Localization-Based Super-Resolution Light Microscopy
Biplane FPALM (BPFPALM) is an extension of FPALM to
3D, which works by splitting the detected fluorescence into two separate paths with different overall lengths. Te two images simultaneously formed on separate regions of the camera correspond to two different focal planes and are later fitted with the measured PSF to determine the x, y, and z coordinates of each molecule. BPFPALM of samples was demonstrated with an axial resolution of ~50–75 nm [21, 33], approximately 10-fold improved over conventional fluorescence microscopy. Optical astigmatism is used to determine both axial
and lateral positions of single molecules in 3D STORM [22]. By inserting a cylindrical lens into the detection path of the microscope, two slightly different focal planes result for the x and y dimensions of each molecular image. As a result, fluorophores appear elliptical in x or y depending on their axial position, allowing that axial position to be determined from the molecular image. Interferometric PALM (iPALM) resolves 3D molecular
coordinates of individual PA-FP tagged proteins with ~10 nm axial resolution and ~20 nm lateral resolution using a 4Pi (two-objective) geometry [23]. Te detected fluorescence is divided by a three-way beam splitter and imaged with three cameras. Te relative intensities of the three images of each molecule are then analyzed to determine the axial position of the molecule. iPALM was recently used to deduce the structure of the focal adhesion complex [34]. In the double-helix point spread function (DH-PSF)
method, a phase modulation pattern in the detection path yields a detected PSF with two lobes whose orientation rotates as a function of the axial position of the detected molecule. DH-PSF has a demonstrated resolution of ~10 nm laterally and 20 nm axially over a sample depth of ~2 µm [24]. Polarization FPALM: imaging molecular orientations
at the nanoscale level. Te localization-based imaging techniques described above are crucial for studies of biological structures, but they do not provide information about the orientation or rotational freedom of single molecules. Polarization FPALM (PolPALM) [25] has extended FPALM to image single molecule anisotropies by simultaneously detecting two spatially separated images of the fluorescence, polarized parallel and perpendicular to a specific axis at the sample. To measure the anisotropy for each localized molecule, PolPALM analyzes the relative intensities of molecules in the two images. Te demonstrated resolution of ~17 nm using PolPALM allows quantification of structure on length scales inaccessible to diffraction-limited techniques. Te anisotropy obtained reflects the range of accessible probe orientations and can also be used to quantify binding between molecules.
Conclusion Localization microscopy provides nanoscale spatial
resolution in living fluorescent specimens using far-field optics. Such capabilities suggest that a multitude of fund- amental but previously inaccessible biological questions can now be addressed. Current limitations such as number of simultaneously distinguishable species and the time resolution of imaging are likely to improve with refined experimental methods and improved probes. Furthermore, using existing localization microscopy methods, the number of compatible
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biological systems is continuously increasing. New biological insights hopefully are imminent.
Acknowledgments Te authors thank M. V. Gudheti and T. J. Gould for
FPALM images included within the article; S.-R. Yin and Vladislav Verkhusha for constructs; and A. Gosse, J. Lake, and J. Gosse for support. Tis work was funded by NIH Career Award K25 AI65459, NIH R15 GM094713, NIH R01 GM094713, NSF MRI CHE-0722759, Maine Technology Institute MTAF 1106 and 2061, UMaine V.P. for Research, and the Maine Economic Improvement Fund (MEIF).
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