Bianca Buchegger

Frequency domain photoacoustic and fluorescence microscopy.

We report on simultaneous frequency domain optical-resolution photoacoustic and fluorescence microscopy with sub-µm lateral resolution. With the help of a blood smear, we show that photoacoustic and fluorescence images provide complementary information. Furthermore, we compare theoretically predicted signal-to-noise ratios of sinusoidal modulation in frequency domain with pulsed excitation in time domain.

Stimulated Emission Depletion Lithography with Mercapto-Functional Polymers

Surface reactive nanostructures were fabricated using stimulated emission depletion (STED) lithography. The functionalization of the nanostructures was realized by copolymerization of a bifunctional metal oxo cluster in the presence of a triacrylate monomer. Ligands of the cluster surface cross-link to the monomer during the lithographic process, whereas unreacted mercapto functionalized ligands are transferred to the polymer and remain reactive after polymer formation of the surface of the nanostructure. The depletion efficiency in dependence of the cluster loading was investigated and full depletion of the STED effect was observed with a cluster loading exceeding 4 wt %. A feature size by λ/11 was achieved by using a donut-shaped depletion beam. The reactivity of the mercapto groups on the surface of the nanostructure was tested by incubation with mercapto-reactive fluorophores.

Streptavidin functionalized polymer nanodots fabricated by visible light lithography

BackgroundTwo-photon polymerization, optionally combined with stimulated emission depletion (STED) lithography, allows two and three dimensional polymer fabrication with structure sizes and resolution below the diffraction limit. Structuring of polymers with photons, whose wavelength is within the visible range of the electromagnetic spectrum, gives new opportunities to a large field of applications e.g. in the field of biotechnology and tissue engineering. In order to create new biotechnological applications, versatile methods are needed to functionalize the polymeric structures.ResultsHere we report the creation of polymer-nanodots with high streptavidin (SA) affinity via two-photon polymerization (TPP). Controlling the size of the polymer dots allows for limiting the number of the SA molecules. TPP dots with a diameter of a few 100 nm show up to 100% streptavidin loading. We can show that most of the dots are loaded by one to two streptavidins on average. Attached streptavidin molecules remain functional and are capable to bind 0.7 biotin molecules on average.ConclusionThe presented functionalized nanostructures may be used as platforms for a multitude of biological experimental setups. Nanoscopic well defined structures, capable of selective binding of streptavin proteins, used as linkers for other biotinylated biomolecules, may also find application in in-vitro sensing, like for example lab on chip devices with limited surface area.

Frequency domain optical resolution photoacoustic and fluorescence microscopy using a modulated laser diode

In this paper a multimodal optical-resolution photoacoustic and fluorescence microscope in frequency domain is presented. Photoacoustic waves and modulated fluorescence are generated in chromophores by using a modulated diode laser. The photoacoustic waves, recorded with a hydrophone, and the fluorescence signals, acquired with an avalanche photodiode, are simultaneously measured using a lock-in technique. Two possibilities to optimize the signal-to-noise ratio are discussed. The first method is based on the optimization of the excitation waveform and it is argued why square-wave excitation is best. The second way to enhance the SNR is to optimize the modulation frequency. For modulation periods that are much shorter than the relaxation times of the excited chromophores, the photoacoustic signal scales linearly with the modulation frequency. We come to the conclusion that frequency-domain photoacoustic microscopy performed with modulation frequencies in the range of 100 MHz can compete with time-domain photoacoustic microscopy regarding the signal-to-noise ratio. The theoretical predictions are confirmed by experimental results. Additionally, images of stained and unstained biological samples are presented in order to demonstrate the capabilities of the multimodal imaging system.

