Resolution enhancement for low-temperature scanning microscopy by cryo-immersion

Abstract

Abstract:Here we report a simple way to enhance the resolution of a confocal scanning microscope under cryogenic conditions. Using a microscope objective (MO) with high numerical aperture (NA = 1.25) and 1-propanol as an immersion fluid with low freezing temperature we were able to reach an imaging resolution at 160 K comparable to ambient conditions. The MO and the sample were both placed inside the inner chamber of the cryostat to reduce distortions induced by temperature gradients. The image quality of our commercially available MO was further enhanced by scanning the sample (sample scanning) in contrast to beam scanning. The ease of the whole procedure marks an essential step towards the development of cryo high-resolution microscopy and correlative light and electron cryo microscopy (cryoCLEM).

Photon Yield Enhancement of Red Fluorophores at Cryogenic Temperatures

“…For example with a fluid immersion lens [18,19] or solid immersion lens. This is a more than hundred-fold increase in photon yield over room temperature, leading to a 10 fold increase in localization precision.…”

“…An additional enhancement in localisation precision could be attained by combining cryogenic sample conditions with high NA objectives. For example with a fluid immersion lens [18,19] or solid immersion lens. [20,21] From this study it seems that JF 646, Alexa Fluor 647, and ATTO 655 give the highest photon yields resulting in the best localization precision.…”

Correlated Cryo Super‐Resolution Light and Cryo‐Electron Microscopy on Mammalian Cells Expressing the Fluorescent Protein rsEGFP2

“…The axial resolution could be improved by, e.g., placing a cylindrical lens in the optical system so changes in location can be estimated by measuring the astigmatism of the detected single‐molecule signals . Alternatively, the resolution of cryoSRM could be further improved by enabling objective lenses with a higher NA, for instance by utilizing immersion lenses . Nevertheless, by employing the reduced photobleaching characteristics of FPs inherent with low temperature imaging, localization accuracies in the sub‐nm range have been achieved …”

“…[39,64] Alternatively, the resolution of cryoSRM could be further improved by enabling objective lenses with a higher NA, for instance by utilizing immersion lenses. [65][66][67] Nevertheless, by employing the reduced photobleaching characteristics of FPs inherent with low temperature imaging, [13,[68][69][70] localization accuracies in the sub-nm range have been achieved. [68,71]…”

Determination of the refractive index of liquids at cryogenic temperature

“…In recent years, different approaches for cryoimmersion microscopy have been implemented, [2][3][4][5] but they all share the same limitation: the refractive index of the immersion fluid at the operating temperature is unknown and could be guessed only from the data available at RT. In our previous work, 5 we performed cryo-immersion microscopy at 133 K by using a water immersion objective, modified to work in cryo-conditions, in combination with the immersion fluid HFE 7200.…”