Localization-Based Super-Resolution Light Microscopy
on both the precision with which molecules can be localized as well as the nearest-neighbor distance between molecules [5, 6]. Fluorescence photoactivation localization microscopy (FPALM) and similar techniques have achieved a resolution approximately ten-fold better than the diffraction limit. In general, any technique that is capable of creating a sparse subset of visible molecules, imaging that subset, and sampling many subsets can be used to generate a super-resolution image. FPALM, PALM, STORM, and dSTORM. Conceptually,
FPALM [2], photoactivated localization microscopy (PALM) [3], stochastic optical reconstruction microscopy (STORM) [4], and direct stochastic optical reconstruction microscopy (dSTORM) [7, 8] are very similar. Each technique relies on photophysical properties of probes to control the number of visible fluorescent molecules in the field of view. FPALM and PALM typically utilize photoactivatable fluorescent proteins (PA-FPs) to allow for direct optical control over the number of fluorescent molecules visible at a given time. Tis is achieved by adjusting the rates of photoactivation and photobleaching. Similarly, STORM uses pairs of combinations of organic fluorophores that photoswitch in the presence of thiol-containing reducing agents to control the number of molecules that are fluorescent at a given time. dSTORM uses single fluorophores that photoswitch in the presence of thiols and can be used in living cells, which contain reducing agents such as glutathione at micromolar to millimolar concentrations [8]. In all of these techniques, images are subsequently reconstructed from the coordinates and intensities of the localized molecules. Probes. Localization-based techniques can be performed
with either certain conventional fluorophores [8–10], PA-FPs, caged or photoswitchable organic dyes, or pairs of probes that act together as a molecular switch [11–15]. Typically, one wave- length (called the activation wavelength) is used to control the density of visible molecules while another wavelength (called the readout wavelength) is used to collect the fluorescence from the activated molecules. In some instances, one wavelength is sufficient to do both tasks; regardless, the choice of wavelength, intensity, and temporal illumination pattern enables active control over the number of visible molecules. In doing so, these methods rely on photophysical properties of the probes to control the number of molecules in the field of view at a given time [2, 16]. Te photophysical properties of probes play a major role in
the spatial resolution and rate of image acquisition. Resolution in localization microscopy is a function of the localization precision and density of localized molecules. As localiza- tion precision and single-molecule detection (against back- ground) are greatly improved with more detected photons per molecule, probes that emit the maximum number of photons possible before bleaching are ideal. Desirable probes have a high rate of photon emission and a low rate of spontaneous or readout-induced activation, are small in size to minimize perturbation of cell structure or function, and have a large extinction coefficient to minimize the illumination intensity necessary for readout. Probes should also have large contrast (the ratio of brightness in active to inactive forms) so that background from inactive molecules is negligible. Probes with longer wavelength emission are beneficial to minimize autofluorescence background levels. For multicolor labeling,
2011 July •
www.microscopy-today.com
optimal probe combinations have these same characteristics and are typically spectrally well separated. Fundamental limitations. One important limitation
is the trade-off between spatial and temporal resolution. Sub-millisecond observation of molecular motions is limited by the intensity of the readout laser, the probe emission rate, and camera readout speed. Terefore, acquisition of super-resolution FPALM images on timescales faster than 0.1 s is currently difficult to achieve. Te density of localized molecules is another limiting factor in image resolution. Because only one molecule (or at best a few molecules) can be localized per diffraction-limited area in any given frame, improved spatial resolution comes at the price of temporal resolution. Encouraging recent work using methods
from
astrophysics has enabled analysis of molecular images with at least ten-fold higher density, which in principle should allow imaging rates that are faster by the same factor [17]. Minimization of background is crucial both for identi-
fication of fluorescent probe molecules and for maximization of the localization precision. Tus, samples should not emit significant autofluorescence in the same spectral region as the probe being used. Use of UV-bleached water for imaging buffers, use of phenol-red-free media for cell growth, and use of a water-immersion objective beaded with bleached water can help. Additionally, if background levels are high initially, activation of probes can be delayed while the background is allowed to bleach. Many available PA-FPs have overlapping emission spectra,
limiting the number of species usable in a typical multicolor imaging application. Ultimately, the continued development of photoactivatable probes will extend the capabilities of localization microscopy. Te improvement of probes relies on better understanding and control of their photophysical properties. Te labeling efficiency of each probe is another factor to consider, as well as the expression level of the protein to which it is fused. As probes are developed with higher peak photon emission rates, localization microscopy will be possible with higher spatial and temporal resolution.
Methods and Applications Recent progress in the field of localization-based
super-resolution microscopy has led to extensions of these techniques to multicolor [8, 18–20], three-dimensional [21–24], and polarization imaging [25]. For instance, using modifications to the FPALM detection path has enabled multi-channel detection on the same camera chip as well as nanoscale imaging of single molecule anisotropies. A typical experimental geometry for two-color three-dimensional imaging is shown in Figure 2. Tese and other advances show great promise for improved understanding of biological systems at the molecular level. Single-species localization. A typical localization micro-
scopy setup is a fluorescence microscope with activation and readout lasers aligned so that they are collinear, a high-NA objective lens, and a sensitive charge-coupled device (camera) capable of imaging single fluorescent molecules. Illumination light is focused in the center (or at the edge) of the objective back aperture to illuminate the sample by widefield (or by TIRF) over an area of ~100–1000 μm2.
13
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72 |
Page 73 |
Page 74 |
Page 75 |
Page 76 |
Page 77 |
Page 78 |
Page 79 |
Page 80 |
Page 81 |
Page 82 |
Page 83 |
Page 84