a fluorescence microscopy technique with an intermediate-to-high optical resolution, but good optical sectioning capabilities and high speed. In contrast to epifluorescence microscopy only a thin slice of the sample is illuminated perpendicularly to the direction of observation. For illumination, a laser light-sheet is used, i.e. a laser beam which is focused only in one direction. A second method uses a circular beam scanned in one direction to create the lightsheet. As only the actually observed section is illuminated, this method reduces the photodamage and stress induced on a living sample. Also the good optical sectioning capability reduces the background signal and thus creates images with higher contrast, comparable to confocal microscopy. Because light sheet fluorescence microscopy scans samples by using a plane of light instead of a point, it can acquire images at speeds 100 to 1,000 times faster than those offered by point-scanning methods.
AFM-IR or infrared nanospectroscopy is one of a family of techniques that are derived from a combination of two parent instrumental techniques. AFM-IR combines the chemical analysis power of infrared spectroscopy and the high-spatial resolution of scanning probe microscopy (SPM). The term was first used to denote a method that combined a tuneable free electron laser with an atomic force microscope equipped with a sharp probe that measured the local absorption of infrared light by a sample with nanoscale spatial resolution.
Tip-enhanced Raman spectroscopy (TERS) is a variant of surface-enhanced Raman spectroscopy (SERS) that combines scanning probe microscopy with Raman spectroscopy. High spatial resolution chemical imaging is possible via TERS, with routine demonstrations of nanometer spatial resolution under ambient laboratory conditions, or better at ultralow temperatures and high pressure.
Micro-spatially offset Raman spectroscopy (micro-SORS) is an analytical technique developed in 2014 that combines SORS with microscopy. The technique derives its sublayer‐resolving properties from its parent technique SORS. The main difference between SORS and micro-SORS is the spatial resolution: while SORS is suited to the analysis of millimetric layers, micro-SORS is able to resolve thin, micrometric-scale layers. Similarly to SORS technique, micro-SORS is able to preferentially collect the Raman photons generated under the surface in turbid media. In this way, it is possible to reconstruct the chemical makeup of micrometric multi-layered turbid system in a non destructive way. Micro-SORS is particularly useful when dealing with precious or unique objects as for Cultural Heritage field and Forensic Science or in biomedical applications, where a non-destructive molecular characterization constitute a great advantage.
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. In 1982 the first CARS microscope was demonstrated. In 1999, CARS microscopy using a collinear geometry and high numerical aperture objective were developed in Xiaoliang Sunney Xie's lab at Harvard University. This advancement made the technique more compatible with modern laser scanning microscopes. 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. CRS can also be used to image samples labeled with Raman tags, 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 and environmental science.
Ultrafast scanning electron microscopy (UFSEM) combines two microscopic modalities, Pump-probe microscopy and Scanning electron microscope, to gather temporal and spatial resolution phenomena. The technique uses ultrashort laser pulses for pump excitation of the material and the sample response will be detected by an Everhart-Thornley detector. Acquiring data depends mainly on formation of images by raster scan mode after pumping with short laser pulse at different delay times. The characterization of the output image will be done through the temporal resolution aspect. Thus, the idea is to exploit the shorter DeBroglie wavelength in respect to the photons which has great impact to increase the resolution about 1 nm. That technique is an up-to-date approach to study the dynamic of charge on material surfaces.
Raman spectroelectrochemistry (Raman-SEC) is a technique that studies the inelastic scattering or Raman scattering of monochromatic light related to chemical compounds involved in an electrode process. This technique provides information about vibrational energy transitions of molecules, using a monochromatic light source, usually from a laser that belongs to the UV, Vis or NIR region. Raman spectroelectrochemistry provides specific information about structural changes, composition and orientation of the molecules on the electrode surface involved in an electrochemical reaction, being the Raman spectra registered a real fingerprint of the compounds.
| Illumination and contrast methods |  | |
|---|---|---|
| Fluorescence methods | ||
| Sub-diffraction limit techniques | ||
