Fluorescence microscopy is often performed on inverted microscopes as this arrangement improves the stability of the optical setup and imposes less limitations on sample weight or height. Here, due to the need for having liquid nitrogen close to the sample to reach cryogenic temperatures, an upright microscope configuration was built above a LINKAM cryo-stage (Fig. ). The laser is coupled in a square-core multimode fibre with vibration motors to obtain a homogeneous intensity beam distribution. After the fibre, an iris (aperture) was placed to adjust the diameter of the beam. The laser beam is collimated with a lens and reflected by mirrors to an additional lens, which is inserted for beam expansion and wide-field imaging. Then, the laser beam is reflected via the dichroic mirror to the objective. We chose an air-immersion objective to minimize heat transfer between the objective and the sample held at cryogenic temperatures. In our experiments, we selected an air immersion objective with a high NA of 0.9 and a long working distance of 2 mm to reach the samples through the cryo-chamber (Fig. ). Focusing along the Z direction is controlled by the motorized translation stage and the XY position is controlled by an internal motorized stage implemented in the LINKAM cryo-stage. The emission beam is collected with the same objective and passes through the dichroic mirror. A rubber tube was inserted between the sample and the covering part of the objective to minimize the temperature difference between the lid of the sample stage and the top side of the objective (Fig. ). Without the rubber tube, the objective's top side was fogged due to the temperature difference leading to weakening of the fluorescence signal over time. In addition, this inserted rubber tube helped avoiding condensation of water vapor on the outside of the objective, thus preventing ice formation or contamination on the samples. Finally, the beam is focused onto an IDS camera using a tube lens. The picture of cryogenic fluorescence microscopy setup is described in the supplementary information (Fig. ). For measuring the beam intensity distribution in absence of the cryo-stage, we used a fluorescent dye (Cy3). In brief, a 20 µl volume of a 1 µM dye solution was placed between two cover glasses and excited using 40 W/cm 2 laser intensity at 520 nm excitation. The uniform spatial distribution of the laser beam intensity was achieved by mode mixing the fibre with a small coin motor. Our homogeneous beam distribution has a width of 60 µm at FW90M (full width at 90% of the maximum intensity) and 83 µm at FWHM (full width at half-maximum), thereby smaller than the size of a single grid square, ensuring that there is no further potential laser damage in other grid squares (Fig. ). The illumination area does not extend over the metal grid bar, ensuring that the beam effectively illuminates only a single grid square. We note that illumination over the metal grid bar potentially causes heating and concomitant devitrification of the samples.