Reflecting microscope system with a 0.99 numerical aperture designed for three-dimensional fluorescence imaging of individual molecules at cryogenic temperatures

We have developed a cryogenic fluorescence microscope system, the core of which is a reflecting objective that consists of spherical and aspherical mirrors. The use of an aspherical mirror allows the reflecting objective to have a numerical aperture (NA) of up to 0.99, which is close to the maximum possible NA of 1.03 in superfluid helium. The performance of the system at a temperature of 1.7 K was tested by recording a three-dimensional fluorescence image of individual quantum dots using excitation wavelengths (λ(ex)) of 532 nm and 635 nm. At 1.7 K, the microscope worked with achromatic and nearly diffraction-limited performance. The 1/e(2) radius (Γ) of the point spread function of the reflecting objective in the lateral (xy) direction was 0.212 ± 0.008 μm at λ(ex) = 532 nm and was less than 1.2 times the simulated value for a perfectly polished objective. The radius Γ in the axial (z) direction was 0.91 ± 0.04 μm at λ(ex) = 532 nm and was less than 1.4 times the simulated value of Γ. The chromatic aberrations between the two wavelengths were one order of magnitude smaller than Γ in each direction.