Raman spectroscopy is the inelastic scattering of photons by molecular bonds, and therefore enables chemically-specific measurements within a sample without the use of external labels. This unique characteristic gives Raman-based techniques the potential to be used in a variety of biological research applications. However, many of the chemical components within the biological material are fluorescent, acting as a background and thus severely limiting the use of Raman spectroscopy for these applications. Although there have been attempts to reduce this background, only an optical time-gated approach can be sufficiently effective. Since the Raman signal occurs almost instantaneously with the excitation light while the fluorescence is delayed by several nanoseconds, lasers with pulses in the range of several picoseconds can be used for his purpose. A time gate that opens only for the duration of the Raman signal and closes before the background fluorescence is produced can be built by using a nonlinear medium and highpower laser pulses. This combination allows for the measurement of Raman spectra in the presence of a strong fluorescence background and opens up new potential applications of Raman spectroscopy for biological research such as (i) non-invasive characterization of the chemical composition of the endogenous fluorophores in bacteria, cells and tissues, (ii) understanding cellular processes and diseases such as cancer, vascular or neurodegenerative diseases by using natural markers, (iii) developing new probes that can be used both as fluorescence and Raman labels, (iv) noninvasive medical sensors for blood analytes such as glucose sensors for diabetes treatment. This technique will have a significant impact on related fields such as analytical chemistry, biomedicine, pharmacology, forensics, food safety, agriculture, biofuel research, environmental monitoring, and bio-defense.Outreach will involve students and educators at all levels, from middle school to postdoctoral, as well as representatives and participants from the biotechnology industry.Further information can be requested from the PI.
Within the frame of this NSF-funded proposal we developed an instrument with the unique capability of detecting time-resolved Raman scattering in the presence of fluorescence background and fluorescence spectra of biological specimens in a non-invasive way. The instrument works on the principle of all-optical, ultrafast gating induced in a nonlinear medium. While other similar instruments have been developed in the past, we focused our efforts on the development of an efficient technique, capable of gating even at low energies per pulse, which allows for the use of the instrument on biological samples, at high repetition rates. Several activities were performed, including optimization of the nonlinear medium, improvements in the gating geometry, and background reduction via novel algorithms. Time-resolved experimental measurements of Raman and fluorescence spectra were then performed on non-biological and biological specimens. The unique capability of this instrument to record Raman spectra in the presence of fluorescence adds to our facility a component that our biologist collaborators are interested to have access to, and will help us form new partnerships with researchers of various backgrounds (biology, physics, chemistry, engineering, etc) and attract students and researchers from underrepresented groups. Learning about the components that make up the endogenous fluorophores in bacteria, cells, and tissues, as well as the added ability to use intrinsic Raman markers, will contribute to our understanding of the mechanisms of diseases, enabling researchers to find better treatments and cures. This instrument will likely have a significant impact not only to the field of biology and medicine, but also to the related fields of analytical chemistry, pharmacology, forensics, food safety, and bio-defense. We also expect that this instrument will continue to have a significant impact beyond its main purpose as a new and innovative research tool, and will serve as a tool for the integration of scientific research, education, and knowledge-transfer. While the technology has been highlighted in many educational settings, such as undergraduate and graduate courses and short courses at conferences, it has also raised the interest of several industry partners.
