Fluorescent vital staining of plant sexual cell nuclei with DNA-specific fluorochromes and its application in gametoplast fusion

DNA-binding fluorochromes are often used for vital staining of plant cell nuclei. However, it is not always sure whether the cells after staining still remain in living state. We chose several criteria to estimate the validity of real vital staining for sexual cell nuclei. These were: the cytoplasmic streaming in pollen tubes whose nuclei were stained, the simultaneous visualization of fluo-rochromatic reaction and nucleus staining in isolated generative cells, and the capability of isolated, prestained generative or sperm cells to fuse with other protoplasts. The results confirmed that 4',6-diamidino-2-phenylindole (DAPI), Hoechst 33258 and mithramycin could be used as real vital stains, though their efficiency varied from case to case; among them DAPI showed best effect. The fluorescent vital staining technique offered a useful means fori-dentification and selection of heterokaryons in gametoplast manipulation studies.

Fluorochrome-labeled monoclonal antibody with characteristic M-shaped spectral peak for optical imaging: Dual-labeling versus mixture of fluorochromes

In this work two different fluorochromes （Alexa 594 and Alexa 680） are conjugated to the same monoclonal antibody （Cetuximab） for obtaining a characteristic M-shaped dual-peak spectrum. Dual-labeling of Cetuximab by mixing both fluorochromes before the conjugation step gives spectral results similar to those of mixing of fluorochrome-labeled Cetuximab after the conjugation step （P 〉 0.05）. In conclusion, both methods may be used equivalently for producing a dual-labeled single-antibody probe. Future studies may test whether the M-shaped spectrum may increase the diagnostic confidence in tumor-targeted multispectral optical imaging.

Mineral Fabrication and Golgi Apparatus Activity in <i>Spirostomum ambiguum</i>: A Primordial Paradigm of the Stressed Bone Cell?

The histological basis for acute osteocyte mechanosensitivity remains uncertain. A novel bone cell model of mechanotransduction and inorganic trafficking may be the powerful, silt-burrowing protozoan Spirostomum ambiguum which when being physically challenged fabricates within vesicles populations of bone-like calcium phosphate microspheres, about 1 μm in diameter. These not only attribute considerable compression-resilience but also resemble the Golgi-directed mineral assemblies we recently reported in osteocytes. Advantageously, calcification in the protozoan (confirmed by ultramicroscopy with EDX elemental microanalysis) enabled Golgi comparison under overt, natural phases of both high (i.e. silt-tunnelling) and low (i.e free-swimming) stress. Established hard-tissue microscopy techniques previously positive in bone cells included quantitative fluorescent tetracycline labelling for bone salt together with the same metazoan Golgi body marker (Green Fluorescent Protein-tagged mannosidase II construct). Organellar modulation was monitored by transfection of live organisms in situ (some post-stained with red nuclear fluorochrome TOPRO-3). Results showed that GFP-tagged Golgi fluorescence increased from swimmers (mean 74.5 ± SD 6.7 AU) to burrowers (mean 104.6 ± SD 2.7;p < 0.0001) synchronous with juxtanuclear tetracycline-labelled mineral fluorescence (swimmers, mean 89.7 ± SD 3.3 AU;burrowers, mean 138.0 ± SD 4.0;p < 0.0001). Intracellular dense microspheres, single or bridged, were harvested as pellets rich in Ca, P (Ca:P 0.98) and Si, their polarised alignment moving from transaxial in swimmers to axial in burrowers. It was concluded that Golgi-directed mineral fabrication in the large, accessible, silt-enclosed ciliate resembles that in the smaller, less-accessible bone cell and may be a conserved early mechanobiological intracellular development predicating force translation into compression-resistant mineral fabrication in loaded segments of the osteocyte syncitium.

