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October 2012

Volume 17, Issue 10 (partial)

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Nanosurgery of cells and chromosomes using near-infrared twelve-femtosecond laser pulses

Aisada Uchugonova, Matthias Lessel, Sander Nietzsche, Christian Zeitz, Karin Jacobs, Cornelius Lemke, and Karsten König

J. Biomed. Opt. 17, 101502 (May 16, 2012); http://dx.doi.org/10.1117/1.JBO.17.10.101502

Online Publication Date: May 16, 2012

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Laser-assisted surgery based on multiphoton absorption of near-infrared laser light has great potential for high precision surgery at various depths within the cells and tissues. Clinical applications include refractive surgery (fs-LASIK). The non-contact laser method also supports contamination-free cell nanosurgery. In this paper we describe usage of an ultrashort femtosecond laser scanning microscope for sub-100 nm surgery of human cells and metaphase chromosomes. A mode-locked 85 MHz Ti:Sapphire laser with an M-shaped ultrabroad band spectrum (maxima: 770  nm/830  nm) and an in situ pulse duration at the target ranging from 12 fs up to 3 ps was employed. The effects of laser nanoprocessing in cells and chromosomes have been quantified by atomic force microscopy. These studies demonstrate the potential of extreme ultrashort femtosecond laser pulses at low mean milliwatt powers for sub-100 nm surgery of cells and cellular organelles.

Metabolism of HeLa cells revealed through autofluorescence lifetime upon infection with enterohemorrhagic Escherichia coli

Tatyana Yu. Buryakina, Pin-Tzu Su, Wan-Jr Syu, C. Allen Chang, Hsiu-Fang Fan, and Fu-Jen Kao

J. Biomed. Opt. 17, 101503 (May 21, 2012); http://dx.doi.org/10.1117/1.JBO.17.10.101503

Online Publication Date: May 21, 2012

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Fluorescence lifetime imaging microscopy (FLIM) is a sensitive technique in monitoring functional and conformational states of nicotinamide adenine dinucleotide reduced (NADH) and flavin adenine dinucleotide (FAD),main compounds participating in oxidative phosphorylation in cells. In this study, we have applied FLIM to characterize the metabolic changes in HeLa cells upon bacterial infection and made comparison with the results from the cells treated with staurosporine (STS), a well-known apoptosis inducer. The evolving of NADH’s average autofluorescence lifetime during the 3 h after infection with enterohemorragic Escherichia coli (EHEC) or STS treatment has been observed. The ratio of the short and the long lifetime components’ relative contributions of NADH increases with time, a fact indicating cellular metabolic activity, such as a decrease of oxidative phosphorylation over the course of infection, while opposite dynamics is observed in FAD. Being associated with mitochondria, FAD lifetimes and redox ratio could indicate heterogeneous mitochondrial function, microenvironment with bacterial infection, and further pathway to cell death. The redox ratios for both EHEC-infected and STS-treated HeLa cells have been observed and these observations also indicate possible apoptosis induced by bacterial infection.

Analysis of the efficiency of hair removal by different optical methods: comparison of Trichoscan, reflectance confocal microscopy, and optical coherence tomography

Monika Kuck, Sabine Schanzer, Martina Ulrich, Natalie Garcia Bartels, Martina C. Meinke, Joachim Fluhr, Martin Krah, Ulrike Blume-Peytavi, Eggert Stockfleth, and Jürgen Lademann

J. Biomed. Opt. 17, 101504 (May 14, 2012); http://dx.doi.org/10.1117/1.JBO.17.10.101504

Online Publication Date: May 14, 2012

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Noninvasive diagnostic tools, such as Trichoscan®, reflectance confocal microscopy (RCM), and optical coherence tomography (OCT), are efficient methods of hair shaft and growth evaluation. The aim of this study was to carry out a comparative assessment of these three medical procedures by measuring the hair shaft and hair growth after hair removal for a defined period of five days. The application of these techniques was demonstrated by measuring hair growth on the lower leg of six female volunteers. After removal of the hair shaft with a shaving system, the hair follicle infundibula and the length of the growing hairs were measured with the Trichoscan®, RCM, and OCT method. All three methods are reliable hair measuring tools after hair removal. Trichoscan® is best suited in the implementation of hair growth measurement and RCM in the analysis of hair follicles, whereas the OCT system can be consulted as an additional measurement for the evaluation of the hair follicle and length.

Effect of ouabain on metabolic oxidative state in living cardiomyocytes evaluated by time-resolved spectroscopy of endogenous NAD(P)H fluorescence

Alzbeta Chorvatova, Fathia Elzwiei, Anton Mateasik, and Dusan Chorvat, Jr.

J. Biomed. Opt. 17, 101505 (May 14, 2012); http://dx.doi.org/10.1117/1.JBO.17.10.101505

Online Publication Date: May 14, 2012

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Time-resolved spectrometry of endogenous nicotinamide dinucleotide phosphate [NAD(P)H] fluorescence is a useful method to evaluate metabolic oxidative state in living cells. Ouabain is a well-known pharmaceutical drug used in the treatment of cardiovascular disease, the effects of which on myocardial metabolism were recently demonstrated. Mechanisms implicated in these actions are still poorly understood. We investigate the effect of ouabain on the metabolic oxidative state of living cardiac cells identified by time-resolved fluorescence spectroscopy of mitochondrial NAD(P)H. Spectral unmixing is used to resolve individual NAD(P)H fluorescence components. Ouabain decreased the integral intensity of NAD(P)H fluorescence, leading to a reduced component amplitudes ratio corresponding to a change in metabolic state. We also noted that lactate/pyruvate, affecting the cytosolic NADH gradient, increased the effect of ouabain on the component amplitudes ratio. Cell oxidation levels, evaluated as the percentage of oxidized NAD(P)H, decreased exponentially with rising concentrations of the cardiac glycoside. Ouabain also stimulated the mitochondrial NADH production. Our study sheds a new light on the role that ouabain plays in the regulation of metabolic state, and presents perspective on a noninvasive, pharmaceutical approach for testing the effect of drugs on the mitochondrial metabolism by means of time-resolved fluorescence spectroscopy in living cells.
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