Nanometer Accuracy in Cryogenic Far-Field Localization Microscopy of Individual Molecules

Abstract

We demonstrate the nanometer accuracy of far-field fluorescence localization microscopy at a temperature of 1.8 K using near-infrared and red fluorophores bonded to double-stranded DNA molecules (10.2 nm length). Although each fluorophore was localized with a 1 nm lateral precision by acquiring an image at one axial position within the focal depth of ±0.7 μm, the distance between the two fluorophores on the lateral plane (D xy ) was distributed from 0 to 50 nm. This systematic error was mainly due to detecting with the large focal depth the dipole emission from orientationally fixed fluorophores. Each fluorophore was localized with precisions of ±1 nm (lateral) and simultaneously ±11 nm (axial) by acquiring images every 100 nm in the axial direction from −900 to 900 nm. By correcting the dipole orientation effects, the distribution of D xy was centered around the DNA length. The average and standard deviation of D xy were 10 and 5 nm.

Polarization-Encoded Colocalization Microscopy at Cryogenic Temperatures

“…With the advent of super-resolution methods, optical microscopy has provided fascinating new insights into the subcellular domain and has become an indispensable tool in elucidating the structure and function of biological systems at the nanoscale. − The high specificity and spatial resolution of fluorescence imaging has the potential to deliver further information on the molecular architecture of proteins and their complexes, even in a native environment, for example, membrane proteins or protein aggregates implicated in diseases. Recently, it has been recognized that super-resolution microscopy performed at cryogenic temperatures can be of great value. − The main advantage of this approach stems from the fact that photochemistry is considerably slowed down at low temperatures. As a result, each fluorophore can emit more than 2 orders of magnitude more photons than at room temperature before it photobleaches.…”

“…Figure 4f shows that, indeed, by selecting bright dipoles and small relative angles between the two fluorophores of the data presented in Figure 4b, we arrive at narrower and more symmetric distributions. We remark that the complication caused by the 3D orientation of the dipole moment could be addressed more rigorously by direct measurement of the complete orientation 11 or by filtering the azimuthal contributions of the PSF with a phase mask. 34 We also remark that the success of polarization selection comes at the cost of a lower signal in each channel since we have to split the emission from single molecules.…”

Roadmap on bio-nano-photonics

“…Short distances to room temperature elements affect the sample temperature due to radiation heating for temperatures below 50 K. Additionally, spatial drift between sample and collection optics is an issue. Using helium bath cryostats, and inserting the collection optics (aspheres or simple multi-element objectives) at low temperatures avoids temperature and NA limitations [66,67,69,70,82], and allows to use superfluid helium to reach 1.4 K. It also suppresses image drift, and dedicated reflective optics have been developed [87]. However, these cryostats require a local liquid helium supply and are laborious to handle.…”

Polarization-encoded co-localization microscopy at cryogenic temperatures

“…Figure 4f shows that, indeed, by selecting bright dipoles and small relative angles between the two fluorophores of the data presented in Figure 4b, we arrive at narrower and more symmetric distributions. We remark that the complication caused by the 3D orientation of the dipole moment could be addressed more rigorously by direct measurement of the complete orientation 8 or by filtering the azimuthal contributions of the PSF with a phase mask. 31 We also remark that the success of polarization selection comes at the cost of a lower signal in each channel since we have to split the emission from single molecules.…”

“…Recently, it has been recognized that super-resolution microscopy performed at cryogenic temperatures can be of great value. [2][3][4][5][6][7][8][9][10][11] The main advantage of this approach stems from the fact that photochemistry is considerably slowed down at low temperatures. As a result, each fluorophore can emit more than two orders of magnitude more photons than at room temperature before it photobleaches.…”