Fluorescence microscopy equipment

Fluorescence microscopy measurements were performed with a Zeiss Axioplan microscope (Zeiss Co. Germany) equipped with a mercury lamp and a 40X objective. Images were acquired by CCD camera CH250 (Photometrix Co., Germany) cooled at -40°C by a liquid cooling unit CH260 (Photometrix Co., Germany). 

Noise can be also introduced by biochemical heterogeneity of the specimen. This can be a major cause of uncertainty in biological imaging. The high (three-dimensional) spatial resolution of fluorescence microscopy results in low numbers of fluorophores in the detection volume. In a typical biological sample, the number of fluorophores in the detection volume can be as low as 2-3 fluorophores for a confocal microscope equipped with a high NA objective at a fluorescent dye concentration of 100 nM. This introduces another source of noise for imaging applications, chemical or molecular noise, related to the inherent randomness of diffusion and the interaction of molecules. 

Wash each coverslip twice in 0.1% saponin-PBS, once in PBS, and finally once in water to remove salts. Drain all liquid onto a Kimwipe and mount coverslips on microscopy slides by inverting them (cells facing down) on 10-pL drops of Mowiol mounting medium. Allow at least 2 h of polymerization before viewing samples on a confocal fluorescence microscope equipped with 488-, 568-, and 643-nm laser lines. 

Figure 9 Formation of giant vesicles by fusion of LUVs equipped with an amphiphilic dipyridine ligand and filled with rhodamine sulfonate (50 xM) in presence of NiCL (0.1 xM) observed by fluorescence microscopy. The time between the first panel (upper left) and the last one (lower right) was 7 s. Scale bar 10 pm. (Reproduced with permission from Ref. 61. National Academy of Sciences, 2004.)...

Antibodies provide a powerful tool to localize antigens in cells or tissues by immunocytochemistiy at the light or electron microscope level. The development of efficient fluorescent dyes which can be coupled to antibodies for their visualization by fluorescence microscopy has pioneered a technology which is known as immunofluorescence microscopy (IFM). IFM is easy to apply to many biological and medical questions, the protocols involved are short, and the development of sophisticated imaging equipment has even made possible the acquisition of quantitative data on the 3D distribution of several antigens in the same specimen. 

In the absence of commercially available simple equipment for TRL microscopy, authors have modified existing instruments. For instance, the group of T. Nagano fitted an Olympus fluorescence microscope equipped with a cooled CCD camera with a 60-W xenon flash lamp and an image intensifier unit located in the emission pathway. The controller of the image intensifier provided the delay (52 or 70 ps) and acquisition (808 ps) times (Fig. 4.7). Time-resolved dual-colour images of cells were obtained by simultaneous use of two LLBs (Eu and Tb ) and changes in intracellular Zn could be monitored with a Eu chemosensor [53]. 

The first two chapters of this work cover theoretical and practical aspects of the emission process, the spectroscopic techniques and the equipment used to characterize the emission. Chapter 3 introduces and reviews the property of circularly polarized emission, while Chapter 4 reviews the use of lanthanide ion complexes in bioimaging and fluorescence microscopy. Chapter 5 covers the phenomenon of two-photon absorption, its theory as well as applications in imaging, while Chapter 6 reviews the use of lanthanide ions as chemo-sensors. Chapter 7 introduces the basic principles of nanoparticle upconversion luminescence and its use for bioimaging and Chapter 8 reviews direct excitation of the lanthanide ions and the use of the excitation spectra to probe the metal ion s coordination environment in eoordination compounds and biopolymers. Finally, Chapter 9 describes the formation of heterobimetallic complexes, in whieh the lanthanide ion emission is promoted through the hetero-metal. 

Electron Beam Techniques. One of the most powerful tools in VLSI technology is the scanning electron microscope (sem) (see Microscopy). A sem is typically used in three modes secondary electron detection, back-scattered electron detection, and x-ray fluorescence (xrf). AH three techniques can be used for nondestmctive analysis of a VLSI wafer, where the sample does not have to be destroyed for sample preparation or by analysis, if the sem is equipped to accept large wafer-sized samples and the electron beam is used at low (ca 1 keV) energy to preserve the functional integrity of the circuitry. Samples that do not diffuse the charge produced by the electron beam, such as insulators, require special sample preparation. 

The introduction and diversification of genetically encoded fluorescent proteins (FPs) [1] and the expansion of available biological fluorophores have propelled biomedical fluorescent imaging forward into new era of development [2], Particular excitement surrounds the advances in microscopy, for example, inexpensive time-correlated single photon counting (TCSPC) cards for desktop computers that do away with the need for expensive and complex racks of equipment and compact infrared femtosecond pulse length semiconductor lasers, like the Mai Tai, mode locked titanium sapphire laser from Spectra physics, or the similar Chameleon manufactured by Coherent, Inc., that enable multiphoton excitation. 

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