Super-Resolution Microscopy - Nanoscopy

For further information, please contact Monika Bäumle (monika.baeumle@sial.com)
 

Growth cone at the tip of the axon of a primary hippocampal neuron at 1 day in vitro imaged with the Abberior Instruments Expert Line STED microscope. Microtubules Tuj1 (red, Abberior STAR580) are bundled in the central-domain suggesting a pausing state. The molecular motor myosin IIB (blue) is enriched at the transition-zone, along the F-actin arcs. In the peripheral domain actin forms bundles in the filopodia (Phalloidin, Abberior STAR635, green).
Sample was prepared by Elisa D’Este @ MPIBPC, Göttingen.

Optical microscopy is a powerful and multipurpose diagnostic tool. Fluorescent labeling techniques, including immunohistochemistry, in situ hybridization, and fluorescent protein tags enable the study of distribution and dynamics of cellular organelles and proteins as well as any genomic sequence of interest. However, the conventional microscopy techniques suffer from low spatial resolution, mainly due to the diffraction of light. This diffraction limit is 200-300 nm in the lateral direction and 500-700 nm in the axial direction. Most macromolecular cellular complexes occur in the size range of ten to a few hundred nanometers, which is not in the reach of conventional light microscopy. In contrast, electron microscopy can obtain more detailed information in the cell because it uses an accelerated beam of electrons. However, the utility of this technique is very limited in a life sciences context because it must be done in a vacuum. Furthermore, it is also technically demanding, costly, and time consuming. Accordingly, different approaches have been used to improve the diffraction limit of optical resolution.

The development of super-resolution fluorescence microscopy techniques addresses some of the difficulties inherent in earlier microscopy techniques. However, before delving into super-resolution microscopy, we will address earlier fluorescent microscopy techniques.

Improving Resolution Using Far-Field and Near-Field Techniques1

Both confocal and multiphoton laser scanning microscopies are far-field microscopy techniques. Both techniques use a focused laser for excitation and allow enhancement of the spatial resolution. Confocal microscopy uses a pinhole for reducing the out-of-focus fluorescence background, whereas multiphoton microscopy depends on the nonlinear interaction between photons and the sample, causing excitation from a confined focal plane.2 In addition, a third far field method is Structured Illumination Microscopy (SIM), which also takes an optical approach to enhance the resolution. The sample field is illuminated with a sinusoidal stripped pattern of light series, which mixes with the spatial pattern of the sample, resulting in an interference pattern (known as a “moiré fringe”).3 The resulting light patterns can provide details below the diffraction limit using computer algorithms. Three Dimensional Structured Illumination Microscopy, (3D)-SIM, a variation on SIM, has been successfully used to check higher order chromatin structure, and the localization of nuclear pores and nuclear lamina in C2C12 myoblast cells.4

Total internal reflection fluorescence (TIRF) microscopy, a near‑field technique, is used for near surface studies. It uses an evanescent wave generated by reflecting light at highly inclined angles relative to a glass-medium interface. The evanescent wave can penetrate about 100 nm in the medium and excite fluorophore molecules in the thin region present at the surface of the sample, resulting in high axial resolution. The interior of the cell is not visible with this technique. Scanning Near-field Optical Microscopy (SNOM), also known as Near-field Scanning Optical Microscopy (NSOM), also uses the properties of evanescent waves. The studied surface is illuminated at a very close range by excitation of laser light through a narrow aperture (smaller than the wavelength of the excitation light). This results in the generation of evanescent waves, which are limited laterally as well as axially to within 20 nm. The fluorescence can be transformed into an optical image of the sample surface, whose resolution depends on the aperture size rather than the wavelength. A resolution of 50 nm or less can be possible with this technique. However, like TIRF microscopy, SNOM and NSOM applications are limited to surface studies.5

It is important to note that higher image resolution in all of the aforementioned microscopy techniques can be obtained through the use of fluorophores, photophysics, and/or photochemistry. However, even these improvements cannot match super-resolution microscopy. Super-resolution microscopy improves the optical resolution up to almost tenfold.

Super-Resolution Fluorescence Microscopy

Super-resolution microscopy includes techniques based on tailored illumination, the localization of individual fluorescent molecules, and nonlinear fluorophore effects to sharpen the point spread function (PSF) of the microscope. PSF defines the resolution of the microscope and in a point object the three‑dimensional intensity distribution of the image is called “PSF”.

In general, the most challenging part of super-resolution live cell microscopy is observing dynamic processes with multi-dimensional time series without altering the physiology of the cell. With the commercialization of super-resolution microscopes, new insights into cellular responses are expected in the near future. The various fluorescent probes offered by us will help bring the potentials of super-resolution microscopy to fruition. Some of these super-resolution microscopy techniques are discussed below.

