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Confocal Raman Microscopy

Since the development of light microscopy in the early 17th century each technical improvement resulted in major scientific breakthroughs. In biophysical studies which depend widely on microscopy techniques, it is therefore important to use a variety of technically different microscopes. Whereas most of our optical microscopes image the fluorescence signal of synthetic or genetic probes, the Raman microscope makes use of the characteristic vibrational spectra of molecules to image the sample. The Raman microscope hence combines optical microscopy with Raman spectroscopy.

In Raman microscopy (Figure 1), characteristic molecular vibrations are excited by a laser beam which lead to a red shifted response by the sample (Stokes signal). The response is measured by a spectrometer and the characteristic red shift is used to identify the molecule. The opposite process where energy is transferred from the molecule to the laser beam (anti-Stokes signal) is generally much lower in intensity and is therefore not used in Raman microscopy.

Fig. 1Figure 1: Excitation of a molecule by a laser beam of frequency ?i and the characteristic Stokes (?St)- and anti-Stokes (?aSt) transitions.

In Raman microscopy, the light-optical image and the local Raman spectra are recorded simultaneously. The combination of optical and spectral signal yields information on the spatial and time distribution of molecules and their conformation. Since Raman microscopy is chemically specific this technique enables to measure also the spatial and time distribution of biomolecules.

At ICS-7 we use a Raman microscope based on the WiTec alpha 300R which is capable of confocal, 3D imaging (Figures 2 and 3). This instrument combines high spatial resolution in three dimensions with high time resolution, i.e. many Raman spectra per pixel and time and low fluorescence background.
Our instrument is a special design which includes an additional atomic force microscope with its cantilever located in the optical axis of the microscope and combines an upright as well as an inverse Raman microscope.


Figure 2Figure 2: Sketch of the confocal Raman microscope (based on the WiTec alpha 300R ) as used at the ICS-7.WiTec



Figure 3Figure 3: Photos of the Raman microscope at ICS-7.

The integrated atomic force microscope is used for high-resolution topographic imaging of the samples and may also be used for tip-enhanced Raman spectroscopy (TERS). In TERS, the relatively weak Raman signal (compared to the fluorescence signal) is enhanced by several orders of magnitudes.

Figure 4 shows an example of a giant unilamelar lipid vesicle made from the phospholipid DOPC in 10 mM succhrose. The first image is an optical bright-field image and the second the respective Raman image. The displayed Raman spectrum is the difference between the succhrose environment and the vesicle membrane. Variations in signal intensity along the vesicle membrane are not due to domain formation but rather due to polarization effects.


Figure 4Figure 4: Bright-field and Raman image of a giant unilamelar vesicle made from DOPC in 10 mM Sucrose and the respective Raman spectrum of the vesicle membrane.

Contact:

Dr. G. Beltramo (g.beltramo@fz-juelich.de, 02461 / 61 – 3907)


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