High-numerical-aperture cryogenic light microscopy for increased precision of superresolution reconstructions

Marc Nahmani, Conor Lanahan, David DeRosier, Gina G. Turrigiano
2017 Proceedings of the National Academy of Sciences of the United States of America  
Superresolution microscopy has fundamentally altered our ability to resolve subcellular proteins, but improving on these techniques to study dense structures composed of single-molecule-sized elements has been a challenge. One possible approach to enhance superresolution precision is to use cryogenic fluorescent imaging, reported to reduce fluorescent protein bleaching rates, thereby increasing the precision of superresolution imaging. Here, we describe an approach to cryogenic photoactivated
more » ... calization microscopy (cPALM) that permits the use of a room-temperature high-numerical-aperture objective lens to image frozen samples in their native state. We find that cPALM increases photon yields and show that this approach can be used to enhance the effective resolution of two photoactivatable/switchable fluorophore-labeled structures in the same frozen sample. This higher resolution, two-color extension of the cPALM technique will expand the accessibility of this approach to a range of laboratories interested in more precise reconstructions of complex subcellular targets. superresolution | single molecule | localization microscopy | photoactivated localization microscopy | cryogenic microscopy S ingle-molecule localization-based microscopy (SMLM) techniques have enhanced our ability to resolve the intricate spatial relationships between identified subcellular proteins (1-3). These techniques routinely achieve an average localization precision of ∼25 nm (4). Recent SMLM studies have begun to fundamentally alter our understanding of cytoarchitecture (5) and the dynamic interplay of cellular proteins (6, 7). Despite this progress, many subcellular structures remain difficult to study because they are composed of a compact network of single-molecule-sized elements. These structures lie beyond current SMLM approaches because the accuracy of SMLM techniques is dependent on the achieved precision in localizing individual fluorophores, which primarily relies on the number of photons collected from each of these molecules (8). Unfortunately, genetically encoded probes visualized at room temperature suffer from relatively low quantal yield and high numbers of unactivatable fluorophores, which limit localization precision and the completeness of SMLM reconstructions (9), respectively. Equally lacking are localization methods based on antibody labeling because antibodies necessarily add ∼10 nm of uncertainty in localizing the molecules of interest (10). To study in detail the native structural relationships of small, densely packed proteins, it would be ideal if one could (i) study frozen, hydrated specimens preserved in their native state, (ii) increase the localization precision of individual molecules to resolve the intricate relationships of small, dense structures and more faithfully reconstruct these objects, and (iii) combine these localization maps of identified objects with the ultrastructural information gained via cryogenic electron microscopy (cryoEM). Examples of the combined use of a cryogenic SMLM method, cryogenic photoactivated localization microscopy (cPALM), and cryoEM have illustrated the utility of combining these methods. However, these studies used cryogenic stages with air-coupled objective lenses that consequently limited the collection of photons and the effective resolution (11-13). Here we developed a cryogenic stage and objective interface that extends the cPALM approach by using a commercially available high-numericalaperture (N.A.) lens (≥1.2 N.A.) , enabling more precise fluorescent imaging of frozen (−140 to −150°C) hydrated specimens and offering significant improvement over room-temperature imaging with identical equipment. Unlike the only other high-N.A. implementation of cPALM in which the objective is held at temperatures below −135°C (14) , we developed an approach that keeps the objective at ambient temperature and therefore can be used on a conventional microscope. Moreover, we find that like photoactivatable GFP [PaGFP (11)], the photoswitchable protein PSmOrange (15) is able to switch at cryogenic temperatures, adding to the repertoire of photoswitchable proteins capable of switching at cryogenic temperatures (13), and enabling simultaneous localization and comparison of two photoactivatable/ switchable proteins within the same sample under cryogenic conditions. These samples can then be imaged using cryoEM to correlate SMLM localization "maps" of identified molecules with ultrastructural information (11, 12) . To image frozen hydrated samples with a short working distance high-N.A. lens at temperatures below the threshold for ice crystal formation (≤−135°C), we built a cryogenic stage that is cooled by the flow of cold nitrogen gas and that maintains samples on electron microscopy grids at stable cryogenic temperatures ( Fig. 1 D and E) . Samples were optically coupled to the coverslip by a cryofluid that freezes below −150°C and does not boil at ambient temperature Significance The precision of localization-based superresolution microscopy techniques fundamentally relies on the point-spread function of the optical system and the number of photons one can collect. Here, we report that by using a high-numericalaperture objective lens and a custom cryogenic stage, we are able to increase photon yield by 2-3-fold over room temperature, thereby achieving more precise superresolution reconstructions of complex subcellular structures. Conflict of interest statement: The authors built successive versions of the cPALM stage and objective collar and eventually patented the current system (Publ US 2015/0248002 A1). Design elements and detailed scaled images for constructing a similar cryogenic stage and objective collar system can be found in this patent, as well as in the annotated computer-aided design (CAD) drawings available as Movies S1 and S2.
doi:10.1073/pnas.1618206114 pmid:28348224 pmcid:PMC5393246 fatcat:q3fwn7ywpbb3nb2izpttkm4zby