Stimulated Emission Depletion Microscopy (STED Microscopy)

The idea behind STED microscopy is to generate an illumination beam with a diameter smaller than the diffraction limit. The technique uses a conventional focused low intensity laser beam for excitation, along with a second high intensity red-shifted doughnut-shaped beam which depletes the emission of the fluorophore outside the central region. The second beam, which is called either a STED or depletion beam, rapidly brings the excited fluorophore molecules back to the vibrational ground state. This method allows light emission only from a small central part of the diffraction-limited focal region. The smaller PSF leads to an approximately tenfold increase in resolution. Increasing the intensity of the depletion beam can further improve resolution, but it can also cause photo-damage in biological samples so caution must be used. Furthermore, it is important to understand that not all fluorophores are appropriate for STED. A STED-appropriate fluorophore should have emission in the range of the depletion beam wavelength and it should have the photostability to withstand the high intensities at the depletion wavelength.

STED Microscopy has been successfully applied to biological samples. For example, it has been used to show neurofilaments in human neuroblastoma and create video-rate imaging of synaptic vesicles in a neuronal axon. Stefan Hell, who developed STED-Microscopy, won the 2014 Nobel Prize in Chemistry for his work.

Reversible Saturable Optically Linear Fluorescence Transition (RESOLFT) and Ground State Depletion (GSD) Microscopy

Both RESOLFT and GSD Microscopy are effective in reducing the STED beam intensity. RESOLFT uses switchable fluorescent probes with both on and off states. The probes are turned off by the donut beam, resulting in detectable fluorescent probes only within the defined focal volume. The on/off states of switchable probes are long-lived states, so the light intensity needed to turn off the probes is lower than that in STED microscopy. GSD microscopy applies the same theory as RESOLFT, but here the fluorophore is sent to the metastable dark state (for instance the triplet state). The lifetime of these dark states is much longer than the fluorescence lifetime. Accordingly, the needed STED beam intensity is much smaller.6-8 It is important to note there are major problems of photobleaching because of the continuous illumination of fluorophores in the dark state. GSD has been used to examine nitrogen vacancy centers in diamond.9

However, the slow switching of the probes is a problem in biological samples for both RESOLFT and GSD, though progress is being made in this regard. Recently, rsEGFP2, a photoswitchable fluorescent protein, has been shown to increase the switching speed and has been used to observe the dynamics of endoplasmic reticulum.10 Nevertheless, the techniques remain unpopular in biological research.

Saturated Structured-Illumination Microscopy (SSIM)

SSIM can be thought of as the inverse of STED microscopy. The illumination pattern in SSIM is similar to SIM. However, the fluorescence excitation is saturated by high excitation intensity. This leads to sharp dark regions in the effective illumination pattern, providing high resolution spatial information. The spatial resolution is limited by the level of fluorescence saturation. For SSIM, fluorophores must have high photostability because the fluorophores are kept in highly reactive excited states for most of the imaging process.11

Single-Molecule Localization Microscopy: Photo-Activated Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM)

Single-molecule microscopy, a technique which produces an image molecule by molecule, is based on the work of Eric Betzig and William Moerner, who both shared in the 2014 Nobel Prize in Chemistry with Stefan Hell. The technique is based on a simple principle of using a weak laser pulse and turning a subgroup of fluorescent molecules on and off in subsequent imaging cycles. With only a few molecules emitting per cycle, a series of images can be processed to obtain a complete high resolution image. Biological samples can be imaged at nanometer resolution by determining the localization of each fluorophore molecule one after another. PALM and STORM are both methods of single molecule localization. PALM microscopy uses photoactivable fluorescent probes and STORM uses photoswitchable chemical fluorophores (mostly pairs of cyanine dyes which cycle between the dark and light states). Direct STORM, or dSTORM, a variation of STORM, exploits the blinking phenomenon by using a single fluorophore which is shifted between long-lived dark states (for instance, triplet state) and bright states. Both STORM and PALM require probes whose state can be controlled. The probes must also be bright and there should be a high contrast ratio between the two states. Additionally, both approaches have applications in the study of biological samples. In particular, PALM has been combined with quantum dot tracking to observe dynamic changes in spine morphology and multicolor STORM has been used for imaging microtubules alongside clathrin coated pits.

Fluorescent probes for super-resolution microscopy

Abberior Dyes

Atto Dyes
 

Factors Affecting the Spatial Resolution in Super-Resolution Microscopy11,16

Labeling Density

There are issues with labeling density in all super-resolution microscopy methods. In general, insufficient labeling can result in artifacts. On the other hand, in the single molecule localization approach high labeling is not advantageous. This difference is because in the dark state most of the fluorophores will not be dark, but either emitting weak fluorescence or switching to the fluorescent state spontaneously despite the absence of activation light. The weak ambient fluorescence can result in the overlap of single-molecule images.

