The use of widefield epifluorescence microscopy is well established and traditionally used for sensitive image acquisition of fixed cell immunofluorescence in many cell biology laboratories. Recent development of fluorescent tools for tagging specific proteins within live cells furthered interest in the use of more advanced imaging techniques such as confocal, and TIRF microscopy. These techniques have been applied to ion channel research and each has its respective advantages and disadvantages, as discussed below.
Widefield epifluorescence imaging of live cells can provide an excellent overview of protein dynamics within a cell, and images can be acquired rapidly. However, at high objective magnification, there is poor vertical spatial resolution and noise from out of plane signals can significantly confound the image. Post-processing mathematical deconvolution techniques can significantly resolve this problem, but at a cost of increasing imaging time with multiple plane acquisitions. Thus, with some exceptions, deconvolution techniques work best for ‘static’ fixed cell preparations. Another drawback of epifluorescence imaging of live cell preparations is the need for entire specimen exposure to the excitation light. This can result in phototoxic effects on the cell and rapid bleaching of fluorophores. Despite these limitations, epifluorescence imaging remains a useful tool applicable to study ion channel movements within the cell cytoplasm. For studies concentrating on protein dynamics at specific compartments of the cell, such as the plasma membrane, higher vertical spatial resolution is required.
Laser scanning and spinning disc confocal microscopy are widely used techniques in the acquisition of images with higher vertical spatial resolution (typically ~ 500 nm). These techniques exploit pinholes which only accept emission from a specific and precisely controlled plane within the sample, the acquisition of z-stacks can generate impressive 3D images. A disadvantage of confocal microscopy, particularly laser scanning microscopy, is that the entire field of view is exposed to excitation laser light during acquisition. As with epifluorescence imaging yet with a laser source, overexposure can result in phototoxicity of live samples and photobleaching effects on fluorophores. Spinning disc confocal microscopes can acquire confocal images of the entire field of view in a shorter time than their laser scanning counterparts, reducing these unwanted effects. The problem remains, however, that dynamic proteins studied in live cell imaging can move in and out of the focal plane. Furthermore, when studying protein movements that exist within a 5 nm plasma membrane, even confocal imaging will capture mainly cytoplasmic signal. It is for these reasons, that we recommend the use of TIRF microscopy in studying the biology of ion channels at the plasma membrane.
The application of TIRF in the study of cell biology was promoted by Daniel Axelrod in the early 1980s [34
]. TIRF microscopy uses specially angled light and exploits the difference in refractive indexes of aqueous medium and a glass coverslip to obtain extremely high vertical spatial resolution. Briefly, an excitation laser beam is directed at the specimen at an angle of incidence greater than or equal to the critical angle of refraction whereby the excitation laser light is reflected. At the point of reflection, an exponentially decaying evanescent wave is generated and penetrates the specimen. Due to exponential decay, only those fluorophores within 10–100 nm of the glass/medium interface are excited, providing high resolution images of the nearest plasma membrane and immediate subcortical regions (). Given the limited depth of fluorophore excitation, signal to noise ratios can be high enough to image single ion channels or clusters. With a sensitive CCD camera and rapid data acquisition techniques, TIRF microscopy can be used to visualize ion channel or other membrane protein insertion and lateral diffusion within the plasma membrane at the single molecule level. One limitation of TIRF microscopy is that only the interface of the cell and coverslip may be imaged, the focal plane can not be moved “upwards” as in confocal microscopy. However, the relatively low level of energy from the evanescent wave causes markedly less phototoxicity and photobleaching, compared to the toxic effects of confocal and epifluorescence imaging. Recent improvements in TIRF technology now allow for fully automated multi-wavelength systems which greatly improve consistency and facilitate the use of techniques such as FRAP and fluorescence resonance energy transfer (FRET) in combination with TIRF microscopy. TIRF microscopy is therefore a highly appropriate tool in the study of ion channel trafficking to, and dynamics within, the plasma membrane [4
Widefield Epifluorescence and TIRF microscopy