Superresolution imaging of biological nanostructures by spectral precision distance microscopy

Abstract

For the improved understanding of biological systems on the nanoscale, it is necessary to enhance the resolution of light microscopy in the visible wavelength range beyond the limits of conventional epifluorescence microscopy (optical resolution of about 200 nm laterally, 600 nm axially). Recently, various far-field methods have been developed allowing a substantial increase of resolution ("superresolution microscopy", or "lightoptical nanoscopy"). This opens an avenue to 'nano-image' intact and even living cells, as well as other biostructures like viruses, down to the molecular detail. Thus, it is possible to combine light optical spatial nanoscale information with ultrastructure analyses and the molecular interaction information provided by molecular cell biology. In this review, we describe the principles of spectrally assigned localization microscopy (SALM) of biological nanostructures, focusing on a special SALM approach, spectral precision distance/position determination microscopy (SPDM) with physically modified fluorochromes (SPDM(Phymod) . Generally, this SPDM method is based on high-precision localization of fluorescent molecules, which can be discriminated using reversibly bleached states of the fluorophores for their optical isolation. A variety of application examples is presented, ranging from superresolution microscopy of membrane and cytoplasmic protein distribution to dual-color SPDM of nuclear proteins. At present, we can achieve an optical resolution of cellular structures down to the 20-nm range, with best values around 5 nm (∼1/100 of the exciting wavelength).

Resolution enhancement techniques in microscopy

“…Generally, localization microscopy (for reviews see [Zhuang 2009;Cremer et al 2011;Cremer 2012]) is based on the optical isolation of point emitters, their subsequent high precision localization, and assignment of these coordinates to a joint position map; the optical isolation is achieved by using a suitable set of optically detectable physical characteristics, such as differences in absorption/emission spectra [Betzig 1995;Ha et al 1996;Cremer et al 1996Cremer et al , 1999Bornfleth et al 1998;van Ojen 1998;Esa et al 2000Esa et al , 2001, fluorescence life times , Heilemann 2002, and time dependent differences in luminescence ; or various methods of photoswitching of single molecules between two optically distinguishable states, e.g. a 'dark' and a 'bright' state Lidke et al 2005;Betzig et al 2006;Hess et al 2006;Rust et al 2006;Andresen et al 2008].…”

Optical Nanoscopy Tools for Biologists:Advancements of Fluorophores and Optics for High Resolution and Live Imaging

“…Other similar methods with different names have been invented such as fluorescence-PALM (f-PALM), spectral precision distance measurement (SPDM), direct-STORM (d-STORM), PALM with independent running acquisition (PALMIRA), etc. [58][59][60] . Many cellular events have been explored with high resolution using localization precision microscopy techniques.…”

Perspectives in Super-Resolved Fluorescence Microscopy: What Comes Next?

“…In this illumination mode, SIE techniques do not completely bypass the Abbe-limit (as the fluorescence response depends linearly on the illumination intensity), they can "stretch" it by a factor of about two to four in the object plane and also along the optical axis: But even a stretching of the 200 nm Abbe limit by a factor of two in all three dimensions can be of substantial importance in Biology and Medicine: In this case the observation volume is smaller by the factor 2 3 = 8, i.e., the 3D sampling can be enhanced by about one order of magnitude. In many cases, this will already be sufficient to obtain multiple "nanoimages" of live cells; these high resolution live cell observations can be followed by a last "snapshot" image taken with one of the SRM methods indicated above [27,28]. This is analogous to the observation of a moving animal at a distance, followed by a close look at the animal after it has been captured; peculiarities of movement can be understood by combining the two aspects, the lower resolution time lapse series and the final, further enhanced superresolution snapshot.…”

“…Such an "Avogadro" microscope (to honor the pioneer of Molecular Physics) would have a 3D resolution millions of times more enhanced than then conventional microscopy technology Since at this level of resolution, antibodies and even F(ab)2-fragments would be too bulky, standard fluorophores engineered to bind to specific amino acid residues may be used [28], provided that the targeting specificity is not compromised. At the genome sequence level, a resolution of 1 nm would correspond to a very few base pairs/nucleotides only.…”