Time Resolution

For both STED and RESOLFT microscopy, a high spatial resolution requires a smaller scanning pixel size and, therefore, a slow acquisition speed. To achieve faster time resolution, it is common practice to reduce the area of the imaging field and use an enlarged pixel size. Time resolution can also be improved by using multiple excitation focal points. In the single molecule localization approach, imaging speed is restricted by the time needed for the accumulation of fluorophore localization density. The rate of accumulation depends on both the switching kinetics of fluorescent probes and the acquisition rate of the camera. With regard to STORM and PALM, imaging speed cannot be increased rapidly by reducing image area.

Super-Resolution Probes

The choice of fluorescent probe is critical in super-resolution microscopy. As we have said, each technique has different criteria for the optimal fluorescent probe. The risk of photobleaching, the state of the fluorescent probe, and the probe’s brightness are some of the factors which are important in choosing a probe for super-resolution. In addition, experiments using multiple labels require more consideration. We offer fluorescent probes optimized for super-resolution microscopy.

All super-resolution techniques have trade-offs in terms of spatial resolution, fluorescent probes, acquisition time, image processing, etc. These factors need to be taken into consideration before deciding which method to use. Table 1 gives a brief overview of the different microscopy techniques discussed in in this article.

Table 1: Fluorescence Microscopy12-15
 

Type Microscope illumination Resolution (nm) Acquisition time Post-image processing Problems
Standard fluorescence microscopy Epi*/TIRF/Confocal 250 - - Limited resolution
SSIM Wide field (epi/TIRF) 50 Long (min) Yes Photobleaching
STED Laser scanning 30-70 Short (s) No Photobleaching
RESOLFT Laser scanning 40-80 Long (min) No Moderate photobleaching
GSD/PALM/ STORM Wide field (epi/TIRF) 10-55 Long (min) Yes Over/under labeling artifacts

*epi stands for epifluorescence

 

 References

  1. Schermelleh, Lothar, Rainer Heintzmann, and Heinrich Leonhardt. "A guide to super-resolution fluorescence microscopy." The Journal of cell biology 190.2 (2010): 165-175.
  2. Ustione, A., and D. W. Piston. "A simple introduction to multiphoton microscopy." Journal of microscopy 243.3 (2011): 221-226.
  3. und Halbach, Oliver von Bohlen. "Cell biology needs super-resolution." Edorium Journal of Cell Biology 2 (2015): 1-3.
  4. Schermelleh, Lothar, et al. "Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy." Science 320.5881 (2008): 1332-1336.
  5. De Lange, Frank, et al. "Cell biology beyond the diffraction limit: near-field scanning optical microscopy." Journal of cell science 114.23 (2001): 4153-4160.
  6. Heilemann, Mike, et al. "Photoswitches: Key molecules for subdiffraction‐resolution fluorescence imaging and molecular quantification." Laser & Photonics Reviews 3.1‐2 (2009): 180-202.
  7. Hell, Stefan W., and M. Kroug. "Ground-state-depletion fluorscence microscopy: A concept for breaking the diffraction resolution limit." Applied Physics B 60.5 (1995): 495-497.
  8. Huang, Bo, Hazen Babcock, and Xiaowei Zhuang. "Breaking the diffraction barrier: super-resolution imaging of cells." Cell 143.7 (2010): 1047-1058.
  9. Bradshaw, Ralph A., Stahl, Philip D. "Encyclopedia of Cell Biology." Academic Press (2015): 81.
  10. Grotjohann, Tim, et al. "rsEGFP2 enables fast RESOLFT nanoscopy of living cells." Elife 1 (2012): e00248.
  11. Galbraith, Catherine G., and James A. Galbraith. "Super-resolution microscopy at a glance." J Cell Sci 124.10 (2011): 1607-1611.
  12. Ehrenberg, M. "Super-resolved fluorescence microscopy." The Royal Swedish Academy of Sciences (2014).
  13. Sydor, Andrew M., et al. "Super-resolution microscopy: from single molecules to supramolecular assemblies." Trends in cell biology 25.12 (2015): 730-748.
  14. Thorley, Jennifer A., Jeremy Pike, and Joshua Z. Rappoport. "Super-resolution microscopy: A comparison of commercially available options." Fluorescence Microscopy: Super Resolution and Other Novel Techniques (2014): 185-197.
  15. Yamanaka, Masahito, Nicholas I. Smith, and Katsumasa Fujita. "Introduction to super-resolution microscopy." Microscopy (2014): dfu007.
  16. Huang, Bo, Mark Bates, and Xiaowei Zhuang. "Super resolution fluorescence microscopy." Annual review of biochemistry 78 (2009): 993.

 

Find more information on STED Microscopes: Quick Guide to STED Sample Preparation, PDF (846 KB)

Product Manager: Monika Baeumle
Author: Chhavi Aggarwal

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