FV1000: The broad range of applications available to laser scanning confocal microscopy includes a wide variety of studies in neuroanatomy and neurophysiology, as well as morphological studies of a wide spectrum of cells and tissues.
In addition, the growing use of new fluorescent proteins is rapidly expanding the number of original research reports coupling these useful tools to modern microscopic investigations. Other applications include resonance energy transfer, stem cell research, photobleaching studies, lifetime imaging, multiphoton microscopy, total internal reflection, DNA hybridization, membrane and ion probes, bioluminescent proteins, and epitope tagging. Many of these powerful techniques are described in the sections listed below.
Many different types of molecular analyses have been successfully performed on cells procured by laser microdissection. These include genomic analyses such as loss of heterozygosity analysis, restriction fragment length polymorphism analysis, DNA methylation analysis, fluorescence in situ hybridization, and comparative genomic hybridization.
Gene expression analysis (i.e., RNA analysis) has been accomplished from laser microdissected samples utilizing reverse transcription PCR, and gene expression microarray analysis after amplification of RNA. Successful proteomic analysis has been accomplished by coupling laser microdissection with a variety of techniques including western immunoblotting, one dimensional and two dimensional polyacrylamide gel electrophoresis (PAGE), and mass spectrometry.
For all these assays, the expected results will depend on the quantity and quality of preservation of the analyte of interest within the sample and upon the number of cells procured for analysis. Selected references demonstrating a variety of applications for laser microdissected samples are provided below.