Dutch researchers have overcome the traditional incompatibility of Raman microscopy with fluorescence microscopy by exploiting the optical properties of semiconductor fluorescent quantum dots (QDs). They have now used their hybrid Raman fluorescence spectral imaging approach in single-cell microscopy applications.

Biophysical engineers Henk-Jan van Manen and Cees Otto of the University of Twente, The Netherlands, have used fluorescent nanoparticles to broaden the scope of single-cell microscopy by combining it with intracellular chemical analysis based on Raman.

According to the researchers, the need for fluorescent nanoparticles, or quantum dots, arises because common fluorescent labels overshadow the intrinsically weak Raman signals from DNA, proteins and lipids in cells. To side-step this problem, the team used fluorescent quantum dots that emit light in a wavelength region that is well-separated from Raman signals.

The group at the University of Twente has pioneered the use of Raman spectroscopy in investigating the chemical make-up of single cells. Van Manen and Otto have now demonstrated successfully the hybrid technique by illuminating white blood cells under UV at 413 nm, and observing the Raman signal from an enzyme critical to the innate immune response. This allowed them to detect and visualize the enzyme across the cell. In a second proof of principle experiment, the team used 647 nm light to investigate cellular proteins and lipids.

Van Manen and Otto say that the fluorescence Raman microscopy combination provides exciting new possibilities because the nanoparticles coated with antibodies raised against cancer biomarkers could be used to guide Raman microscopy to certain areas in tissues and allow the spread of cancer in a patient to be detected with molecular precision.
"We have exploited the optical properties of nanosized quantum dots to develop two new strategies for combining confocal Raman microscopy with confocal fluorescence microscopy on QD-labelled biological cells," conclude the researchers. The first approach uses resonant Raman microspectroscopy and imaging of enzymes, in this case flavocytochrome b558, and is, the team says, fully compatible with linear fluorescence microscopy of intracellular QDs.

The second method involves continuous-wave two-photon-excited fluorescence of the QDs in white blood cells and does not interfere with non-resonant Raman signals produced by proteins or lipids in the cells. "As these hybrid Raman fluorescence strategies should be applicable to any cell type that is amenable to labelling with QDs," the researchers add, "we envision that hybrid Raman fluorescence microscopy will offer new prospects for integrating QD imaging on cells with spatially resolved, detailed intracellular chemical analysis by broadband Raman microspectroscopy."

Related links:

• Nano Lett, 2007, in press
• Otto Departmental Page

Article by David Bradley