Describing the various methods of cell imaging

1) Green flourescent protein (GFP) was isolated from Aequorea victoria in the 1960s, but was not widely used until the 1990s when its nucleotide sequence was revealed and derivatives began to be genetically engineered. The first major one (a single point mutation in S65T) improved the flourescence and shifted the major excitation peak to 488 nm. Later developments led to blue, yellow, and cyan as new flourescent colors, and changed the folding efficiency which allowed it to be inserted into mammalian cells. The molecule containing GFP is then imaged under one of many different flourescent microscopes, which take advantage the fact that flourophores absorb light of a low wavelength and emit light of either a different wavelength or color.  Applications to the brain: Hechler et al (2006) used GFP to study the axon regeneration of large numbers of neurons in vitro following lesion. Other applications utilize its ability to be inserted into living cells, such as the incredible detail available from brainbow experiments where neurons are each given different amounts of the different colors of GFP. Additionally, genes expressing GFP can be inserted into mice, from which researchers can remove a slice of brain tissue, and use proteins found in bacteria to manipulate the ion flow (and thus the potential) of individual neurons in vitro. The surface of the potential uses for GFP is just now be touched upon. 2) Calcium imaging is based on indicator molecules that bind to calcium ions after carboxylic acid groups chelate them. These indicators are all derived from the general GFP. It can either be inserted as a dye, the genes encoding for the proteins can be transfected to cell lines (in vitro), or you can create transgenic animals expressing the genes for the proteins (either globally or selectively).  The spectral properties of the dye changes after binding to calcium, which can be analyzed under a flourescence microscope. Applications to the brain: Calcium ion flows are a crucial part of ion flows during action potentials and the process can be analyzed in detailed in vitro using these methods. Also, using two-photon calcium imaging, Stosieck et al (2003) was able to record through intact mice skulls in vivo and stained cells up to 150-200 micrometers below the cortical surface. Using this technique they were able to stain in the range of tens to hundreds of neurons. It is conceivable that if neurons were stained and imaged, and then that area of the brain was removed, the process could be iterated until the whole brain would be at least partially imaged. 3) Laser capture microdissection is a purely in vitro technique where a tissue section is collected, and a transparent tissue film is applied. When the cell or cluster of cells identified for isolation is found under microscope, a close to infrared laser activates a precise spot on the film which fuses with the selected underlying cells. The tissue can be removed, leaving all of the other cells behind (and intact), and the process does not damage the chemistry and structure of the cells attached. Applications to the brain: Bi et al (2002) used the technique to analyze interneurons from tissues of frozen rat brains and conduct real-time PCR. They used it to analyze the cellular aspects of glutamate receptors. It can also be used in the study of neurodegenerative disorders, which often are manifested in highly specific regions of the brain (ie, dopaminergic neurons of the ventral tier of the substantia nigra pars compacta in Parkinson’s disease). In these cases, laser capture microdissection allows very specific cells in brain tissues to be targeted, separated, and studied (Standaert, 2005). The ability to isolate specific neurons from general tissue is very impressive, although its super long-run applicability might be limited simply because it is an in vitro technique. But as I am fond of saying, in the long-run we are all cryogenically frozen. 4) Evanescent-wave microscopy has an especially high numerical aperture in its objective lens that allows for high resolution imaging. It allows for the visualization of flourescent objects under evanescent wave light. Note that I do not have a high understanding of the physics behind this machine, but it does allow for flourescently labeled cells to be viewed in vitro at an extremely high resolution. Applications to the brain: The ability to visualize living flourescent cells with high accuracy is tremendously advantageous to researchers. Oheim et al (1999) used it to elucidate some novel observations of vescicle movement in bovine chromaffin cells. Terekawa et al (2005) suggests that it can be used in “living cells”, but I believe that they are referring to cultured neurons in vitro that are technically “alive”, but not actually in the brains of mammals. Nevertheless, this is obviously an important technique, which also piggybacks on the valuable tool of GFP. 5) Pulse-chase analysis is a process where a labeled (often by being radioactive) compound is inserted into a cell or a group of cells, and then about 5 minutes later an unlabeled portion of the compound is inserted into the same cell or cells. The idea is that the radioactive detection will detect the first part (the pulse), but not the second part (the chase), allowing for the pathway of the whole process to be easily visualized using radioactive detection methods. Applications to the brain: Leuchtenberger et al (2008) used a pulse chase analysis in order to help determine the mechanism of nonsteroidal anti-inflammatory drugs on the shedding of amyloid precursor protein in chicken neurons. This is a valuable technique for determining the pathway of an agonist acting on a group of cells in vivo, which could be used to evaluate the effect of a given drug or chemical. References Hechler D, Nitsch R, Hendrix S. 2006 Green-fluorescent-protein-expressing mice as models for the study of axonal growth and regeneration in vitro. Brain Research Reviews 52: 160-169. doi:10.1016/j.brainresrev.2006.01.005. Stosieck C, Garaschuk O, Holthoff K, Konnerth A. 2003 In vivo two-photo calcium imaging of neuronal networks. PNAS 100: 7319-7324. doi: 10.1073/pnas.1232232100. Bi WL, Keller-McGandy C, Standaert DG, Augood SF. 2002 Identification of nitric oxide synthase neurons for laser capture microdissection and mRNA quantification. BioTechniques 33: 1274-1283. Standaert DG. 2005 Applications of laser capture microdissection in the study of neurodegenerative disease. Archive of Neurological Review 62: 203-205. Oheim M, Loerke D, Chow RH, Stuhmer W. 1999 Evanescent-wave microscopy: a new tool to gain insight into the control of transmitter release. Philos Trans R Soc Lond B Biol Sci 354: 307–318.

Leucthenberger S, Maler J, Czirr E, Ness J, Lictenthaler SF, Esselmann H, Pietrzik CU, Wiltfang J, Weggen S. 2008 Nonsteroidal anti-inflammatory drugs and ectodomain shedding of the amyloid precursor protein. Neurodegenerative Diseases 6: 1-8. doi: 10.1159/000121391.

Terakawa S, Sakurai T, Tsuboi T, Wakazono Y, Zhou J-P, Yamamoto S. 2005 Dynamics of the cell membrane Observed under the wave microscope and the confocal microscope. Springer US, pp. 15-23. In the book Biophotonics. doi: 10.1007/0-387-24996-6_2.