Raman microscope

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Photo of a confocal Raman imaging microscope
Confocal Raman imaging microscope
Photo of a Raman microscope, with a sample enclosure
Raman microscope

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.

Imaging modes[edit]

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, has very high spatial resolution [7]. For example, the lateral and depth resolutions were 250 nm and 1.7 µm, respectively, using a confocal Raman microspectrometer with the 632.8 nm line from a helium–neon laser with a pinhole of 100 µm diameter. Since the objective lenses of microscopes focus the laser beam to several micrometers in diameter, the resulting photon flux is much higher than achieved in conventional Raman setups. This has the added benefit of enhanced fluorescence quenching. However, the high photon flux can also cause sample degradation, and for this reason some setups require a thermally conducting substrate (which acts as a heat sink) in order to mitigate this process.

Raman imaging[edit]

Chemical Imaging of a pharmaceutical emulsion with confocal Raman microscopy.
Chemical image of a pharmaceutical emulsion acquired by confocal Raman microscopy (alpha300 microscope, WITec; blue: Active pharmaceutical ingredient, green: Oil, red: Silicon impurities).

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 [8], J-aggregated dyes inside carbon nanotubes and multiple other 2D materials such as MoS2[9] 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 vivotime- 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, in vivotime- and space-resolved Raman spectroscopy is suitable to examine proteins, cells and organs.

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 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 and ceramics and polymers [10], can use a broader range of excitation wavelengths.

Correlative Raman imaging[edit]

Correlative Raman-SEM imaging of a hematite.
Correlative Raman-SEM imaging of a hematite (taken with RISE microscope, WITec). The Raman image is overlaid over the SEM image.

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[11] [12], Raman-SNOM[13], and Raman-SEM[14].

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[15]. 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[16] [17]. 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.

Biological Applications[edit]

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 [18][19][20][21]. 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-B-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]. Using confocal Raman microspectroscopy essentially as a single-cell mass spectrometer is enabled by the fact that the vibrational frequency of any molecular bonds is a function of the masses of the bound atoms. Thus, incorporation of heavy isotopes will cause quantitative "red shifts" in diagnostic Raman peaks.

See also[edit]


  1. ^ a b Microscopical techniques in the use of the molecular optics laser examiner Raman microprobe, by M. E. Andersen, R. Z. Muggli, Analytical Chemistry, 1981, 53 (12), pp 1772–1777 [1]
  2. ^ Krishnan, K. S.; Raman, C. V. (1928). "A New Type of Secondary Radiation". Nature. 121 (3048): 501–502. doi:10.1038/121501c0. ISSN 1476-4687.
  3. ^ Matthäus, Christian; Krafft, Christoph; Dietzek, Benjamin; Brehm, Bernhard R.; Lorkowski, Stefan; Popp, Jürgen (2012-10-16). "Noninvasive Imaging of Intracellular Lipid Metabolism in Macrophages by Raman Microscopy in Combination with Stable Isotopic Labeling". Analytical Chemistry. 84 (20): 8549–8556. doi:10.1021/ac3012347. ISSN 0003-2700.
  4. ^ Baranska, Malgorzata; Chlopicki, Stefan; Fedorowicz, Andrzej; Kachamakova-Trojanowska, Neli; Kaczor, Agnieszka; Majzner, Katarzyna (2012-12-10). "3D confocal Raman imaging of endothelial cells and vascular wall: perspectives in analytical spectroscopy of biomedical research". Analyst. 138 (2): 603–610. doi:10.1039/C2AN36222H. ISSN 1364-5528.
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  7. ^ Toporski, Jan; Dieing, Thomas; Hollricher, Olaf, eds. (2018). "Confocal Raman Microscopy". Springer Series in Surface Sciences. doi:10.1007/978-3-319-75380-5. ISSN 0931-5195.
  8. ^ Shen, Zexiang; Yu, Ting; Wang, Yingying; Ni, Zhenhua (2008-10-01). "Raman spectroscopy and imaging of graphene". Nano Research. 1 (4): 273–291. doi:10.1007/s12274-008-8036-1. ISSN 1998-0000.
  9. ^ Li, Hai; Lu, Gang; Yin, Zongyou; He, Qiyuan; Li, Hong; Zhang, Qing; Zhang, Hua (2012-03-12). "Optical Identification of Single- and Few-Layer MoS2 Sheets". Small. 8 (5): 682–686. doi:10.1002/smll.201101958. ISSN 1613-6829.
  10. ^ Schmidt, U.; Hild, S.; Ibach, W.; Hollricher, O. (2005-12-01). "Characterization of Thin Polymer Films on the Nanometer Scale with Confocal Raman AFM". Macromolecular Symposia. 230 (1): 133–143. doi:10.1002/masy.200551152. ISSN 1521-3900.
  11. ^ "A novel approach to investigate vascular wall in 3D: Combined Raman spectroscopy and atomic force microscopy for aorta en face imaging". Vibrational Spectroscopy. 75: 39–44. 2014-11-01. doi:10.1016/j.vibspec.2014.09.004. ISSN 0924-2031.
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  13. ^ Stark, Robert W.; Hillenbrand, Rainer; Ziegler, Alexander; Bauer, Michael; Huber, Andreas J.; Gigler, Alexander M. (2009-12-07). "Nanoscale residual stress-field mapping around nanoindents in SiC by IR s-SNOM and confocal Raman microscopy". Optics Express. 17 (25): 22351–22357. doi:10.1364/OE.17.022351. ISSN 1094-4087.
  14. ^ "An overview of emerging hyphenated SEM-EDX and Raman spectroscopy systems: Applications in life, environmental and materials sciences". TrAC Trends in Analytical Chemistry. 77: 156–166. 2016-03-01. doi:10.1016/j.trac.2015.12.001. ISSN 0165-9936.
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  17. ^ Wille, G.; Lerouge, C.; Schmidt, U. (2018-06-01). "A multimodal microcharacterisation of trace-element zonation and crystallographic orientation in natural cassiterite by combining cathodoluminescence, EBSD, EPMA and contribution of confocal Raman-in-SEM imaging". Journal of Microscopy. 270 (3): 309–317. doi:10.1111/jmi.12684. ISSN 1365-2818.
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