Microscopy wide-field

In wide field microscopy, spatial information of the entire image is acquired simultaneously thus providing comparatively short acquisition times compared with scanning microscopy implementations. Combining TCSPC with wide field microscopy is not straightforward. However, a four quadrant anode multichannel plate (MCP) has been used for time- and space-correlated SPC experiments [25, 26]. This detector has excellent timing properties that make it very suitable for FLIM. Unfortunately, it can be operated only at low count-rates ( 105-106 Hz) therefore, it requires comparatively long acquisition times (minutes). 

Note that parameters ft and 5 depend on signal amplifications in the utilized detectors and on the elements in the optical path (optical filter, spectral detection bands) only, while a and y are additionally influenced by relative excitation intensity. This is usually a fixed constant in wide-field microscopy but in confocal imaging laser line intensities are adjusted independently. Furthermore, note that the a factor equals 5 multiplied by y (see Appendix for further detail). 

Keywords DNA/RNA transfection, Fluorescence wide-field microscopy, Gene carriers, Gene therapy, Single-particle tracking, Trajectory analysis... 

Fig. 27.1. Comparison between transmission electron microscopy (TEM) and single-molecule tracking (SMT) by optical wide-field microscopy, (a) High-resolution TEM gives the landscape of a channelar structure of a hexagonal mesoporous system and (b) SMT gives the trajectories of the movement of single molecules as guests in the nanoporous host...

Confocal Laser Scanning Microscopy (CLSM). Prior to the invention of confo-cal microscopy, conventional wide-field microscopy has a fundamental limitation for providing three-dimensional imaging due to blurring of the image caused by... 

Usually, in conventional wide-field microscopy the images from the focal plane and out-of-focus parts of the sample are superimposed. This problem can be overcome in different ways ... 

Optical sectioning in wide-field microscopy may be achieved using the method of structured illumination. For example, a diffraction grating can be imaged in the plane of the sample, and the intensity resulting from this plane can be described by... 

What is generally meant by wide-field microscopy is any microscope whereby image formation takes place by the optics without scanning the lens directly forms an image, which can be projected on a camera or observed through the eye piece. Before the development of confocal microscopy and other scanning microscopy modes, this was the only way to perform microscopy. In the biomedical sciences, wide-field fluorescence microscopy is still widely used and offers a number of advantages over confocal and other advanced microscopy modes. 

Keywords Deconvolution Wide-field microscopy Confocal microscopy ... 

In wide-field microscopy, lateral sampling is achieved by the CCD chip and is a function of the CCD pixel size Pj, camera binning b, and objective magnification M, as follows ... 

Turner JN, Szarowski DH, Tmner TJ et al (1994) Three-dimensional imaging and image analysis of hippocampal nemons con-focal and digitally enhanced wide field microscopy. Microsc Bm Tech 29 269-278... 

Swedlow JR, Platani M (2002) Live cell imaging using wide-field microscopy and deconvolution. Cell Struct Funct 27 335-341... 

Pichaandi J., J. C. Boyer, K. R. Delaney and F. C. J. M. van Veggel, Two-Photon Upconversion Laser (Scanning and Wide-Field) Microscopy Using Ln(3-l-)-Doped NaYF4 Upconverting Nanocrystals A Critical Evaluation of their Performance and Potential in Bioimaging, J. Phys. Chem. C, 115, 19054-19064 (2011). 

Within the wide-fleld methods, there are two different excitation schemes possible, namely epi-illumination and total internal reflection (TIR) illumination. The latter category has been subdivided into TIR-prism and TIR-through-objective illuminations. The three different excitation schemes within wide-field microscopy are shown below in Figure 6. 

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