Formalin-Induced Fluorescence Reveals Cell Shape and Morphology in Biological Tissue Samples

Ultramicroscopy is a powerful tool to reveal detailed three-dimensional structures of large microscopical objects. Using high magnification, we observed that formalin induces fluorescence more in extra-cellular space and stains cellular structures negatively, rendering cells as dark objects in front of a bright background. Here, we show this effect on a three-dimensional image stack of a hippocampus sample, focusing on the CA1 region. This method, called FIF-Ultramicroscopy, allows for the three-dimensional observation of cellular structures in various tissue types without complicated staining techniques

Resolution of Ultramicroscopy and Field of View Analysis

In a recent publication we described a microscopical technique called Ultramicroscopy, combined with a histological procedure that makes biological samples transparent. With this combination we can gather three-dimensional image data of large biological samples. Here we present the theoretical analysis of the z-resolution. By analyzing the cross-section of the illuminating sheet of light we derive resolution values according to the Rayleigh-criterion. Next we investigate the resolution adjacent to the focal point of the illumination beam, analyze throughout what extend the illumination beam is of acceptable sharpness and investigate the resolution improvements caused by the objective lens. Finally we conclude with a useful rule for the sampling rates. These findings are of practical importance for researchers working with Ultramicroscopy to decide on adequate sampling rates. They are also necessary to modify deconvolution techniques to gain further image improvements

6- and 8-Prenylnaringenin, Novel Natural Histone Deacetylase Inhibitors Found in Hops, Exert Antitumor Activity on Melanoma Cells

Background/Aims: Prenylnaringenins are natural prenylflavonoids with anticancer properties. However, the underlying mechanisms have not been elucidated yet. Here we report a novel mode of action of 6- and 8-prenylnaringenin (PN) on human melanoma cells: Inhibition of cellular histone deacetylases (HDACs). Methods: We performed in silico and in vitro analyses using 6-PN or 8-PN to study a possible interaction of 6-PN or 8-PN with HDAC as well as Western blot and FACS analyses, real-time cell proliferation and cell viability assays to assess the impact of 6-PN and 8-PN on human metastatic melanoma cells. Results: In silico, 6-PN and 8-PN fit into the binding pocket of HDAC2, 4, 7 and 8, binding to the zinc ion of their catalytic center that is essential for enzymatic activity. In vitro, 100 µmol/L of 6-PN or 8-PN inhibited all 11 conserved human HDAC of class I, II and IV. In clinical oncology HDAC inhibitors are currently investigated as new anticancer compounds. In line, treatment of SK-MEL-28 cells with 6-PN or 8-PN induced a hyperacetylation of histone complex H3 within 2 h. Further, 6-PN or 8-PN mediated a prominent, dose-dependent reduction of cellular proliferation and viability of SK-MEL-28 and BLM melanoma cells. This effect was apoptosis-independent and accompanied by down-regulation of mTOR-specific pS6 protein via pERK/pP90 in SK-MEL-28 cells. Conclusion: The identification of a broad inhibitory capacity of 6-PN and 8-PN for HDAC enzymes with antiproliferative effects on melanoma cells opens the perspective for clinical application as novel anti-melanoma drugs and the usage as innovative lead structures for chemical modification to enhance pharmacology or inhibitory activities

Light Sheet Microscopy for Single Molecule Tracking in Living Tissue

Single molecule observation in cells and tissue allows the analysis of physiological processes with molecular detail, but it still represents a major methodological challenge. Here we introduce a microscopic technique that combines light sheet optical sectioning microscopy and ultra sensitive high-speed imaging. By this approach it is possible to observe single fluorescent biomolecules in solution, living cells and even tissue with an unprecedented speed and signal-to-noise ratio deep within the sample. Thereby we could directly observe and track small and large tracer molecules in aqueous solution. Furthermore, we demonstrated the feasibility to visualize the dynamics of single tracer molecules and native messenger ribonucleoprotein particles (mRNPs) in salivary gland cell nuclei of Chironomus tentans larvae up to 200 µm within the specimen with an excellent signal quality. Thus single molecule light sheet based fluorescence microscopy allows analyzing molecular diffusion and interactions in complex biological systems

Ultra-schnelle Zweiphotonenmikroskopie für in vivo Gehirnuntersuchungen

Two-photon microscopy is an imaging technique for optical investigations in deep layers of the biological tissue (~0.5-1 mm), but the acquisition of full-frame images is relatively slow (~30 images/sec), because most microscopes use revolving mirrors for the scanning process. This work describes the development of a high-speed two-photon microscope (~1000 images/sec), using acousto-optical deflectors (AOD) as scanning devices. AODs induce several beam distortions, and this work investigates their compensation. A special focus is on beam distortions from the ‘cylindrical lens effect’, an artifact when AODs are used for highest line-scan frequencies. The newly designed microscope has a highly increased sensitivity, allowing for the detection of calcium-transients in individual spines in vivo and in vitro. This is demonstrated in measurements of an acute slice preparation of a Purkinje-cell, and in vivo in the auditory cortex.Zweiphotonenmikroskopie ist eine optische Technik um tiefe Schichten biologischen Gewebes abzubilden (~0.5-1mm Tiefe), aber die Aufnahme ganzer Bilder ist relativ langsam, hauptsächlich weil zum Scannen rotierende Spiegel benützt werden. Diese Arbeit beschreibt die Entwicklung eines zweiphotonen-Hochgeschwindigkeitsmikroskops (~1000 Bilder/sec), welches akusto-optische Deflektoren (AOD) als Scanner benützt. AODs erzeugen einige Strahlverformungen, und diese Arbeit untersucht deren Kompensation. Spezieller Fokus liegt dabei auf dem ‘Zylinderlinsen-Effekt’, der bei AODs bei hohen Linienscannfrequenzen auftritt. Das neu entwickelte Mikroskop hat eine deutlich erhöhte Empfindlichkeit, und das ermöglicht die Detektion von Kalziumsignalen in einzelnen dendritischen Spines in vitro und in vivo. Dies wird mit Messungen an Purkinje-Zellen in einem Hirnschnitt, und an einem in vivo Präparat des auditorischen Cortex gezeigt

The principle of illumination in Ultramicroscopy: Light (e.g. 488 nm Argon laser) is being focused quoin-like by a cylinder lens.

<p>In the area where the beam is focused maximally a quoin-like description is not appropriate and the profile of the illumination beam stays relatively constant. The sample is immersed in the same liquid used to gain transparency. This assures that the illumination beam is not diffracted on the surface of the sample and propagates straight, even inside the sample. By moving the sample, different z-positions can be illuminated and recorded afterwards. In this way we acquired three-dimensional tomographical data.</p

Modified setup for the measurement of the cross-section of the illumination beam.

<p>The illumination beam was focused towards the microscope and a fluorescent cover slip was placed in its way. By observing the fluorescence of the cover slip, the cross-sections of the illumination beam was measured. By moving the cylindrical lens, cross-sections of various positions from the focal point can be observed.</p

Focusing the illumination beam through a glass window into a different medium with a higher refractive index.

<p>The cross-section and thickness stay the same, but the illumination beam is stretched lengthwise by a factor equal to the new refractive index <i>n</i>₂ (deduction in the text).</p