Modern bio-imaging techniques such as light-sheet and PALM/STORM are now aiming to image more complex biological samples at larger depth and therefore face larger-amplitude and more complex aberrations. Here we will present our newly-developed deformable mirror which addresses these challenges. It is a continuous-membrane deformable mirror with 91 centro-symmetrically-arranged electromagnetic actuators, which is much more efficient for correction of both low and high-order aberrations compared to square layout, used in the past. Mirror has 99.7% linearity and almost no hysteresis, which ensures exceptional precision and speed in closed-loop mode as well as accuracy and unrivaled temporal stability in open-loop mode.
PSF engineering has been widely used over the past years to enable 3D localization in Single Molecule Localization Microscopy (SMLM) techniques, such as PALM/STORM superresolution methods. It can make use of a cylindrical lens, a phase mask, a Spatial Light Modulator, a Deformable Mirror (DM) to encode depth on a spatial variation of the PSF along the Z axis. Among these techniques, we demonstrate that a DM-based approach – such as implemented in our MicAO-3DSR system - provides the best versatility/performance combination
The determination of 3-dimensional arrangement of subcellular assemblies has become a necessary requirement in cellular biology. Unfortunately, the size of most assemblies lies beyond the diffraction limit and therefore they cannot be visualized using conventional fluorescence microscopy techniques. Photoactivation localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) enable the localization of fluorescent molecules with nanometric resolution. We used these microscopy methods together with MicAO 3D-SR – the first adaptive optics device which introduces the three-dimensional imaging capability for PALM/STORM. MicAO also corrects various types of aberrations induced by optical elements inside the microscope and by the biological sample. The correction of these aberrations almost doubles the number of detected photons, increasing the localization precision of PALM/STORM by 40%. At 1000 detected photons the localization precision of our setup is 8 nm in lateral and 16 nm in axial directions. The separate optimization performed for two different colors delivers superb imaging quality, as demonstrated by dual-color 3-dimentional imaging of two centrosomal proteins in HeLa cells.
KEYWORDS: Dendrites, Spine, Neurons, In vivo imaging, Fluorescent proteins, Absorption, Two photon excitation microscopy, Two photon imaging, Green fluorescent protein, Brain
Two-photon microscopy has been used to perform high spatial resolution imaging of spine plasticity in the intact
neocortex of living mice. Multi-photon absorption has also been used as a tool for the selective disruption of cellular
structures in living cells and simple organisms. In this work we exploit the spatial localization of multi-photon excitation
to perform selective lesions on the neuronal processes of cortical neurons in living mice expressing fluorescent proteins.
This methodology was applied to dissect single dendrites with sub-micrometric precision without causing any visible
collateral damage to the surrounding neuronal structures. The spatial precision of this method was demonstrated by
ablating individual dendritic spines, while sparing the adjacent spines and the structural integrity of the dendrite. The
morphological consequences were then characterized with time lapse 3D two-photon imaging over a period of minutes to
days after the procedure. Here we present the results of our systematic study of the morphological response of cortical
pyramidal neurons to nanosurgical perturbations. Dendritic branches were followed after transecting distal segments,
whilst the plasticity and remodeling of individual dendritic spines on a given branch was also followed after removing of
a subset of spines.
KEYWORDS: Dendrites, Spine, Neurons, In vivo imaging, Fluorescent proteins, Two photon excitation microscopy, Absorption, Two photon imaging, Neuroscience, Multiphoton processes
Two-photon microscopy has been used to perform high spatial resolution imaging of spine plasticity in the intact neocortex of living mice. Multiphoton absorption has also been used as a tool for the selective disruption of cellular structures in living cells and simple organisms. In this work, we exploit the spatial localization of multiphoton excitation to perform selective lesions on the neuronal processes of cortical neurons in living mice expressing fluorescent proteins. Neurons are irradiated with a focused, controlled dose of femtosecond laser energy delivered through cranial optical windows. The morphological consequences are then characterized with time lapse 3-D two-photon imaging over a period of minutes to days after the procedure. This methodology is applied to dissect single dendrites with submicrometric precision without causing any visible collateral damage to the surrounding neuronal structures. The spatial precision of this method is demonstrated by ablating individual dendritic spines, while sparing the adjacent spines and the structural integrity of the dendrite. The combination of multiphoton nanosurgery and in vivo imaging in mammals represents a promising tool for neurobiology and neuropharmacology research.
The interaction of haematoporphyrin (Hp), dimethoxyhematoporphyrin IX (DMHp), meso-tetraphenylporphine tetrasulphonate (TPPS4), photofrin II (PF), chlorin e6 (Cle6) and aluminum phthalocyanine tetrasulphonate (AlPcS4) with human serum albumin (HSA) in aqueous solution and the influence of complexation on the photostability of sensitizers have been investigated by means of steady state absorption spectroscopy. By using the binding isotherm method and graphical Scatchard plot the sequence of affinity of sensitizers to HSA was obtained: TPPS4 greater than DMHp, Cle6, Hp, AlPcS4 greater than PF. The influence of aggregation on the binding ability of sensitizers is discussed. It was observed that complexation with HSA decreases the photostability of sensitizers. The increased rates of sensitizer photobleaching in the presence of photo-oxidizable substrates can be explained by the changes of photophysical properties of sensitizers caused by the interaction with substrates or by the attack of the biomolecules photo- oxidized by the type I mechanism involving electron transfer from excited porphyrin molecule or by the type II mechanism via singlet oxygen on porphyrin microcycle. The sequence of photostability of sensitizers in the presence and in the absence of HSA was established.
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