Mineral Fabrication and Golgi Apparatus Activity in the Mouse Calvarium

There is diverse opinion about the mechanism of bone mineralization with only intermittent reports of any direct organellar role played by the golgi apparatus (juxtanuclear body). Light and laser confocal microscopy was combined with electron microscopy and elemental EDX (energy dispersive X-ray microanalysis) in comparing “young” osteocytes in situ in fresh and “slam” frozen developing mouse calvarium, with similar cells (MC3T3-E1) maintained in vitro. The distribution of “nascent” electron dense mineral was examined histochemically (von Kossa, GBHA), including tetracycline (TC) staining as a fluorescent complex with bone salt, while golgi body activity was demonstrated by transfection with a specific green fluorescent construct (GFP/mannosidase II). In tissue culture golgi body activity and mineralization were both blocked by brefeldin A (an established golgi inhibitor) and restored by forskolin, enabling an association with mineral fabrication to be quantified as changing fluorescence intensity (AU) of GFP or TC markers. Results from osteocytes in situ supported previous descriptions of intracellular electron dense objects (microspheres and nanospheres) in a juxtanuclear pattern, containing Ca, P and transitory Si, in a spectrum recapitulated in the calcifying culture after 10 days, when GFP fluorophore surged from 71.7 ± 1.4SD to 133.7 ± 2.7SD AU by 14 days (p < 0.0001). At this stage TC fluorophore mean intensity was 23.8 ± 3.7SD AU (14 days) rising to 45.0 ± 5.1SD AU by 17 days, compared to its stationary 21.7 ± 3.6SD when treated 3 days previously with BFA golgi inhibitor (p < 0.0001), until forskolin reversal. It was concluded from the changing juxtanuclear morphology, Si mineralization mediation and the variably controlled activity versus stasis that the inorganic phase of bone is a complex golgi-directed fabrication with implications for bone matrix biology and evolution.

Comparison of Direct Microbial Count Procedures for Planktonics and Sessiles Enumeration

In the present investigation, the sensitivity of different direct microbial count procedures applied on systems containing both planktonics and sessiles was tested. The direct count pour plate was compared with direct epifluorescent microscopic enumerations in order to evaluate the efficiency of the studied techniques in giving information about microbial activity or viability. Our results indicate that the standard plate count procedure is the most sensitive method to estimate viable and cultivable planktonic cells. On the other hand, direct enumeration by epifluorescent microscopy may become an interesting alternative to count sessile cells.

Theoretical investigation of fluorescence changes caused by methanol bridge based on ESIPT reaction

The different fluorescence behavior caused by the excited state proton transfer in 3-hydroxy-4-pyridylisoquinoline(2a)compound has been theoretically investigated.Our calculation results illustrate that the 2a monomer in tetrahydrofuran solvent would not occur proton transfer spontaneously,while the 2a complex in methanol(MeOH)solvent can undergo an asynchronous excited state intramolecular proton transfer(ESIPT)process.The result was confirmed by analyzing the related structural parameters,infrared vibration spectrum and reduced density gradient isosurfaces.Moreover,the potential curves revealed that with the bridging of single MeOH molecular the energy barrier of ESIPT was modulated effectively.It was distinctly reduced to 4.80 kcal/mol in 2a-MeOH complex from 25.01 kcal/mol in 2a monomer.Accordingly,the ESIPT process induced a fluorochromic phenomenon with the assistant of proton-bridge.The elucidation of the mechanism of solvent discoloration will contribute to the design and synthesis of fluorogenic dyes as environment-sensitive probes.

The mechanodonor-acceptor coupling (MDAC) approach for unidirectional multi-state fluorochromism

Uni-directional multi-state fluorochromic scaffolds are valuable photofunctional molecules and yet scarce. We report a general approach for their design, i.e., mechanodonor-acceptor coupling(MDAC). A photochromic molecule is a mechanodonor, due to its capability to convert photonic energy into mechanical force. Upon proper coupling, it can be used to drive a mechanochromic molecule for uni-directional multi-state fluorochromism. The embodiment of this approach is a rhodamine-dithienylethylene hydride(RDH), which has been successfully employed in super-resolution localization microscopy.