A novel approach for detecting nucleic acid in solution has been adopted for real-time imaging of native mRNAs in living cells. This method utilizes hybridization probes, called “molecular beacons,” that generate fluorescent signals only when they are hybridized to a complementary ta ...

Three-dimensional electron microscopy of single macromolecular assemblies has made large strides forward over the last decade. A large number of image processing techniques have been developed and many have found general distribution. For the proper usage of the wide range of availa ...

Molecules that have no center of symmetry are able to convert light to its second harmonic, at twice the frequency and half the wavelength. This only happens with any efficiency at very high light intensities such as are given by a pulsed laser, and because the efficiency of the process depends on the squa ...

A microbiopsy system was developed to overcome long sampling times for tissues before they are cryo-fixed by high-pressure freezing. A commercially available biopsy gun was adapted to the needs of small-organ excisions, and biopsy needles were modified to allow small samples (0.6 mm k 1.2 mm � 0.3 m ...

Calcium (Ca2+) is a fundamentally important component of cellular signal transduction. Dynamic changes in the concentration of Ca2+ () in the cytoplasm and within organelles are tightly controlled and regulate a diverse array of biological activities, including fertilization, ce ...

Lung tissue consists of more than 40 individual cell types that might interact to produce adverse pathologies. After injury, a number of signaling proteins expressed in various epithelial and other cell types have been linked to the advent of apoptosis, compensatory proliferation, and ad ...

The confocal laser scanning microscope (CLSM) has enormous potential in many biological fields. When tests are made to evaluate the performance of a CLSM, the usual subjective assessment is accomplished by using a histological test slide to create a “pretty picture.” Without the use of funct ...

Comparative quantitation has become an increasingly desirable tool in determining compositional differences of aortic plaque lesion in transgenically altered mice. To this end, methodology has been developed to identify lipid, cellularity, collagen, and elastin componen ...

Gene therapy has become an extremely important and active field of biomedical research. Microscopy is an integral component of this effort. This chapter presents an overview of imaging techniques used in our facility in support of cystic fibrosis gene therapy research. Instrumentati ...

The laser scanning cytometer (LSC) is the microscope-based cytofluorometer that offers a plethora of analytical capabilities. Multilaser-excited fluorescence emitted from individual cells is measured at several wavelength ranges, rapidly (up to 5000 cells/min), with high se ...

Biological samples from human tissues are characterized by complexity and heterogeneity. The ability to make rapid, reliable, quantitative fluorochromatic measurements on clinical samples allows the development of new and practical assays that could influence diagnosis a ...

High-pressure freeze fixation is the method of choice to arrest instantly all dynamic and physiological processes inside cells, tissues, and small organisms. Embedded in vitreous ice, such samples can be further processed by freeze substitution or directly analyzed in their fully hyd ...

In this chapter we discuss the latest developments in the field of high-pressure freezing (HPF). The Leica HPF machine EM HPM100 is discussed in detail due to significant changes compared to its predecessor model. Its centerpiece is a multipart polymer cartridge which holds the specimen carr ...

Freeze fracture depends on the property of frozen tissues or cells, when cracked open, to split along the hydrophobic interior of membranes, thus revealing broad panoramas of membrane interior. These large panoramas reveal the three-dimensional contours of membranes making the meth ...

Metal shadowing of bacteria, viruses, isolated molecules, and macromolecular assemblies is another high-resolution method for observing the ultrastructure of biological specimens. The actual procedure for producing a metal shadow is relatively simple; a heavy metal is evapor ...

Post-staining of ultrathin sections and/or en bloc staining of specimens is necessary for differential contrast and improved resolution of cellular structures. Often specimens are fixed and stained with osmium tetroxide during fixation, but additional contrast is the result of ad ...

The identification of environmental processes and mechanisms often requires information on the organochemical and inorganic composition of specimens at high spatial resolution. X-ray spectroscopy (XAS) performed in the soft X-ray range (100–2,200 eV) provides chemical spec ...

With its low detection limits and the ability to analyze most of the elements in the periodic table, secondary ion mass spectrometry (SIMS) represents one of the most versatile in situ analytical techniques available, and recent developments have resulted in significant advantages for t ...

Secondary ion mass spectrometry (SIMS) is capable of providing detailed atomic and molecular characterization of the surface chemistry of (bio)molecular samples. It is one of a range of mass spectrometry imaging techniques that combine the high sensitivity and specificity of mass sp ...

The techniques of electron energy-loss spectroscopy (EELS) and energy-filtered TEM (EFTEM) are routinely applied in the physical sciences to map the distribution of elements at the nanoscale. EELS can also provide details of the bonding/valence of elements through variations in the fi ...