Fast 100-nm resolution three-dimensional microscope reveals structural plasticity of mitochondria in live yeast

Abstract

By introducing beam-scanning multifocal multiphoton 4Pi-confocal microscopy, we have attained fast fluorescence imaging of live cells with axial super resolution. Rapid scanning of up to 64 pairs of interfering high-angle fields and subsequent confocal detection enabled us to perform three to five times finer optical sectioning than confocal microscopy. In conjunction with nonlinear image restoration, we demonstrate, to our knowledge for the first time, three-dimensional imaging of live eukaryotic cells at an equilateral resolution of Ϸ100 nm. This imaging mode allowed us to reveal the morphology and size of the green fluorescent protein-labeled mitochondrial compartment of live Saccharomyces cerevisiae (bakers' yeast) growing on different carbon sources. Our studies show that mitochondria of cells grown on medium containing glycerol as the only carbon source, as opposed to glucose-grown cells, exhibit a strongly branched tubular reticulum. We determine the average tubular diameter and find that it increases from 339 ؎ 5 nm to 360 ؎ 4 nm when changing from glucose to glycerol, that is, from a fermentable to a nonfermentable carbon source. Moreover, this change is associated with a 2.8-fold increase of the surface of the reticulum, resulting in an average increase in volume of the mitochondrial compartment by a factor of 3.0 ؎ 0.2. F ocused light is the only means by which it is possible to visualize and quantify biochemical processes in a live cell. Catalyzed by the advent of specific exogenous and endogenous markers, such as the green fluorescent protein (GFP) and its mutants, fluorescence-based far-field microscopy has evolved into a powerful tool for elucidating the relationship between cellular structure and function. This fact particularly applies to confocal and multiphoton fluorescence microscopes, which, by providing three-dimensional (3D) images, facilitate quantitative studies in the interior of cells (1). Despite its obvious advantages, the application of far-field microscopy has been seriously restricted. A major reason is the limited spatial resolution that has precluded 3D imaging and the quantitative 3D analysis of submicron-scale cellular compartments.In Fourier space, where the imaging process is expressed in terms of spatial frequencies, the limited resolution of light microscopes is attributed to their inability to grasp and transfer object frequencies higher than about one-half of the reciprocal wavelength (2). In real space, the resolution limit is described by the minimal fluorescence spot size, which for a standard highaperture confocal microscope is Ϸ180 and Ϸ500 nm in the transverse and axial directions, respectively (1). The demand for higher resolution in biological imaging has spurred a number of interesting developments, such as scanning 4Pi-confocal microscopy (3), stimulated emission depletion fluorescence microscopy (4, 5), wide-field I 5 M (6), and combinations of structured illumination with image restoration (7,8).In fluorescence imaging, the mathematical function describing th...

Mdm30 Is an F-Box Protein Required for Maintenance of Fusion-competent Mitochondria in Yeast

“…On nonfermentable carbon sources, such as glycerol, mitochondria are much more elaborate and ramified ( Figure 2A) (Egner et al, 2002). The ⌬mdm30 deletion mutant grown on glucose-containing medium harbors highly aggregated mitochondria with some fragmented organelles ( Figure 2A and Table 1) (Dimmer et al, 2002).…”

“…Yeast mitochondria form an extended tubular network located below the cell cortex (Hoffmann and Avers, 1973). Mitochondria undergo gross structural changes during adaptation to nutritional conditions and growth phase (Stevens, 1981;Pon and Schatz, 1991;Egner et al, 2002). During vegetative growth, the continuity of the mitochondrial network is maintained by a balanced frequency of opposing fusion and fission events (Nunnari et al, 1997).…”

Quantitative 3-D imaging of eukaryotic cells using soft X-ray tomography

“…Thus, the organelles with the highest x-ray absorption coefficients-colored red in In comparison with other microscopy techniques, soft x-ray tomography has several advantages for studying the size, distribution, and density of organelles. New 'super-resolution' techniques such as stimulated emission depletion microscopy (STED) (Hell, 2007), saturated structured illumination microscopy (SSIM) (Gustafsson, 2005), stochastic optical reconstruction microscopy (STORM) (Rust et al, 2006), photoactivated localization microscopy (PALM) (Betzig et al, 2006), and 4PI confocal microscopy (Egner et al, 2002), can match the resolution of soft x-ray tomography in two dimensions, but in the third dimension they have significantly lower spatial resolutions (~100 nm), or are limited to thin sections. In addition, these techniques are limited to information from the fluorescent labels, and do not have access to full structural information on the cell.…”

“…In many types of cells, the number and organization of certain organelles can change quickly in response to environmental factors such as cell density, temperature, oxygen tension, and the availability of nutrients (Conibear and Stevens, 1995;Egner et al, 2002;Weisman et al, 1987). Yeast is a particularly good model system for studying the size, shape, and distribution of organelles such as mitochondria as a means of coping with changes in their environment (Anesti and Scorrano, 2006;Hales, 2004;Hermann and Shaw, 1998;Hoog et al, 2007;Jakobs et al, 2003;Jensen, 2005;Logan, 2003;Logan, 2006;Sun et al, 2007;Yaffe et al, 1996).…”

Comparing video‐rate STED nanoscopy and confocal microscopy of living neurons

“…A notable exception, though, was (diffraction-limited) 4Pi microscopy yielding superresolution of dynamic living samples along the optic axis [15][16][17][18]. While the possibility of overcoming the diffraction limit in living cells was demonstrated a decade ago [19], it was not before 2007 that dynamic imaging with nanoscale resolution in the focal plane was demonstrated at 80 frames per second [20] and in 2008 that video-rate imaging was achieved in living cells [21].…”