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MICROSCOPY & IMAGING


with the corresponding confocal Raman image. Particles are identified according to their size and shape and further characterised by their molecular properties through confocal Raman imaging. Te chemical analysis revealed anatase and boron nitride particles in an oil matrix (Raman spectra shown in Fig. 1B). Further evaluation of the results determines the quantitative prevalence of the molecular sample components in the particles (Fig. 1C) and also the distribution of chemical compounds correlated to particle size (Fig. 1D). In extended analyses the chemical characteristics of particles could also be linked to parameters such as area, perimeter, bounding box, Feret diameter, aspect ratio, equivalent diameter and many others.


RAMAN AND FLUORESCENCE Fluorescence microscopy has been a widespread imaging method for the analysis of biological cells and organisms for decades. Samples are stained with fluorescent dyes or organisms are genetically engineered to express fluorescent proteins. Te fluorescence signal is usually much stronger than the Raman signal. Nevertheless, correlative Raman fluorescence measurements are possible with an appropriate system. Te investigation described below was carried out using a WITec alpha300 Ri inverted confocal Raman imaging microscope to examine the cells in their aqueous cell culture medium in the Petri dish. Fig. 2 shows a Raman fluorescence image of a live cell culture of eukaryotic cells. Te cell nuclei were stained with the fluorescent dye DAPI. An excitation


Fig. 1. Particles in a cosmetic peeling cream sample. A: Optical bright field image overlaid with the confocal Raman image. B: Corresponding Raman spectra of the molecular components in the sample. C: Pie chart of the quantitative compound distribution in the sample. D: Graphical representation of the correlation between chemical characteristics and particle size


wavelength of 532nm was used for the Raman measurement. An image with 50 x 40 µm² and 150 x 120 pixels was acquired. A Raman spectrum was recorded at each pixel. In the correlative Raman fluorescence image, the nuclei are shown in blue (recorded with fluorescence


microscopy), the nucleoli in green and the endoplasmic reticula in red (recorded with Raman microscopy). Te corresponding Raman spectra are shown in the same colours.


SUMMARY Raman microscopy is a versatile and powerful tool for identifying the chemical components present in a sample. Te speed and precision it offers, along with its ease of use and the minimal sample preparation it requires, makes it appealing to researchers in many fields. A Raman microscope of modular design that permits combinations with specialised software tools and other techniques extends its analytical range and utility.


Fig. 2. A: Correlative Raman fluorescence image of primate cells in a cell culture. Blue: Nuclei imaged with fluorescence microscopy; Red: Endoplasmic reticula, Green: Nucleoli imaged with Raman microscopy. B: Corresponding Raman spectra


66 www.scientistlive.com


Damon Strom is with WITec. www.witec.de


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