Illumination light microscopy

Figure 3.11. Light microscopy illumination for low magnification. The condenser lens is weak. Because a point on the image is illuminated by a restricted region of the filament, a diffuser (ground glass) is used to make the illumination more even.

Light microscopy allows, in comparison to other microscopic methods, quick, contact-free and non-destmctive access to the stmctures of materials, their surfaces and to dimensions and details of objects in the lateral size range down to about 0.2 pm. A variety of microscopes with different imaging and illumination systems has been constmcted and is conunercially available in order to satisfy special requirements. These include stereo, darkfield, polarization, phase contrast and fluorescence microscopes. 

Analysts use reflected light microscopy to examine the surface of polymers. By changing the angle of illumination they can accentuate surface texture and other features of interest. Reflected light microscopy is well suited to the examination of opaque and pigmented samples. Polymer scientists make extensive use of reflected light microscopy when examining the fracture surfaces of failed samples. 

The structure (e.g., number, size, distribution) of fat crystals is difficult to analyze by common microscopy techniques (i.e., electron, polarized light), due to their dense and interconnected microstructure. Images of the internal structures of lipid-based foods can only be obtained by special manipulation of the sample. However, formation of thin sections (polarized light microscopy) or fractured planes (electron microscopy) still typically does not provide adequate resolution of the crystalline phase. Confocal laserscanning microscopy (CLSM), which is based on the detection of fluorescence produced by a dye system when a sample is illuminated with a krypton/argon mixed-gas laser, overcomes these problems. Bulk specimens can be used with CLSM to obtain high-resolution images of lipid crystalline structure in intricate detail. 

Since cells are so small, visualizing them requires powerful microscopes. Most detailed pictures of cells are obtained by electron microscopy, in which electrons are used instead of light for illumination. 

Hildenbrand G, Rapp A, Spori U, Wagner C, Cremer C, Hausmann M (2005). Nano-Sizing of Specific Gene Domains in Intact Human Cell Nuclei by Spatially Modulated Illumination Light Microscopy. Biophysical Journal 88 4312-4318. 

Transmission electron microscopy (TEM) has been used extensively in biology for direct visualization of ultrastructural details and platinum deposits in cells. The underlying physics behind TEM is similar to that of ordinary light microscopy however, the resolutions achieved by TEM can be some 400-fold greater than that of light microscopy (38). Briefly, the mechanics behind TEM involves an illuminating source, the electron gun, that sends a beam of electrons through a vacuum and onto the... 

For overcoming the limit of light microscopy and further improvement in spatial resolution, the implementation of scaiuiing near-held microscopy (SNOM) by means of a local illumination probe is an interesting approach [33-35]. The method is based on the held enhancement in the cavity between a sharp metal dp and the sample. In combination with Raman spectroscopy, this scanning probe technique is called tip-enhanced Raman spectroscopy (TERS) and enables high-resolution spatial microscopy with a lateral resolution of 50 nm [35]. Bouhelier [36] has reviewed advances in this held. 

Several far-field light microscopy methods have recently been developed to break the diffraction limit. These methods can be largely divided into two categories (1) techniques that employ spatially patterned illumination to sharpen the point-spread function of the microscope, such as stimulated emission depletion (STED) microscopy and related methods using other reversibly saturable optically linear fluorescent transitions (RESOLFT) [1,2], and saturated structured-illumination microscopy (SSIM) [3], and (2) a technique that is based on the localization of individual fluorescent molecules, termed Stochastic Optical Reconstruction Microscopy (STORM [4], Photo-Activated Localization Microscopy (PALM) [5], or Fluorescence Photo-Activation Localization Microscopy (FPALM) [6]. In this paper, we describe the concept of STORM microscopy and recent advances in the imaging capabilities of STORM. 

Figure 1.43 Optical arrangement for fluorescence microscopy with epi-illumination. The dotted line indicates the path of excitation light, and the solid line indicates the path of fluorescent light. (Reproduced with permission from D.B. Murphy, Fundamentals of Light Microscopy and Electronic Imaging, Wiley -Liss. 2001 John Wiley Sons Inc.)...

Compare light microscopy, transmission electron and scanning electron microscopy in terms of optical arrangement, illumination source, working environment, imaging formation mechanism and specimen preparation discuss their similarities and differences. 

Then, microscopic examinations follow optical research microscopes allow to determine the number, thickness, and color sequence of layers in paint fragments, and to recognize the textures as well as fundamental features of pigment and extender mixtures. Bright field and dark field illuminations, polarized light microscopy (incident and transmitted), particularly the differential interference contrast (DIC) procedure, and fluorescence microscopy are necessary for paint examinations (see Figure 3(A)-3(E)). 

Microscopy AU morphological and marker screening utilizes simple light microscopy via a dissecting scope. We use Zeiss Stani SR microscopes. However, any microscope with the capability of translucent illumination and up to 5X objectives would be adequate. Translucent lighting is essential for clear optical inspection of structures of the embryo. 

Light Microscopy Earliest form of microscopy, also called optical microscopy, it uses light to illuminate small objects under a series of magnifying glass... 

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