Confocal Raman imaging is well suited to investigating biological specimens. The inverted beam path of the Raman microscope alpha300 Ri is especially useful for imaging samples in liquids such as cells and tissues.
Optical microscopy using fluorescence labeling cannot employ more than a handful of labels in one experiment, restricting the number of molecules that can be analyzed. This limitation does not apply to the label-free Raman imaging as the Raman effect is caused by the inelastic scattering of monochromatic light by the sample's molecules. Each type of molecule in a sample can be identified unambiguously by its individual Raman spectrum. By combining Raman spectroscopy with confocal microscopy the spatial distribution of the compounds within a sample can be visualized (1). It has been successfully applied for the investigation of organic materials (2–6).
The confocal microscope setup enables the generation of 3D Raman images by stacking 2D images of a series of focal planes. Here, a pressed piece of banana pulp mixed with water was investigated with an alpha300 Ri inverted confocal Raman microscope (WITec). The scan range was 300 × 200 × 90 µm3 with 450 × 300 × 45 pixels. At each pixel a complete Raman spectrum was acquired. The integration time was 34 ms/spectrum. The 3D reconstruction of the Raman image stack (Figure 1) shows starch grains (green) and cell wall components (red).
Figure 1: Raman image of banana pulp with starch grain marked in green, cell wall material in red.
Though strong fluorescence is the "natural enemy" of the weaker Raman signal, correlative fluorescence–Raman imaging using one microscope is possible by choosing a suitable laser wavelength, to avoid signal overlap. Kidney cells (Vero ATCC CCL-81) were grown in a petri dish. Nuclei were stained with DAPI. Cells were imaged in aqueous solution under the alpha300 Ri microscope with a laser wavelength of 532 nm. An image of 50 × 40 µm2 with 150 × 120 pixels was generated. The acquisition time was 0.2 s/spectrum. The resulting correlative Raman–fluorescence image shows the nuclei (blue) imaged by fluorescence microscopy and the nucleoli (green) and endoplasmic reticula (red) imaged by confocal Raman microscopy based on their Raman spectra (Figure 2). The fluorescent dye does not interfere with the Raman measurement.
Figure 2: Correlative Raman-fluorescence image showing DAPI-stained nuclei (blue). Nucleoli (green) and endoplasmic reticula (red) were identified by their Raman spectra.
(1) J. Toporski, T. Dieing and O. Hollricher (eds.), Confocal Raman Microscopy, 66, Springer International Publishing (2018).
(2) S. Richter, J. Müssig and N. Gierlinger, Planta 233, 763–772 (2011).
(3) B. Prats-Mateu et al., Biotechnology Journal 12, 1600037 (2017).
(4) P. Heraud, et al., Scientific reports 7, 8949–8954 (2017).
(5) Kallepitis et al., Nat. Commun. 8, 14843–18851, (2017).
(6) H.K. Yosef et al., Anal. Chem.89, 6893-6899 (2017).
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