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Harvard Team Says New Imaging Method Offers Label-Free View of Drug Distribution

Investigators at Harvard University have developed what they claim is a highly sensitive microscopy technique that could allow for real-time tracking of drugs and drug metabolites in live cells without the use of fluorescent labels. Their work was published two weeks ago by Christian Freudinger, Wei Min, and their colleagues in the journal Science.
Drug developers want to know how a compound gets distributed inside a tissue or cell, but “previously, no other methods of doing this existed,” said Sunney Xie, a professor of chemistry and chemical biology at Harvard University and the corresponding author on the paper. 
Xie said that the new method is an improvement over vibrational microscopy techniques based on infrared absorption and Raman scattering, which have been used as label-free contrast imaging mechanisms, but are not suitable for biological applications for several reasons.
One drawback with IR microscopy is that it is not very sensitive when the sample is very small because the wavelength of IR light is long, and “you cannot do high-resolution imaging,” said Xie. In addition, IR microscopy works best with dry samples, which means that investigators cannot look at live cells.
As for spontaneous Raman scattering, it has a higher spatial resolution versus IR microscopy due to its shorter excitation wavelengths, but it is insensitive and has limited imaging speed. To compensate, scientists often use long averaging times and high laser power, which can damage biological samples, said Xie.
Another alternative, coherent anti-Stokes Raman scattering, or CARS, microscopy, is more sensitive than spontaneous Raman microscopy, but has a different spectrum compared to its corresponding Raman spectrum, due to a nonresonant background, which causes difficulties in image interpretation, according to Xie and colleagues.
The new technique is based on stimulated Raman scattering, or SRS, which is complementary to IR, Xie explained.
“We want a fast and real time observation,” he said. “We do not want an average taken over hours.”
Instead of using one laser beam, as in spontaneous Raman scattering, “we use two different laser beams at two different frequencies, both in the near IR, or visible region,” said Xie. He added that when the difference between the two input frequencies matches the frequency of a particular molecule’s vibrations, and all are oscillating in place, a strong signal is generated.

“We want a fast and real time observation. We do not want an average taken over hours.”

“SRS gives us a spectrum that is background free,” Xie said. “With SRS we have something where the spectrum is the same, but the sensitivity is orders of magnitude higher [than spontaneous Raman scattering microscopy and CARS]. This opens up a method for imaging by vibrational contrast with chemical selectivity.”
The SRS-based technique has several potential applications, according to Xie and colleagues. In their paper, they describe the use of the method to study the distribution of metabolites in live cells. The researchers looked at the fish oil omega-3, to understand its distribution and determine why it lowers the blood’s triglyceride levels and has other health benefits.     
The investigators also looked at the distribution of lipids in living cells, and imaged brain and skin tissues based on intrinsic lipid contrast.
SRS is still in the process of being commercialized, said Xie. “We are still negotiating with the microscopy companies,” he said, but did not provide the names of any potential commercialization partners. 
Xie added that he believes that pharmaceutical companies would want to take advantage of SRS microscopy once an instrument is commercially available, and cited Pfizer’s interest as an example. Jason Tsai, a researcher with Pfizer, is a co-author on the Science paper.  
Compared to fluorescence microscopy, the most widely used technique in live sample imaging, the major advantage of SRS imaging is that it does not need labeling or staining, Bo Huang, a postdoctoral fellow in the department of chemistry and chemical biology at Harvard University, told CBA News this week in an e-mail. Instead, it obtains its signal from molecules using their characteristic properties.
Huang is a co-developer of stochastic optical reconstruction microscopy, or STORM, fluorescence microscopy, and presented his group’s work in a poster at the 48th annual meeting of the American Society for Cell Biology, held in San Francisco last month. He is not affiliated with the SRS study published in Science.
Said Xie, “You do not want to label small drug molecules with dyes or fluorophores, because the dyes or fluorophores could be bigger than the small drug molecules or metabolites, and perturb the molecules’ function and could be toxic.” Imaging of these molecules or metabolites is best done without labeling.
One drawback of SRS imaging, however, is that its sensitivity is not as high as fluorescence microscopy, and it cannot specifically identify a large molecule such as a protein unless Raman labeling is used, which compromises the advantages of label-free imaging. “In general, I think SRS imaging is best at visualizing the distribution of small molecules, such as lipids and certain drugs, in live tissue/animals,” said Huang.
He added that for drug discovery, SRS imaging should be a very useful tool for examining in vivo the physiological effects of interfering with a potential drug target or applying a drug.  

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