Widefield imaging

Fig. 3 Representative example of the microtubule network in a proximal branch of a DIV4 hippocampal primary neuron stained with a primary antibody against a-tubulin and visualized via a secondary antibody labeled with Alexa Fluor 647. (a) Widefield overview, (b) Super-resolved SMLM image. Enlarged view of the area in the white teshown in lower panel, (c) Intensity profile of the cross section marked by the white line in (b). Dashed line corresponds to the widefield image, continuous line indicates the profile of the super-resolved image. Note that individual microtubules can only be distinguished in the reconstructed SMLM image...

Figure 12. Metaphase cell hybridized with FISH probes, (a) Widefield fluorescence microscopy without deconvolution and (b) Widefield fluorescence microscopy with deconvolution. The chromosomes are seen in blue. The chromosome that is labeled with FISH probes shows green and red spots which are sharper in the deconvolved image (right) compared to the image without deconvolution (left). Image courtesy, Peter Franklin, Applied Precision Inc., Issaquah, WA, USA.

Figure 11. Analysis of FISH signals using a widefield deconvolution microscope. Seen in this image are xy (top), xz (bottom), and yz (right) projections of deconvolved data. The colocalized FISH signals (yellow) are easily seen in XY and XZ images. Image courtesy. Peter Franklin. Applied Precision Inc, Issaquah, WA, USA.

M.G. Gustafsson, D.A. Agard, J.W. Sedat. 3D widefield microscopy with two objective lenses experimental verification of improved axial resolution, in Three-Dimensional Microscopy Image Acquisition and Processing 111, Proceedings of SPIE, 1996... 

Fig. 1. Wound healing and inflammation in Drosophila embryos, (a) A laser ablation wound to a moesinGFP expressing Drosophila embryo will, by a purse-string mechanism, (b) seal shut over a period of a few hours, (c) This same laser wound will also recmit hemo-cytes that, by driving GFP specifically in these cells, can be imaged by widefield microscopy. (d) Alternatively, the wound site can be imaged by Differential Interference Contrast microscopy to give a general idea of wound size (dotted line) and hemocyte presence ( ). (e) The insertion of a heparin bead into the wound site will also lead to the recruitment of hemocytes that will surround and encapsulate the bead, (f) A hemocyte undergoing developmental dispersal viewed by confocal microscopy.

For basic quantification, imaging can be carried out on a widefield microscope, but for better resolution and accurate quantification we recommend imaging using a confocal microscope as outlined in Subheading 3.4.1. 

Fig. 3 A schematic shows a white-light microsphere nanoscope (a microsphere superlens integrated with a classical widefield optical microscope) with A/8 imaging resolution. The spheres collect the near-field object information and form virtual images that are then...

By setting properly all the aforementioned parameters, a proper imaging of the sample will be obtained. At those conditions, lateral or xjy resolution (minimum spacing that can just be resolved) is about 1.4 times better than in widefield fluorescence microscopy (180-200 run are typical values), while the improvement in viewing axis or z resolution at optimal conditions is about 3.0 times poorer than the lateral resolution (500-800 nm). This represents a marked improvement compared with conventional microscopy, which arises from the rejection of fluorescence light from out-of-focus regions of the specimen. 

Spectral imaging has been implemented in several ways on widefield or con-focal microscopes. Some of the solutions require specific instrumentation, but the method is also very generally applicable since any multi-channel fluorescence image can be considered as a series of spectral images. On a standard widefield fluorescence microscope, spectral separation of overlapping fluorophores was shown to be improved by determining and correcting for the crossover of individual fluorophores into different filter sets [15,16]. Fourier... 

Excitation-based unmixing is being used in commercially available setups for widefield as well as for two-photon imaging. In contrast to the parallel detection possibility for emission unmixing, excitation unmixing is unavoidably based on sequentially acquired data. 

Widefield FT-IR 2D and 3D Imaging at the Microscale Using Synchrotron Radiation... 

The capabiUties afforded by IRENI, namely high SNR, widefield illumination, and spatial oversampling, have lead to significant advances in characterization of IR microscope optics and imaging performance. Here, the spatial resolution and wavelength-dependent point spread functions (PSFs) for a 74x objective (NA = 0.65) are discussed. 

These approaches have led to advances in 2D projection imaging. Results presented from polymers, biological, and inorganic materials imaging exemplify the power of the synchrotron source for widefield spectromicroscopy applied to a variety of disciplines. In addition to the impressive spatial resolution achieved using the multibeam FPA illumination, the unparalleled SNR provided by the synchrotron enables time-resolved studies of systems ranging from live-cell chemical imaging to reactions at surfaces. 

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