Biophotonics provides - amongst others - "all-optical" technology for clinical diagnostics. Here we focus on the description of a novel biophotonic tool to assist in the diagnosis and localization of brain tumors. Currently, staining microscopy is the gold standard used in histopathology to determine tumor properties, but it is virtually impossible to apply this technique in vivo.

All For One

During neurosurgery precise tumor border detection presents a central challenge. By combining three nonlinear microscopic imaging techniques to a multimodal microscopy approach, similar information on the morphology and chemical composition of unprocessed tissue can be extracted. Much greater depth penetration can be achieved in comparison to conventional light microscopy. This all-optical approach fuses coherent anti-Stokes Raman scattering (CARS), second-harmonic generation (SHG) and two-photon excited fluorescence (TPEF) imaging into a single microscopic setup. These three optical spectroscopic methods and their implementation into a single instrument are presented here. The setup has been used to study the morphology and chemical composition of (ex vivo) brain tissue of a domestic pig as a model for human brain tissue and human brain tumor samples from biopsies. The experimental techniques presented are contact-free and label-free all-optical techniques. Thus, they are potentially applicable in vivo, opening the door to label-free diagnostic and surgical guidance for significant improvements in online tumor border detection.

Deeper

Due to the availability of stable laser sources for ultra short pulses the development of nonlinear imaging techniques was intensified, resulting in the introduction of TPEF, SHG and CARS microscopy in life sciences in the last decade of the 20^th century. Using near infrared light, the depth penetration in tissue is greatly enhanced to several hundred µm, enabling the investigation of thick tissue specimens as opposed to just "scratching the surface" with visible light. Furthermore, the nonlinear nature of signal generation with low energy NIR photons greatly reduces phototoxic effects and improves the 3D resolution.

This is due to the signal being solely generated within the laser focal region, where the photon density is high enough to cause nonlinear signals.

In contrast to normal fluorescence, TPEF is based on nonlinear absorption, i.e. two photons are simultaneously absorbed to electronically excite a molecule, which emits the fluorescence photon to be detected (fig. 1A). Relying on the fluorescence properties of the analyte molecules, TPEF is restricted to imaging autofluorescent species, which include, for instance, NADH, flavins, keratin, retinol and elastin in biological samples. However, SHG and CARS are coherent nonlinear scattering processes. In SHG two NIR photons are fused to a single scattered photon of twice the photon energy (fig. 1B). With SHG, molecular structures lacking inversion geometry can be visualized. This is especially true for the structural protein collagen in connective tissue, but also of acto-myosin and tubulin, or in general (hidden) interfaces. CARS is the most informative and generally applicable imaging process. In principle, all types of molecules can be visualized by CARS, in contrast to SHG and TPEF, which are limited to certain molecular species. CARS requires two pulsed lasers of different wavelengths to simultaneously illuminate the sample. If the frequency difference of both lasers matches a vibrational resonance, this vibrational level becomes selectively populated. Further photons are then coherently scattered off this vibrational state, generating scattered light at the anti-Stokes frequency (fig. 1C). In our study, CARS was used to display the spatial distribution of lipids in tissue, which is an important marker used to differentiate between cancerous and normal brain tissue, by focusing on the CH-stretching vibration.

One For All

Figure 1D displays the basic layout of the multimodal nonlinear imaging setup, allowing for joint TPEF, SHG and CARS imaging. The output of a pulsed Ti:Sa laser is split to pump an optical parametric oscillator to generate the tunable pump wavelength for CARS, while the other fraction serves as the Stokes beam. For SHG and TPEF either the pump or the Stokes laser alone can be used, and a suitable detection window can be selected by the appropriate choice of filters. After recombining both pulse trains spatially and temporally, the lasers are fed into a commercial laser scanning microscope. Dichroic beamsplitters and filters are used to separate the three signals, which are detected by photomultiplier tubes.

Looking Inside