Stain-free enucleation of mouse and human oocytes with a 1033 nm femtosecond laser.

Significance: Preparation of a recipient cytoplast by oocyte enucleation is an essential task for animal cloning and assisted reproductive technologies in humans. The femtosecond laser is a precise and low-invasive tool for oocyte enucleation, and it should be an appropriate alternative to traditional enucleation by a microneedle aspiration. However, until recently, the laser enucleation was performed only with applying a fluorescent dye. Aim: This work is aimed to (1) achieve femtosecond laser oocyte enucleation without applying a fluorescent dye and (2) to study the effect of laser destruction of chromosomes on the structure and dynamics of the spindle. Approach: We applied polarized light microscopy for spindle visualization and performed stain-free mouse and human oocyte enucleation with a 1033 nm femtosecond laser. Also, we studied transformation of a spindle after metaphase plate elimination by a confocal microscopy. Results: We demonstrated a fundamental possibility of inactivating the metaphase plate in mouse and human oocytes by 1033 nm femtosecond laser radiation without applying a fluorescent dye. Irradiation of the spindle area, visualized by polarized light microscopy, resulted in partly or complete metaphase plate destruction but avoided the microtubules impairment. After the metaphase plate elimination, the spindle reorganized, however, it was not a complete depolymerization. Conclusions: This method of recipient cytoplast preparation is expected to be useful for animal cloning and assisted reproductive technologies.

Understanding the Mechanism of Light-Induced Age-Related Decrease in Melanin Concentration in Retinal Pigment Epithelium Cells.

It is known that during the process of aging, there is a significant decrease in the number of melanosomes in the retinal pigment epithelium (RPE) cells in the human eye. Melanosomes act as screening pigments in RPE cells and are fundamentally important for protection against the free radicals generated by light. A loss or change in the quality of melanin in melanosomes can lead to the development of senile pathologies and aggravation in the development of various retinal diseases. We have previously shown that the interaction between melanin melanosomes and superoxide radicals results in oxidative degradation with the formation of water-soluble fluorescent products. In the present study, we show, using fluorescence analysis, HPLC, and mass spectrometry, that visible light irradiation on melanolipofuscin granules isolated from RPE cells in the human eye results in the formation of water-soluble fluorescent products from oxidative degradation of melanin, which was in contrast to lipofuscin granules and melanosomes irradiation. The formation of these products occurs as a result of the oxidative degradation of melanin by superoxide radicals, which are generated by the lipofuscin part of the melanolipofuscin granule. We identified these products both in the composition of melanolipofuscin granules irradiated with visible light and in the composition of melanosomes that were not irradiated but were, instead, oxidized by superoxide radicals. In the melanolipofuscin granules irradiated by visible light, ions that could be associated with melanin oxidative degradation products were identified by applying the principal component analysis of the time-of-flight secondary ion mass spectrometry (ToF-SIMS) data. Degradation of the intact melanosomes by visible light is also possible; however, this requires significantly higher irradiation intensities than for melanolipofuscin granules. It is concluded that the decrease in the concentration of melanin in RPE cells in the human eye with age is due to its oxidative degradation by reactive oxygen species generated by lipofuscin, as part of the melanolipofuscin granules, under the action of light.

Formation of a supramolecular charge-transfer complex. Ultrafast excited state dynamics and quantum-chemical calculations.

The formation of a supramolecular complex of bis(18-crown-6)stilbene (1) and 4,4'-bipyridine with two ammoniopropyl N-substituents (3) and the substitution reaction between 1·3 and alkali and alkaline-earth metal perchlorates have been studied using absorption, steady-state fluorescence, and femtosecond transient absorption spectroscopy. The formation of 1·(Mn+)2 complexes in acetonitrile was demonstrated. The weak long-wavelength charge-transfer absorption band of 1·3 completely vanishes upon complexation with metal cations because of disruption of the pseudocyclic structure. The spectroscopic and luminescence parameters, stability and substitution constants were calculated. The relaxation scheme of the 1·3 singlet state excited by a 25 fs laser pulse was proposed. It includes very fast vibrational relaxation and direct (τCT-d = 0.32 ps) and back (τCT-b = 0.51 ps) electron transfer resulting in complete fluorescence quenching. The quantum-chemistry calculations revealed the species taking part in the ET process and elucidated the mechanism of relaxation of the excited complex.

Femtosecond laser surgery of two-cell mouse embryos: effect on viability, development, and tetraploidization.

The effect of the laser pulse energy and total expose of the energy incident on the embryo blastomere fusion probability was investigated. The probability of the four different events after laser pulse was determined: the fusion of two blastomeres with the following formation of tetraploid embryo, the destruction of the first blastomere occurs, the second blastomere conservation remains intact, the destruction and the death of both cells; two blastomeres were not fused, and no morphological changes occurred. We report on viability and quality of the embryo after laser surgery as a function of the laser energy incident. To characterize embryo quality, the probability of the blastocyst stage achievement was estimated and the blastocyst cells number was calculated. Blastocoel formation is the only event of morphogenesis in the preimplantation development of mammals, so we assumed it as an indicator of the time of embryonic "clocks" and observed it among fused and control embryos. The blastocoel formation time is the same for fused and control embryos. It indicates that embryo clocks were not affected due to blastomere fusion. Thus, the analysis of the fluorescence microscopic images of nuclei in the fused embryo revealed that nuclei fusion does not occur after blastomere fusion.

Complexation of Donor-Acceptor Substituted Aza-Crowns with Alkali and Alkaline Earth Metal Cations. Charge Transfer and Recoordination in Excited State.

Complexation between two aza-15-crown-5 ethers bearing electron donor and acceptor fragments and alkali and alkaline earth perchlorates has been studied using absorption, steady-state fluorescence and femtosecond transient absorption spectroscopy. The spectral-luminescent parameters, the stability and dissociation constants of the complexes were calculated. The intramolecular charge transfer reaction takes place both in the excited state of the crowns and their complexes 1:1; the latter is subjected to photorecoordination resulting in a weakening or a complete disruption of coordination bond between nitrogen atom and metal cation, disposed within a cavity of the crown. The compounds investigated can be viewed as novel optical molecular sensors for alkali and alkaline-earth metal cations. The photoejection of a metal cation into the bulk was not observed.

Passive mode locking in a Ti:sapphire laser using a single-walled carbon nanotube saturable absorber at a wavelength of 810 nm.

We report mode locking in a Ti:sapphire (Ti:Sa) laser at the wavelength of 810 nm using a polymer film with single-walled carbon nanotubes (SWNTs) applied as a saturable absorber. Pulses with 600 fs duration and 0.4 nJ energy were generated from the Ti:Sa laser with polymer-SWNT composite film for cw passive mode locking.

Nonlinear optical absorption of single-wall carbon nanotubes in carboxymethylcellulose thin polymerfilm and its application as a saturable absorber for mode-locking in pulsed Nd:glass laser.

Nonlinear optical absorption of single-wall carbon nanotubes in carboxymethylcellulose (CMC) thin polymer film has been studied by the Z-scan method. Nonlinear saturated absorption at lambda=1080 nm was registered with saturation intensity of 170 MW/cm(2). Ultrashort pulses with duration of 3 ps were generated at lambda=1055 nm using this composite polymer film as a saturable absorber for passive mode locking in a neodymium glass laser.