ABSTRACT
Cryogenic fluorescent light microscopy of flash-frozen cells stands out by artifact-free fixation and very little photobleaching of the fluorophores used. To attain the highest level of resolution, aberration-free immersion objectives with accurately matched immersion media are required, but both do not exist for imaging below the glass-transition temperature of water. Here, we resolve this challenge by combining a cryoimmersion medium, HFE-7200, which matches the refractive index of room-temperature water, with a technological concept in which the body of the objective and the front lens are not in thermal equilibrium. We implemented this concept by replacing the metallic front-lens mount of a standard bioimaging water immersion objective with an insulating ceramic mount heated around its perimeter. In this way, the objective metal housing can be maintained at room temperature, while creating a thermally shielded cold microenvironment around the sample and front lens. To demonstrate the range of potential applications, we show that our method can provide superior contrast in Escherichia coli and yeast cells expressing fluorescent proteins and resolve submicrometer structures in multicolor immunolabeled human bone osteosarcoma epithelial (U2OS) cells at [Formula: see text]C.
Subject(s)
Histological Techniques/methods , Microscopy/methods , Cell Line , Equipment Design , Escherichia coli/cytology , Escherichia coli/genetics , Fluorescent Dyes/chemistry , Freezing , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Image Processing, Computer-Assisted , Microscopy/instrumentation , Microscopy, Fluorescence/methods , Photobleaching , Refractometry , Yeasts/cytology , Yeasts/geneticsABSTRACT
Stable passive Q-switching of a Tm: LiYF4 laser is obtained using polycrystalline Cr2+: ZnS as a saturable absorber. The achieved maximum pulse energy of 0.9 mJ and peak power of 65 kW for a pulse duration of â¼14 ns represent substantial improvement and highest values for a passively Q-switched diode-pumped Tm laser operating at â¼1.9 µm.