Photoacoustic microscopy of single cells employing an intensity-modulated diode laser

In this work, we employ frequency-domain photoacoustic microscopy to obtain photoacoustic images of labeled and unlabeled cells. The photoacoustic microscope is based on an intensity-modulated diode laser in combination with a focused piezo-composite transducer and allows imaging of labeled cells without severe photo-bleaching. We demonstrate that frequency-domain photoacoustic microscopy realized with a diode laser is capable of recording photoacoustic images of single cells with sub-µm resolution. As examples, we present images of undyed human red blood cells, stained human epithelial cells, and stained yeast cells.

Frequency-domain photoacoustic and fluorescence microscopy: application on labeled and unlabeled cells

In this paper, multimodal optical-resolution frequency-domain photoacoustic and fluorescence scanning microscopy is presented on labeled and unlabeled cells. In many molecules, excited electrons relax radiatively and non-radiatively, leading to fluorescence and photoacoustic signals, respectively. Both signals can then be detected simultaneously. There also exist molecules, e.g. hemoglobin, which do not exhibit fluorescence, but provide photoacoustic signals solely. Other molecules, especially fluorescent dyes, preferentially exhibit fluorescence. The fluorescence quantum yield of a molecule and with it the strength of photoacoustic and fluorescence signals depends on the local environment, e.g. on the pH. Therefore, the local distribution of the simultaneously recorded photoacoustic and fluorescence signals may be used in order to obtain information about the local chemistry.

Proteins on supported lipid bilayers diffusing around proteins fixed on acrylate anchors

Mobility of proteins and lipids plays a major role in physiological processes. Platforms which were developed to study protein interaction between immobilized and mobile proteins suffer from shortcomings such as fluorescence quenching or complicated fabrication methods. Here we report a versatile platform comprising immobilized histidine-tagged proteins and biotinylated proteins in a mobile phase. Importantly, multiphoton photolithography was used for easy and fast fabrication of the platform and allows, in principle, extension of its application to three dimensions. The platform, which is made up of functionalized polymer structures embedded in a mobile lipid bilayer, shows low background fluorescence and allows for mobility of arbitrary proteins.

Nanostructured functional polymers for selective protein binding

Two-Photon Polymerization enables fabrication of two and three dimensional polymer structures. For structuring, a highly cross-linking acrylate monomer is mixed with a photo-initiator. Using femtosecond IR light as an excitation source, feature sizes down to 100 nm can be achieved. Applying stimulated emission depletion (STED) reduces the feature size even further [1, 2]. Recently, our group developed functional polymer structures below the diffraction limit by mixing metal oxo clusters into the acrylate-based photoresist [3].

Multimodal fluorescence and photoacoustic microscopy in the frequency domain

In this paper a multimodal optical resolution microscope in frequency domain is introduced. Fluorescence and photoacoustic signals are generated simultaneously using an amplitude modulated diode laser. The resulting signals are recorded by a lock-in amplifier. By scanning the sample, two-dimensional plots of the fluorescence and photoacoustic signals can be generated. In this paper, the signal-to-noise ratios of amplitude modulated signals are compared to those expected for pulsed excitation under the assumption that the maximum permissible exposure limits are fulfilled. Furthermore, it is described how to determine the excited state life-times from the measured frequency responses of the fluorescence or photoacoustic signals, respectively.

Functional photoresists for sub-diffraction stimulated emission depletion lithography

Two novel photoresists were developed for the fabrication of sub-diffractionally sized polymeric nanostructures with chemically reactive surfaces. Using multiphoton polymerization as well as stimulated emission depletion (STED) lithography, chemically functional monomers were copolymerized with highly crosslinking triacrylate monomers to yield stable nanostructures. The polymer structure was thereby supplemented with chemical functionalities for further covalent modification reactions. The reactivity of mercapto- and carboxylate groups on the surface of the nanostructures was proved by orthogonally labeling them with reactive fluorophores. The photoresists can be used for stimulated emission depletion lithography and lateral line widths down to 55 nm were achieved. A three-dimensional structure is shown that is made up with three compounds: a frame out of a un-functionalized acrylate photoresist, and two intermediate networks made up with thiol- and carboxyl functional photoresists.