We developed a dual-beam optical tweezers setup with video-based force detection to precisely determine the mechanic properties of living cells in suspension with superior sensitivity compared to other techniques like optical stretchers and atomic force microscopy (AFM). With high time resolution we are able to determine both the elastic and the viscous properties of the cells. This novel setup is combined with custom-designed microfluidic cartridges to automatically and reliably pattern cells and beads at specific positions. The beads and cells are trapped and coupled to yield bead-cell-bead complexes.
First results of the elasticity of HEK293 (human embryonic kidney) cells and skin fibroblasts are presented. The latter contain TMEM43-p.S358L mutation, which is linked to arrhythmogenic right ventricular cardiomyopathy.
We present a new detection method for multifocal two-photon laser scanning microscopy (TPLSM) that allows a fast
and easy access to spectrally resolved, three-dimensional images. In our setup eight fluorescent foci are directed through
a descanned tube lens combination and a straight vision prism. This prism spectrally splits up the fluorescence beamlets,
resulting in eight parallel spectral fluorescence lines. These lines are imaged onto a slit block array in front of a 8x8 multi
anode PMT. Each PMT row detects different spectral characteristics from a special point in the sample whereas each
column represents one focus. The eight exciting foci are scanned in the region of interest inside the sample by the two
scanning mirrors in x- and y-direction. As a result of this imaging technique eight spectrally resolved images of slightly
shifted sample regions are generated simultaneously and added up after the measurement, maintaining the spectral
information. We present spectrally resolved 3D-data of various biological samples like pollen grains, tobacco cells and
orange peel cells.
We have developed a new descanned parallel (32-fold) pinhole and photomultiplier detection array for multifocal multiphoton microscopy that effectively reduces the blurring effect originating from scattered fluorescence photons in strongly scattering biological media. With this method, we achieve a fourfold improvement in photon statistics for detecting ballistic photons and an increase in spatial resolution by 21% in the lateral and 35% in the axial direction compared to single-beam non-descanned multiphoton microscopy. The new detection concept has been applied to plant leaves and pollen grains to verify the improvements in imaging quality.
The influence of metal surfaces and nanoparticles on the fluorescence emission of fluorophores in close proximity is of
particular interest for biophysical applications, near field optics and biosensing. For instance, the quenching of
fluorophores by gold nanoparticles can be used for the investigation of biomolecular conformational changes or
interactions and silver coated metal tips are potent scanning near field optical microscopy tips. Apart from the
quenching effects, nanoparticles are used for fluorescence enhancement in biosensor applications.
Here we use a setup combining total internal reflection fluorescence microscopy (TIRFM) with the piezo-controlled
nanometer-sensitive movement of an atomic force microscope (AFM) in order to measure and quantify the fluorescence
emission as a function of distance between single fluorophores and metal nanoparticles or tiny metal tips. By using
CdSe/ZnS nanocrystals as fluorophores and gold as metal we observed significant fluorescence quenching as well as
enhancement due to exciton-plasmon coupling. In the future, these experiments will be extended to metal nanoparticles
of different elements, alloys, sizes and shapes, giving insight into the related energy transfer processes and quenching
mechanisms.
Single molecules can nowadays be investigated by means of optical, mechanical and electrical methods. Fluorescence imaging and spectroscopy yield valuable and quantitative information about the optical properties and the spatial distribution of single molecules. Force spectroscopy by atomic force microscopy (AFM) or optical tweezers allows addressing, manipulation and quantitative probing of the nanomechanical properties of individual macromolecules. We present a combined AFM and total internal reflection fluorescence (TIRF) microscopy setup that enables ultrasensitive laser induced fluorescence detection of individual fluorophores, control of the AFM probe position in x, y and z-direction with nanometer precision, and simultaneous investigation of optical and mechanical properties at the single molecule level. Here, we present the distance-controlled quenching of semiconductor quantum dot clusters with an AFM tip. In future applications, fluorescence resonant energy transfer between single donor and acceptor molecules will be investigated.
In our experiments 2-Photon laser scanning microscopy (2PLSM) has been used to acquire 3-dimensional structural information on native unstained biological samples for tissue engineering purposes. Using near infrared (NIR) femtosecond laser pulses for 2-photon excitation and second harmonic generation (SHG) it was possible to achieve microscopic images at great depths in strongly (light) scattering collagen membranes (depth up to 300 μm) and cartilage samples (depth up to 460 μm). With the objective of optimizing the process of chondrocyte growth on collagen scaffolding materials for implantation into human knee joints, two types of samples have been investigated. (1) Both arthritic and non-arthritic bovine and human cartilage samples were examined in order to differentiate between these states and to estimate the density of chondrocytes. In particular, imaging depth, fluorescence intensity and surface topology appear promising as key information for discriminating between the non-arthritic and arthritic states. Human chondrocyte densities between 2-106/cm3 and 20-106/cm3, depending on the relative position of the sample under investigation within the cartilage, were measured using an automated procedure. (2) Chondrocytes which had been sown out on different types of I/III-collagen membranes, were discriminated from the scaffolding membranes on the basis of their native fluorescence emission spectra. With respect to the different membranes, either SHG signals from the collagen fibers of the membranes or differences in the emission spectra of the chondrocytes and the scaffolding collagenes were used to identify chondrocytes and membranes.
Native hyaline cartilage from a human knee joint was directly investigated with laser scanning microscopy via 2-photon autofluorescence excitation with no additional staining or labelling protocols in a nondestructive and sterile manner. Using a femtosecond, near-infrared (NIR) Ti:Sa laser for 2-photon excitation and a dedicated NIR long distance objective, autofluorescence imaging and measurements of the extracellular matrix (ECM) tissue with incorporated chondrocytes were possible with a penetration depth of up to 460 μm inside the sample. Via spectral autofluorescence separation these experiments allowed the discrimination of chondrocytes from the ECM and therefore an estimate of chondrocytic cell density within the cartilage tissue to approximately 0.2-2•107cm3. Furthermore, a comparison of the relative autofluorescence signals between nonarthritic and arthritic cartilage tissue exhibited distinct differences in tissue morphology. As these morphological findings are in keeping with the macroscopic diagnosis, our measurement has the potential of being used in future diagnostic applications.
The identification of specific binding molecules is of increasing interest in the context of drug development based on combinatorial libraries. Scanning Probe Microscopy (SPM) is the method of choice to image and probe individual biomolecules on a surface. Functional identification of biomolecules is a first step towards screening on a single molecule level. As a model system we use recombinant single- chain Fv fragment (scFv) antibody molecules directed against the antigen fluorescein. The scFv's are covalently immobilized on a flat gold surface via the C-terminal cysteine, resulting in a high accessibility of the binding site. The antigen is immobilized covalently via a long hydrophilic spacer to the silicon nitride SPM-tip. This arrangement allows a direct measurement of binding forces. Thus, closely related antibody molecules differing in only one amino acid at their binding site could be distinguished. A novel SPM-software has been developed which combines imaging, force spectroscopic modes, and online analysis. This is a major prerequisite for future screening methods.
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