Crisp images of cold cells

March 26, 2018

Microscopy at cryogenic temperature has many exciting qualities. Cryofixed or vitrified, cells can be imaged in a near‑native state with a combination of light, electron, and X-ray microscopy. A particular advantage of cryogenic cooling in fluorescent light microscopy is that bleaching is strongly reduced. However, cryofluorescence microscopy is technologically challenging. To avoid damage due to re-crystallization, the sample must be maintained below the glass transition temperature of water (‑135 °C). At the same time, achieving the highest levels of resolution and contrast in light microscopy requires the use of immersion objectives. Until now, neither high-quality objectives nor any well-matched immersion media for the required temperature range existed. Here, we have overcome this challenge by a new type of objective combined with the discovery of a new immersion medium that matches the refractive index of room temperature water at cryogenic temperature. We expect that this work will be of great value for correlative light and electron microscopy and for pushing the limits of superresolution light microscopy at cryogenic temperature.

Cryofluorescence imaging is of great interest in biological microscopy. Using special preparation techniques, cells and biomolecules can be frozen to the temperature of liquid nitrogen without ice crystallization. In this glassy – or vitrified – state, frozen samples are free from fixation artifacts, highly photostable, and allow direct correlation with electron cryomicroscopy. A long-standing challenge in cryogenic light microscopy is the lack of high numerical aperture (NA) microscope objectives. The NA of an objective is the primary figure of merit that dictates its light-collection efficiency and diffraction-limited resolution.

Air objectives, which do not directly engage with the object, are fundamentally limited to NA values less than 1. Immersion objectives can surpass this limit by making physical contact with the sample via an immersion medium of a refractive index greater than 1. At room temperature, immersion objectives constitute a cornerstone of practically all high-resolution light microscopy. But for imaging below the glass transition of water (‑135 °C), no satisfactory counterpart exists.

We have recently established a new method that for the first time enables high-quality immersion light microscopy below the glass transition temperature of water. During imaging, our objective is not in thermal equilibrium. Instead, an electrical heating system near the connection between a ceramic front lens mount and the housing of the objective adjusts its power to maintain a stable temperature gradient across the lens mount (Fig. 1). Surprisingly, the small front lens is able to withstand the deep temperature cycles without damage due to the precise matching of thermal expansion coefficients in our system and because of the small size of the front lens itself.

Figure 1. (a) Immersion microscopy at cryogenic temperature is enabled by an objective in which the front lens and the objective body are not in thermal equilibrium. An electrical heater maintains a temperature gradient across the ceramic front lens mount. (b) This principle was implemented using a standard Zeiss LD C-Apochromat 63/1.15 water immersion lens design.

As the metal housing of the objective is always at room temperature during imaging, there is no risk of damaging the numerous larger lenses composing the bioimaging objective. At the same time, a thermally shielded microenvironment is created around the sample and front lens. An important advantage of our approach is that refractive index gradients due to temperature variations in the immersion liquid are small and not likely to distort the wavefront.

Equally important to the mechanical design of the objective is the choice of the immersion medium. Aberration-free imaging requires the refractive index to be within at least ~10‑3 refractive index units of the design value for the lens system. In addition, the immersion medium needs to be optically clear, nonfluorescent, and nontoxic, and needs to have low vapor pressure at the imaging temperature. Facile storage and handling, moreover, require that the liquid range should extend above room temperature.

Searching for a suitable immersion medium, we discovered that the partially fluorinated liquid ethoxynonafluorobutane (3M HFE-7200), has a surprisingly low refractive index (1.28) at room temperature and a liquid range from >70 °C to below ‑140 °C. HFE-7200 is also inexpensive, nontoxic, and safe for the environment. As the refractive index increases with decreasing temperature, HFE-7200 was a good candidate for matching the design of our prototype based on a Zeiss LD C-Apochromat 63/1.15 water immersion objective.

To find the optimal working temperature, we compared the point spread function (PSF) of our objective with HFE-7200 immersion to the PSF at room temperature using the standard medium (Zeiss W2010, n = 1.334). The overall shape and symmetry of the PSFs are very similar at ‑140 °C (HFE-7200) and at 23 °C (Zeiss W2010), indicating that the refractive index of HFE-7200 is well matched to the design of the objective at this temperature. These results were consistent across the entire field of view.

One of the key advantages of immersion objectives over air objectives is their light collection efficiency, which grows as ~NA². Indeed, we measured an increase in brightness of 5.7 ± 0.6 times from a 63/0.75 air objective to our 63/1.15 immersion objective at ‑140 °C. This was in agreement with the expected scale factor of ~NA⁴ for widefield fluorescence imaging.

The benefits of cryofixation and imaging in cryoconditions are evident when comparing the bleaching rates at room temperature and at ‑140 °C for GFP in yeast cells (Fig. 2a). A considerable improvement of both signal-to-noise ratio and image quality was achieved in cryoconditions where the photobleaching was suppressed by nearly two orders of magnitude. We also established the possibility of resolving submicrometer cell structures by multicolor cryofluorescence microscopy using our method. Figure 2b illustrates the attainable image quality in widefield fluorescence of immunostained U2OS cells at ‑140 °C. Networks of mitochondria (Tom20 immunolabeled with Alexa Fluor 594-decorated antibodies) and vimentin filaments (Alexa Fluor 488) were well resolved simultaneously.

Figure 2. (a) Bleaching is significantly reduced at ‑140 °C. This is shown here using yeast cells expressing GFP as an example. (b) Three-color widefield cryofluorescence image of plunge frozen U2OS cells labeled with Alexa Fluor 488 (vimentin cytoskeleton), Alexa Fluor 594 (Tom20 mitochondrial protein), and DAPI (cell nuclei). [Images: R. Faoro, M. Bassu. Cells and immunostaining: T. Stephan, S. Jakobs]

In conclusion, we demonstrated a concept to approach diffraction-limited performance in high-NA cryofluorescence microscopy with commercially available immersion objectives. To achieve this, we created a thermally shielded microenvironment around the sample by replacing the metallic front lens mount of the objective with an insulating ceramic mount that was heated around its perimeter. A further enabling step was our discovery of an immersion medium, HFE-7200, that provided accurate index matching at a sample temperature below the glass transition of water. In the future, we believe that this method will enable the combination of advanced light microscopy, including total internal reflection fluorescence or STED, with electron cryomicroscopy to help elucidate connections between structure and function at the subcellular and molecular scale.

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