Martin O. Lenz

Multiphoton multispectral fluorescence lifetime tomography for the evaluation of basal cell carcinomas.

We present the first detailed study using multispectral multiphoton fluorescence lifetime imaging to differentiate basal cell carcinoma cells (BCCs) from normal keratinocytes. Images were acquired from 19 freshly excised BCCs and 27 samples of normal skin (in & ex vivo). Features from fluorescence lifetime images were used to discriminate BCCs with a sensitivity/specificity of 79%/93% respectively. A mosaic of BCC fluorescence lifetime images covering >1 mm2 is also presented, demonstrating the potential for tumour margin delineation. Using 10,462 manually segmented cells from the image data, we quantify the cellular morphology and spectroscopic differences between BCCs and normal skin for the first time. Statistically significant increases were found in the fluorescence lifetimes of cells from BCCs in all spectral channels, ranging from 19.9% (425–515 nm spectral emission) to 39.8% (620–655 nm emission). A discriminant analysis based diagnostic algorithm allowed the fraction of cells classified as malignant to be calculated for each patient. This yielded a receiver operator characteristic area under the curve for the detection of BCC of 0.83. We have used both morphological and spectroscopic parameters to discriminate BCC from normal skin, and provide a comprehensive base for how this technique could be used for BCC assessment in clinical practice.

3-D stimulated emission depletion microscopy with programmable aberration correction

We present a stimulated emission depletion (STED) microscope that provides 3-D super resolution by simultaneous depletion using beams with both a helical phase profile for enhanced lateral resolution and an annular phase profile to enhance axial resolution. The 3-D depletion point spread function is realised using a single spatial light modulator that can also be programmed to compensate for aberrations in the microscope and the sample. We apply it to demonstrate the first 3-D super-resolved imaging of an immunological synapse between a Natural Killer cell and its target cell.

Photoisomerization in Proteorhodopsin Mutant D97N

The first steps of the photocycle of the D97N mutant of proteorhodopsin (PR) have been investigated by means of ultrafast transient absorption spectroscopy. A comparison with the primary dynamics of native PR and D85N mutant of bacteriorhodopsin is given. Upon photoexcitation of the covalently bound all‐trans retinal the excited state decays biexponentially with time constants of 1.4 and 20 ps via a conical intersection, resulting in a 13‐cis isomerized retinal. Neither of the two‐deactivation channels is significantly preferred. The dynamics is slowed down in comparison with native PR at pH 9 and reaction rates are even lower than for native PR at pH 6, where the primary proton acceptor (Asp97) is protonated. Therefore, the ultrafast isomerization is not only controlled by the charge distribution within the retinal binding pocket. This study shows that in addition to direct electrostatics other effects have to be taken into account to explain the catalytic function of Asp97 in PR on the ultrafast isomerization reaction. This may include sterical interactions and/or bound water molecules within the retinal binding pocket.

A STED-FLIM microscope applied to imaging the natural killer cell immune synapse

We present a stimulated emission depletion (STED) fluorescence lifetime imaging (FLIM) microscope, excited by a microstructured optical fibre supercontinuum source that is pumped by a femtosecond Ti:Sapphire-laser, which is also used for depletion. Implemented using a piezo-scanning stage on a laser scanning confocal fluorescence microscope system with FLIM realised using time correlated single photon counting (TCSPC), this provides convenient switching between confocal and STED-FLIM with spatial resolution down to below 60 nm. We will present our design considerations to make a robust instrument for biological applications including a comparison between fixed phase plate and spatial light modulator (SLM) approaches to shape the STED beam and the correlation of STED and confocal FLIM microscopy. Following our previous application of FLIM-FRET to study intercellular signalling at the immunological synapse (IS), we are employing STED microscopy to characterize the spatial distribution of cellular molecules with subdiffraction resolution at the IS. In particular, we are imaging cytoskeletal structure at the Natural Killer cell activated immune synapse. We will also present our progress towards multilabel STED microscopy to determine how relative spatial molecular organization, previously undetectable by conventional microscopy techniques, is important for NK cell cytotoxic function. Keywords: STED, Stimulated Emission Depletion Microscopy, Natural Killer (NK) cell, Fluorescence lifetime imaging, FLIM, Super resolution microscopy.

Primary Reaction of Sensory Rhodopsin II Mutant D75N and the Influence of Azide

The early steps in the photocycle of sensory rhodopsin II mutant D75N are investigated in a comprehensive study using femtosecond visible pump/probe spectroscopy. An overall slower response dynamics after photoexcitation is observed compared to wild-type sensory rhodopsin II, which is assigned to changed electrostatics and an altered hydrogen-bonding network within the retinal binding pocket. Furthermore, the influence of azide on the primary reaction is analyzed. The addition of azide accelerates the sub-10 ps dynamics of the D75N mutant nearly to reaction rates found in wild-type. Moreover, a further reaction pathway becomes observable in the investigated time range, which is assigned to a previously described K(1) to K(2) transition. The specific acceleration of the early steps seems to be a unique feature of the D75N mutant as similar azide effects do not emerge in analogous azide measurements of wild-type sensory rhodopsin II, bacteriorhodopsin, and the bacteriorhodopsin mutant D85N.

Primary Reaction of Sensory Rhodopsin II Mutant D75N

The primary reaction of the sensory rhodopsin II mutant D75N has been investigated using femtosecond transient absorption spectroscopy. A reaction mechanism taking into account all observations including the slower photoresponse has been worked out.