In cryo-FM, the laser illuminates the samples on a TEM grid via the objective (Fig. ). The TEM grid is fixed in a grid holder connected to a metal bridge that is partially suspended in the coolant to maintain cryogenic temperatures. The TEM grid consists of many grid squares (Fig. ), covered with a carbon film that contains further holes where the samples are located (Fig. ). The width and height of a single 200 mesh TEM grid square is 90 µm, with the spacing of the metal grid being 30 µm wide. A side view of the EM grid shows the grid bar, the holey carbon film and the position of the vitreous ice layer containing the sample (Fig. ). The laser beam illuminates the sample, where care should be taken that heat transfer does not cause local devitrification of the sample. Based on the laser beam intensity distribution (Fig. ), the expected heat transfer can be estimated. Here, we have a homogenous intensity distribution that is convenient for estimating the total impact of heat on the samples. With a typical Gaussian beam profile, the region of high laser intensity is confined to the central area, which limits the field of view (FOV) to that area where fluorophores can be effectively illuminated. In contrast, with a homogeneous beam distribution, the laser intensity is uniform across the entire FOV, allowing for a larger area to be illuminated effectively. It was previously shown that high laser power could lead to the devitrification of the cryo-samples . The authors numerically calculated the expected temperature distribution using a finite element method (FEM) . Together with the notion that the devitrification of samples occurs at local temperatures above 136 K the authors showed that devitrification is more dependent on laser power than the illumination time. To prevent devitrification of the ice layer and damage from the local heating induced by the laser, we experimentally determined the optimal condition for our cryo-upright microscopy setup.