Development of novel techniques for in vivo imaging of dynamic systems
Real-time, in vivo imaging of biological samples enhances our understanding of the molecular machinery in living systems, such as cells, organs and even whole organisms. Heretofore, imaging methods have been predominantly limited to approaches that require labeling of the system, which often distorts their normal mechanism of action. Moreover, resolving the temporal dynamics of the quantized vibrations of chemical bonds within a specific molecular species, i.e., acquiring so-called »molecular fingerprints«, has so far been hampered by the limited dynamic range of the current methodology, which is incapable of detecting small amplitude changes near the noise floor.
To tackle these limitations, we are pursuing two routes:
1) Development of a novel, broadband, high-repetition rate, laser source for label-free imaging of biological samples and temporal detection of their molecular fingerprints by means of stimulated Raman spectroscopy.
2) Development of a new field-resolved detection scheme based on nonlinear photoconductive sampling to enhance the dynamic range of detection beyond the current state of the art.
To study the spatio-temporal dynamics of biological systems, we employ the above-mentioned developments, together with »pattern recognition methodology«, for label-free detection and analysis of molecular fingerprints. Here, the aim is to accurately examine the patterns of energy exchange between the Stokes laser field and the sample, due to stimulated Raman scattering.
By resolving the exact temporal evolution of the Stokes field on the shortest observable timescales - within a single light cycle - by means of electro-optic sampling or nonlinear photoconductive sampling, one obtains accurate and detailed information about the evolution of the spectral phase and amplitude of the Stokes field, which yields the required molecular fingerprint.
Tools, Techniques & Labs
Vibrational imaging and spectroscopy can be conceptually compared to the approach used in the children’s book »Where is Waldo?«, in which children are challenged to locate Waldo in a crowded image, based solely on pattern recognition and without the need for labels.
Vibrational spectra of molecules measured by means of:
1) Infrared (IR) spectroscopy, or 2) Raman spectroscopy follow the same concept.
Here, a given »Waldo« molecule can be located and monitored in a dynamic system by detecting its vibrational fingerprint. However, strong absorption in the infrared spectral range by water limits the sensitivity of IR spectroscopy, and the spatial resolution of images obtained by this method is dictated by the diffraction limit of the long IR wavelength. In contrast, the Stokes field utilized for stimulated Raman spectroscopy lies in the near infrared spectral range, far away from the water absorption region, and is free of non-resonant background, all being desired requirements for field resolved spectroscopy.
In this context, we are developing a laser source based on Yb:YAG thin-disk laser technology, which delivers broadband pulses in the near infrared spectral range, with carrier-to-envelope phase stability and at MHz repetition rates, based on Yb:YAG thin-disk laser technology.[Oleg Pronin]
The beating note between the broad spectral bandwidth of the Stokes from 1.1 µm to 2 µm and the pump pulses at 1030 nm, covers the entire vibrational spectrum of molecules in spectral region beyond 2.1 µm. Therefore, complete picture of the molecular fingerprint of a sample can be extracted by field resolved detection of the transmitted Stokes pulses.
In parallel, we are developing a high-energy field synthesizer by combining the two existing technologies of:
1) optical parametric chirped pulse amplification and 2) coherent synthesis.
The synthesizer operates over several optical octaves, from visible to far-infrared part of the electromagnetic spectrum, with the possibility of delivering high-energy, few-cycle pulses at different carrier frequencies or high-energy sub-cycle light transients. Combining this unique laser source with novel field sampling methods such as nonlinear photoconductive sampling, will enable us to study light-matter interaction with unprecedented temporal resolution and dynamic range.
The two new technologies will be directly applied to real-life problems, specifically for detecting cancerous cells’ fingerprint at their early stage in collaboration with.[Mihaela Zigman]