Illumination systems microscopy

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. 

Figure Bl.18.4. The most frequently used illumination system in bright-field microscopy.

Schott (www.schott.com) currently supply this type of illumination system for microscopy. 

Vesicles were observed by phase contrast microscopy using a Zeiss Axiovert 135 TV microscope equipped with a Plan Neofluar objective (40 x Ph2 nA = 0.75). The epi-illumination system of the microscope (100 W mercury arc lamp) was used as a light source to initiate the photochemical reaction in the sample. Images were recorded by a Hamamatsu C5985 CCD camera connected to the microscope, a videorecorder, and a workstation. 

One of the most critical aspects of observation using any optical microscope is the specimen illumination. Two illumination systems are commonly used in optical microscopy transmitted light and reflected light (Fig. 2). Transmitted, also called diascopic illumination, requires the specimen to be transparent. It is used primarily to examine thin sections of biological or material samples. Reflected light, or episcopic illumination (epi-illumination), is most commonly used for fluorescence microscopy, where fluorphores inside the specimen are excited to produce fluorescent light. The fluorescence is then emitted, or reflected back to the objective and collected by the detector (eyes, or camera). The reflected light is also used to study the surfaces of opaque specimens, which is the focus of this session. 

The gels contain inhomogeneities with a characteristic size of about 1 um Under favorable circumstances, this structure can be visualized in the wet gel by epi-illumination microscopy. When it can be visualized, the structure has a "spongy appearance, consistent with what one might expect of a system that has undergone spinodal decomposition. So far, however, there is no concrete evidence to support this mechanism of phase separation. 

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. 

Wet samples can be analyzed without a previous preparation by the so-called environmental scanning electron microscopy (ESEM). In this technique, instead of the vacuum conditions, the sample chamber is kept in a modest gas pressure (Bache and Donald, 1998). The upper part of the column (illumination source) is kept in high vacuum conditions. A system of differential pumps allows to create a pressure gradient through the column (Bache and Donald, 1998 Stokes and Donald, 2000). The choice of the gas depends on the kind of food hydrated food is kept under water vapor. 

In TERS microscopy and spectroscopy, the tip enhancement due to the SPP resonance plays the most essential role both for signal sensitivity and spatial resolution. However, the tip-enhancement effect is not the only one affecting Raman spectra. There coexist other interaction mechanisms between a metal tip and sample molecules, chemical interactions similar to SERS [120-122], and, in addition, mechanical interactions (see Sect. 5.4.1). The latter two interactions show up only when sample molecules are in a close vicinity of a tip. In the TERS system using a ccaitact mode AFM, an experimentally observed TERS spectrum is a complex combination of the contributions of these three interactions, which makes it difficult to interpret experimental TERS spectra. Therefore, elucidation and discrimination of the tip-sample interactions are of scientific and practical importance. This can be realized by measuring a tip-sample distance dependence of TERS, since those three interaction mechanisms have different dependencies on the tip-sample distance. The active control of the distance between the tip and sample is a unique feamre only possible in TERS not in SERS. Two system configurations, time-gated detection and timegated illumination, are described below. 

The second model is usually known as the hydrone-phrotic rat kidney model, and was developed for in vivo visualization of the microcirculation [275] and involves 60 min renal artery occlusion combined with 3 weeks of ligation of the ureter. Atrophy of tubular structures leaves the cortical vasculature relatively intact and visualizable using planar microscopy in an illuminated observation chamber with nerve and blood supply left intact. Absolute and relative changes in lumen diameter of the major resistance vessels-inter-lobular arteries, afferent and efferent arterioles can be monitored in reponse to vasoactive stimuli. This model was adapted for in vitro perfusion by Loutzenhiser et al [276], removing systemic neurohumoral influences. 

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