Laser microbeam

Isenberg, G., Rathke, P.C., Hulsmann, N., Franke, W.W., Wolfarth-Botterman, K.E. (1976). Cytoplasmic actomyosin fibrils in tissue culture cells. Direct proof of contractility by visualization of ATP-induced contraction in fibrils isolated by laser microbeam dissection. Cell Tiss. Res. 166, 427-444. 

Aist JR, Berns MW 1981 Mechanics of chromosome separation during mitosis in Fusarium (Fungi imperfectly, new evidence from ultrastructural and laser microbeam experiments. J Cell Biol 91 446-458... 

Aist JR, Liang H, Berns MW 1993 Astral and spindle forces in PtK2 cells during anaphase B a laser microbeam study. J Cell Sci 104 1207-1216 Bohm H, Brinkmann V, Drab M, Henske A, Kurzchalia TV 1997 Mammalian homologues of C. elegant PAR-1 are asymmetrically localized in epithelial cells and may influence their polarity. Curr Biol 7 603-606... 

Overview of Laser Microbeam Appiications as Reiated to Antibody Targeting... 

The following discussion provides an overview of several applications selected for their use of (or potential use of) antibodies in combination with a laser microbeam to investigate some aspect of molecular or cellular biology. 

Laser microbeams offer several advantages over other fluorescence excitation techniques. In spectrofluorometry, observations are often made on a population of cells in a cuvette, resulting in a combined signal that lacks information about individual cellular responses. In flow cytometry, many individual cells are measured, but there is no temporal resolution since each cell is observed only once, and there is no spatial resolution since the entire cell is illuminated as it passes through the laser beam (see Chapter 30). In conventional fluorescence microscopy, individual cells can be monitored over time, and information about the two-dimensional spatial distribution of fluorescence can be obtained. However, some samples may be more susceptible to photobleaching by the arc lamps used for excitation, and the temporal resolution is limited to video-rate data acquisition (30 frames/s) (see Chapter 14). 

Fluorescence excitation with a laser microbeam allows for a smaller region to be illuminated. Monitoring fluorescence with a sensitive photomultiplier tube also permits the use of lower intensities of irradiation for shorter periods of time. Therefore, unwanted photobleaching can be significantly reduced. If the spot size is adjusted to illuminate an entire cell, information analogous to spectrofluorometry or flow cytometry can be obtained on an individual cell basis with a high degree of temporal resolution. If the spot size is smaller than the cell, similar information can be obtained from a particular location within the cell. 

The photobleaching method has been adapted for use with a laser microbeam system (6). The advantage of this system is that it allows for the selection of individual cells to be analyzed. The laser beam can be tuned to the wavelength closest to the excitation maximum of the donor (the 488-nm fine of an argon ion laser is used for FITC excitation), and the beam can be optically expanded to irradiate an entire cell. A cell density should be used that... 

Recently, infrared laser microbeams (wavelengths >700 nm) have been used to produce electromagnetic fields capable of exerting a sufficient force on cells... 

The techniques discussed in this chapter are intended as an overview of how laser microbeams might be used in conjunction with antibodies to address various aspects of molecular and cellular biology. There may be other applications that were not covered, and there will likely be additional approaches developed as laser microbeams become increasingly available. Although microscopes are already a common tool of biological research, lasers are becoming so. With decreases in their size, complexity, and cost, lasers may become a standard accessory to the research microscope. 

Microdissection of a tumor was initially carried out using a standard syringe (5). The tumor was viewed under a microscope so that it could be separated from the surrounding normal tissues. For this method to be effective, tumors needed to be easily defined under the microscope, which limited the number of samples that were compatible. Microdissection techniques have further evolved to include techniques such as laser microbeam microdissection (EMM) and laser capture microdissection (LCM). These... 

Fluorescence-activated cell sorters (FACS) have been used to separate subpopulations of cells for subsequent treatment or analysis see Chapter 26). However, this approach requires that the cells be in suspension. In the case of adherent cells, some cannot be easily suspended, or the treatments used to suspend them may interfere with subsequent analysis. In these situations, a laser microbeam system capable of fluorescence imaging can serve two purposes. At low power, the laser can excite fluorescence to produce an image, and at a higher power, the laser can be used to kill the undesired cells. 

Weber, G. and Greulich, K. O (1992) Manipulation of cells, organelles, and genomes by laser microbeam and optical trap Int. Rev. Cytol. 133, 1—41... 

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