Nonlinear Microscopy
Microscopy techniques have greatly influenced the evolution of modern sciences, most prominently, of life sciences. The growing demand has boosted the development of novel and better microscopy techniques for micron and submicron level resolution. One technique, which is commonly used in the life sciences today, is fluorescence microscopy, which typically needs specific markers to achieve a high degree of contrast. In many cases, however, this fundamentally modifies the functionality of living samples, or is even toxic.
Fig.1:Two-photon excited auto-fluorescence of horse chestnut. The bark contains Aesculin, a natural fluorescent dye.
In our group, we investigate new techniques for optical microscopy. Our work focuses on label-free imaging, which is often required when samples (e.g., living cells) cannot be stained. Some materials show auto-fluorescence and can thus be imaged without introducing labels. Here, fluorescence excitation via two-photon absorption processes is advantageous due to a reduction of absorption and scattering, and an improved tissue penetration depth when using near-infrared light.
Independent of the presence of auto-fluorescence, label-free imaging can be realized using nonlinear microscopy techniques, such as second- and third harmonic generation, or coherent anti-Stokes Raman scattering. In order to investigate new nonlinear microscopy techniques and related novel light sources, we have set up a laser scanning microscope, where a focused laser beam is scanned across the sample in order to obtain 3D images with sub-micron resolution.
Coherent anti-Stokes Raman scattering (CARS) microscopy
In addition to the common advantages of label-free nonlinear microscopy, CARS also offers a high chemical specificity. CARS directly probes the vibrational levels of large (bio)molecules, and the generated CARS signal is as specific for a certain molecule, as a fingerprint is specific for humans. To achieve this specific CARS signal, a pump beam of angular frequency ωp and a Stokes beam of angular frequency ωS are overlapped and focused in a sample to excite a vibrational level at ωp-ωS, which is detected at the anti-Stokes angular frequency 2ωp-ωS. The excitation of a specific vibrational level at Ω=ωp-ωS is what leads to the chemical specificity of CARS microscopy. The figure below shows two examples, where vibrational resonances of polystyrene and of lipids have been utilized to image polystyrene beads in water and a lipid emulsion, respectively.
Fig.2: a) CARS images. Polystyrene beads in water. The vibrational resonance of polystyrene is at a frequency of 3066 cm-1. b) Fat droplets that formed in an emulsion (salad dressing). To obtain this image, the pump and Stokes frequencies were tuned to a difference of 2845 cm-1, which is the resonance frequency of the symmetric CH2-stretch.
Light sources for CARS
We are developing laser sources, which provide synchronized pulses with wavelengths that can be tuned to the required vibrational frequencies for CARS microscopy. One such light source is a femtosecond synchronously pumped optical parametric oscillator (OPO) based on the nonlinear crystal lithium triborate (LBO) [see C. Cleff et al., 2011]. This OPO generates two trains of pulses, which are intrinsically synchronized with the femtosecond pump laser pulses. The wavelengths of these subharmonic pulses can be tuned by changing the LBO crystal temperature, and in total a range of vibrational frequencies between 1200 and 6600 cm-1 is accessible (wavelength range 1,5 - 8,3 µm). With this, the OPO offers great flexibility for fundamental research, e.g. for the acquisition of complete CARS spectra. A second laser source for CARS was realized by using a Titanium:Sapphire femtosecond laser and by deriving a second wavelength via supercontinuum generation in a microstructured fiber, also yielding two trains of pulses with intrinsic synchronization [see P. Groß et al., 2010]. Within the supercontinuum, a soliton at a wavelength of 1038 nm was formed, which could be amplified to more than 400 mW in ytterbium-doped fiber, and which was very suitable as the second pulse train for CARS. The advantages of this supercontinuum-based light source are its simplicity and robustness.
Fig.3: Photograph of the supercontinuum generated in the
microstructured fiber. The white arrow indicates the
soliton at 1038 nm used as the Stokes wave for CARS.
Fig.4: Raman spectrum of eugenol. The inset shows the spontaneous Raman spectrum for comparsion.
Spectral resolution
The use of femtosecond pulses for CARS typically results in a poor spectral resolution. This can be counteracted by chirping the pulses: the spectral contents of the pulses are distributed over time, resulting in a reduced instantaneous bandwidth and hence improved spectral resolution. We have used fiber stretchers to accomplish the pulse chirp, and have applied this to both laser systems described above. With the improved spectral resolution it was possible, for example, to record a CARS spectrum of eugenol, where all six resonances between 2700 and 3100 cm-1 can be clearly dintinguished.
Spatial resolution
Within the CARS project, we are collaborating with the group of Laser Physics and Nonlinear Optics and the group of Optical Sciences in order to improve the spatial resolution of CARS microscopy. Calculations based on the density matix model show that it should be possible to suppress CARS excitation by disturbing the build-up of coherence, e.g. by incoherently populating the vibrational level. Employing this scheme in a focus-engineering approach should enable the improvement of spatial resolution [see Beeker et al., 2009].
Journal articles
- C. Mallidis, J. Wistuba, B. Bleisteiner, O.S. Damm, P. Groß, F. Wübbeling, C. Fallnich, M. Burger, S. Schlatt, In situ visualisation of damaged DNA in human sperm by Raman microspectroscopy, Hum. Reprod. 26, 1641 (2011)
Download - W.P. Beeker, C.J. Lee, K.-J. Boller, P. Groß, C. Cleff, C. Fallnich, H.L. Offerhaus, J.L. Herek, A theoretical investigation of superresolution CARS imaging via coherent and incoherent saturation of transitions, DOI: 10.1002/jrs.2949
Download - C. Cleff, J. Epping, P. Groß, C. Fallnich, Femtosecond OPO based on LBO pumped by a frequency-doubled Yb-fiber laser-amplifier system for CARS spectroscopy, Appl. Phys. B., DOI 10.1007/s00340-011-4465-8 (2011)
Download - P. Groß, L. Kleinschmidt, S. Beer, C. Cleff and C. Fallnich, Single-laser light source for CARS microscopy based on soliton self-frequency shift in a microstructured fiber, Appl. Phys. B, 101, 167 (2010)
Download - W. P. Beeker, P. Groß, C. J. Lee, C. Cleff, H. L. Offerhaus, C. Fallnich, J. L. Herek, and K.-J. Boller, Spatially dependent Rabi oscillations: An approach to sub-diffraction-limited coherent anti-Stokes Raman scattering microscopy, Phys. Rev. A 81, 012507 (2010)
Download - W.P. Beeker, P. Groß, C.J. Lee, C. Cleff, H.L. Offerhaus, C. Fallnich, J.L. Herek, and K.-J. Boller, A route to sub-diffraction-limited-CARS microscopy, Opt. Expr. 17, 22632 (2009).
- S. Postma, A.C.W. van Rhijn, J.P. Korterik, P. Groß, J.L. Herek, and H.L. Offerhaus, Application of spectral shaping to high resolution CARS spectroscopy, Opt. Expr. 16, 7985 (2008).
