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Foils Label-free tissue image of a low grade glioma (LGG) acquired with the Invenio imaging system ➤
SRS microscopy with colleagues at the Xie lab. Invenio Imaging was founded in 2012 based on Professor Xie’s research, and over the last two years the company has produced a research-based system that can be operated in a similar way to a confocal or two- photon fluorescence microscope, but obtains an SRS contrast. The mechanism of the contrast,
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Freudiger explained, is based on the vibrational frequencies of the molecules: ‘If you have two atoms, they are connected with a spring, and they’ll have a resonance frequency determined by how heavy the atoms are and how stiff the spring is in between,’ he said. ‘By looking at where the vibration is coming from, you know what the molecule is.’ What is specific to coherent Raman scattering is that, instead of using a single wavelength laser as with spontaneous Raman scattering, two-wavelength lasers are used. ‘If you fire these two wavelengths into the sample, the intensity that results is the beat frequency – the difference in frequency of the two laser wavelengths,’ explained
Freudiger. ‘If you now match this beat frequency to the vibrational frequency then you can “shake” the molecules – so, you very actively drive the vibrations. What you measure in CARS is the emission that then originates from these vibrational frequencies. What one does in SRS is look at the energy transferred to the sample that results from exciting these vibrations.’ In an area of crossover between
fluorescence and label-free techniques, scientists are working on developing dyes for use with this traditionally label-free approach. Professor Wei Min from Columbia University has invented new types of Raman label, which provide the specificity advantage of labelling, but that are much smaller (a few atoms in size) so eliminate some of the drawbacks of labelling molecules. ‘They are still not as bright as typical fluorophores, but they are less disruptive to the system, because it is just very small molecules that you would add,’ Freudiger remarked. ‘So, there is this overlap region where people do use labels, together with Raman
imaging, and the motivation is the same – to perturb the system that they are trying to study the least.’
Minimising tissue damage Although label-free imaging leaves the samples largely unaffected, the technique acts more as an alternative for when labelling is not practical, as Freudiger pointed out, rather than as a complete replacement to fluorescence imaging. Fluorophores are still a vital part of imaging live tissue and, for most fluorescence microscopy techniques, it is almost impossible to eliminate damage to the sample. ‘If you use a lamp, laser or LED there will always be some sample damage – phototoxicity – as well as photobleaching because once fluorophores are hit with light they not only emit fluorescence, but they also lose efficiency to release fluorescence and eventually photobleach,’ said Dr Kavita Aswani, senior applications scientist for life sciences, marketing at light source provider Excelitas. ‘The question is how to minimise photobleaching, because there is
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Dr Dan Orringer and Spencer Lewis
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