Advanced Fluorescence Microscopy Techniques-FRAP, FLIP, FLAP, FRET and FLIM

Fluorescence microscopy provides an efficient and unique approach to study fixed and living cells because of its versatility, specificity, and high sensitivity. Fluorescence microscopes can both detect the fluorescence emitted from labeled molecules in biological samples as images or photometric data from which intensities and emission spectra can be deduced. By exploiting the characteristics of fluorescence, various techniques have been developed that enable the visualization and analysis of complex dynamic events in cells, organelles, and sub-organelle components within the biological specimen. The techniques described here are fluorescence recovery after photobleaching (FRAP), the related fluorescence loss in photobleaching (FLIP), fluorescence localization after photobleaching (FLAP), Forster or fluorescence resonance energy transfer (FRET) and the different ways how to measure FRET, such as acceptor bleaching, sensitized emission, polarization anisotropy, and fluorescence lifetime imaging microscopy (FLIM). First, a brief introduction into the mechanisms underlying fluorescence as a physical phenomenon and fluorescence, confocal, and multiphoton microscopy is given. Subsequently, these advanced microscopy techniques are introduced in more detail, with a description of how these techniques are performed, what needs to be considered, and what practical advantages they can bring to cell biological research

Correlated Multimodal Imaging in Life Sciences:Expanding the Biomedical Horizon

International audienceThe frontiers of bioimaging are currently being pushed toward the integration and correlation of several modalities to tackle biomedical research questions holistically and across multiple scales. Correlated Multimodal Imaging (CMI) gathers information about exactly the same specimen with two or more complementary modalities that-in combination-create a composite and complementary view of the sample (including insights into structure, function, dynamics and molecular composition). CMI allows to describe biomedical processes within their overall spatio-temporal context and gain a mechanistic understanding of cells, tissues, diseases or organisms by untangling their molecular mechanisms within their native environment. The two best-established CMI implementations for small animals and model organisms are hardware-fused platforms in preclinical imaging (Hybrid Imaging) and Correlated Light and Electron Microscopy (CLEM) in biological imaging. Although the merits of Preclinical Hybrid Imaging (PHI) and CLEM are well-established, both approaches would benefit from standardization of protocols, ontologies and data handling, and the development of optimized and advanced implementations. Specifically, CMI pipelines that aim at bridging preclinical and biological imaging beyond CLEM and PHI are rare but bear great potential to substantially advance both bioimaging and biomedical research. CMI faces three mai

Lensfree on-chip microscopy over a wide field-of-view using pixel super-resolution.

We demonstrate lensfree holographic microscopy on a chip to achieve approximately 0.6 microm spatial resolution corresponding to a numerical aperture of approximately 0.5 over a large field-of-view of approximately 24 mm2. By using partially coherent illumination from a large aperture (approximately 50 microm), we acquire lower resolution lensfree in-line holograms of the objects with unit fringe magnification. For each lensfree hologram, the pixel size at the sensor chip limits the spatial resolution of the reconstructed image. To circumvent this limitation, we implement a sub-pixel shifting based super-resolution algorithm to effectively recover much higher resolution digital holograms of the objects, permitting sub-micron spatial resolution to be achieved across the entire sensor chip active area, which is also equivalent to the imaging field-of-view (24 mm2) due to unit magnification. We demonstrate the success of this pixel super-resolution approach by imaging patterned transparent substrates, blood smear samples, as well as Caenoharbditis Elegans

Compressed sensing in fluorescence microscopy.

Compressed sensing (CS) is a signal processing approach that solves ill-posed inverse problems, from under-sampled data with respect to the Nyquist criterium. CS exploits sparsity constraints based on the knowledge of prior information, relative to the structure of the object in the spatial or other domains. It is commonly used in image and video compression as well as in scientific and medical applications, including computed tomography and magnetic resonance imaging. In the field of fluorescence microscopy, it has been demonstrated to be valuable for fast and high-resolution imaging, from single-molecule localization, super-resolution to light-sheet microscopy. Furthermore, CS has found remarkable applications in the field of mesoscopic imaging, facilitating the study of small animals' organs and entire organisms. This review article illustrates the working principles of CS, its implementations in optical imaging and discusses several relevant uses of CS in the field of fluorescence imaging from super-resolution microscopy to mesoscopy

The morphology of the ejecta in Supernova 1987A: a study over time and wavelength

We present a study of the morphology of the ejecta in Supernova 1987A based on images and spectra from the HST as well as integral field spectroscopy from VLT/SINFONI. The HST observations were obtained between 1994 - 2011 and primarily probe the outer hydrogen-rich zones of the ejecta. The SINFONI observations were obtained in 2005 and 2011 and instead probe the [Si I]/[Fe II] emission from the inner regions. We find a strong temporal evolution of the morphology in the HST images, from a roughly elliptical shape before ~5,000 days, to a more irregular, edge-brightened morphology thereafter. This transition is a natural consequence of the change in the dominant energy source powering the ejecta, from radioactive decay before ~5,000 days to X-ray input from the circumstellar interaction thereafter. The [Si I]/[Fe II] images display a more uniform morphology, which may be due to a remaining significant contribution from radioactivity in the inner ejecta and the higher abundance of these elements in the core. Both the H-alpha and the [Si I]/[Fe II] line profiles show that the ejecta are distributed fairly close to the plane of the inner circumstellar ring, which is assumed to define the rotational axis of the progenitor. The H-alpha emission extends to higher velocities than [Si I]/[Fe II] as expected. There is no clear symmetry axis for all the emission and we are unable to model the ejecta distribution with a simple ellipsoid model with a uniform distribution of dust. Instead, we find that the emission is concentrated to clumps and that the emission is distributed somewhat closer to the ring in the north than in the south. This north-south asymmetry may be partially explained by dust absorption. We compare our results with explosion models and find some qualitative agreement, but note that the observations show a higher degree of large-scale asymmetry.Comment: Accepted for publication in Ap