We present numerical simulations and experiments employing two-photon excited fluorescence in a sum-frequency mixing scheme which could be used for colocalization experiments in biophysics. By means of numerical calculations using the Debye approximation, the point spread functions (PSF) of each focused laser beam as well as the resulting PSF of the two-color two-photon (2C2P) excitation are calculated and discussed. Experiments are performed on Streptococcus pneumoniae and on surface filaments (“pili”) of the bacteria. Two different fluorescent labels were used for staining the bacteria themselves as well as the surface filaments as structure of interest. Since one fluorophore is excited by one single laser and the other label only in the combination of both lasers, intrinsic colocalization of the signals on the nanometer scale is ensured. The two-color two-photon excitation is performed by an ultrashort fiber laser system with synchronized emission wavelengths at λ1 = 780 nm and λ2 = 1030 nm and pulse durations around 100 fs. The multiphoton microscopy approach provides high resolution and allows for three-dimensional imaging of bacteria in a volume of (5 x 5 x 5) μm3.
This presentation focuses on ultrafast fiber lasers as excitation light-source for advanced microscopy. We will present our latest innovations and show how multiphoton microscopy can benefit from these developments. This will propel imaging techniques like TPEF, SHG, CARS or STED among others that excel conventional microscopy techniques in a deeper penetration depth, label-free imaging capability or higher spatial resolution. Wavelengths around 900 nm for addressing certain applications with fluorescent proteins and pulse durations as short as 100 fs remain a challenge for fiber lasers. TOPTICA has now launched the third generation of ultrafast fiber lasers overcoming these difficulties. The novel FF ultra laser platform is capable of generating such short pulses at not only the wavelengths 780 nm and 1050 nm but most recently at 920 nm too. This enables the imaging of the popular green fluorescent protein (GFP) and its derivatives with lowest possible power level because of the high efficiency. In the course of this work, we present selected applications proving the suitability of this industrial grade laser family for a broad variety of microscopic applications.
Stereotactic biopsy procedure is performed to obtain a tissue sample for diagnosis purposes. Currently, a fiber-based mechano-optical device for stereotactic biopsies of brain tumors is developed. Two different fluorophores are employed to improve the safety and reliability of this procedure: The fluorescence of intravenously applied indocyanine green (ICG) facilitates the recognition of blood vessels and thus helps minimize the risk of cerebral hemorrhages. 5- aminolevulinic-acid-induced protoporphyrin IX (PpIX) fluorescence is used to localize vital tumor tissue. ICG fluorescence detection using a 2-fiber probe turned out to be an applicable method to recognize blood vessels about 1.5 mm ahead of the fiber tip during a brain tumor biopsy. Moreover, the suitability of two different PpIX excitation wavelengths regarding practical aspects was investigated: While PpIX excitation in the violet region (at 405 nm) allows for higher sensitivity, red excitation (at 633 nm) is noticeably superior with regard to blood layers obscuring the fluorescence signal. Contact measurements on brain simulating agar phantoms demonstrated that a typical blood coverage of the tumor reduces the PpIX signal to about 75% and nearly 0% for 633 nm and 405 nm excitation, respectively. As a result, 633 nm seems to be the wavelength of choice for PpIX-assisted detection of high-grade gliomas in stereotactic biopsy.
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