Lars Klembt Andersen

Direct Optical Detection of Singlet Oxygen from a Single Cell

Abstract Singlet oxygen has been detected in single nerve cells by its weak 1270 nm phosphorescence (a1Δg→X3Σg−) upon irradiation of a photosensitizer incorporated in the cell. Thus, one can now consider the application of direct optical imaging techniques to mechanistic studies of singlet oxygen at the single-cell level.

The structure of the Ni–Fe site in the isolated HoxC subunit of the hydrogen‐sensing hydrogenase from Ralstonia eutropha

The regulatory Ni–Fe hydrogenase (RH) from Ralstonia eutropha which forms a [HoxBC]2 complex functions as a hydrogen sensor under aerobic conditions. We have studied a novel Strep‐tag isolate of the RH large subunit, HoxCST, which lacks the Fe–S clusters of HoxB, allowing for structure determination of the catalytic site by X‐ray absorption spectroscopy both at the Ni and, for the first time, also at the Fe K‐edge. This technique, together with Fourier‐transform infrared spectroscopy, revealed a Ni–Fe site with [O1(CysS)2NiII(μ‐SCys)2FeII(CN)2(CO)] structure in about 50% of HoxCST and a [(CysS)2FeII(CN)2(CO)] site lacking Ni in the remainder protein. Possibly both sites may be intermediates in the maturation process of the RH.

Time-resolved Detection of Singlet Oxygen in a Transmission Microscope¶

The time‐resolved absorption spectrum of singlet oxygen [O2(a1Δg) → O2(b1Σg+)] has been recorded in the region ∼5100–5300 cm−1 from air‐saturated polystyrene samples using a microscope attached to a step‐scan Fourier transform IR spectrometer. Singlet oxygen signals were observed with a time resolution of ∼160 ns from sample volumes of ∼20 nL using moderate data‐acquisition times. These data indicate that it is reasonable and worthwhile to consider the further development of a transmission microscope as a viable tool to create singlet oxygen images of inhomogeneous samples including samples of biological importance.

Spatial and temporal changes in the morphology of preosteoblastic cells seeded on microstructured tantalum surfaces

It has been widely reported that surface morphology on the micrometer scale affects cell function as well as cell shape. In this study, we have systematically compared the influence of 13 topographically micropatterned tantalum surfaces on the temporal development of morphology, including spreading, and length of preosteoblastic cells (MC3T3-E1). Cells were examined after 0.5, 1, 4, and 24 h on different Ta microstructures with vertical dimensions (heights) of 0.25 and 1.6 mum. Cell morphologies depended upon the underlying surface topography, and the length and spreading of cells varied as a function of time with regard to the two-dimensional pattern and vertical dimension of the structure. Microstructures of parallel grooves/ridges caused elongated cell growth after 1 and 4 h in comparison to a flat, nonstructured, reference surface. For microstructures consisting of pillars, cell spreading was found to depend on the distance between the pillars with one specific pillar structure exhibiting a decreased spreading combined with a radical change in morphology of the cells. Interestingly, this morphology on the particular pillar structure was associated with a markedly different distribution of the actin cytoskeleton. Our results provide a basis for further work toward topographical guiding of cell function.

A nanosecond near-infrared step-scan Fourier transform absorption spectrometer: Monitoring singlet oxygen, organic molecule triplet states, and associated thermal effects upon pulsed-laser irradiation of a photosensitizer

A step-scan Fourier transform (FT) near infrared (IR) spectrometer has been used to record nanosecond time-resolved absorption spectra of singlet molecular oxygen (a1Δg→b1Σg+) created by pulsed-laser irradiation of a photosensitizer dissolved in a solvent. In addition to oxygen’s a→b transition at ∼5200 cm−1, other laser-induced changes in absorbance can be monitored over the spectral range ∼4000–11 000 cm−1. These include transients derived from the singlet oxygen sensitizer, and examples are provided with triplet state absorption spectra of several molecules in benzene as well as water. In this report, issues pertinent to the use of a step-scan FT spectrometer to monitor weak laser-induced near-IR signals are discussed. Specifically, key modifications to a commercially available instrument are described, and the relevant sources of noise from electronic components are outlined. Transient laser-induced thermal effects such as thermal lenses and temperature-dependent shifts in solvent absorption bands can...

Rapid Communication: Direct Optical Detection of Singlet Oxygen from a Single Cell ¶

Singlet oxygen has been detected in single nerve cells by its weak 1270 nm phosphorescence (a1Δg→X3Σg−) upon irradiation of a photosensitizer incorporated in the cell. Thus, one can now consider the application of direct optical imaging techniques to mechanistic studies of singlet oxygen at the single‐cell level.