Thiem Daniel B, Szabo Greta, Burg Thomas P
Integrated Micro-Nano-Systems Laboratory, Technische Universität Darmstadt, 64283 Darmstadt, Germany.
Centre for Synthetic Biology, Technische Universität Darmstadt, 64289 Darmstadt, Germany.
Micromachines (Basel). 2024 Aug 24;15(9):1069. doi: 10.3390/mi15091069.
Cryofixation by ultra-rapid freezing is widely regarded as the gold standard for preserving cell structure without artefacts for electron microscopy. However, conventional cryofixation technologies are not compatible with live imaging, making it difficult to capture dynamic cellular processes at a precise time. To overcome this limitation, we recently introduced a new technology, called microfluidic cryofixation. The principle is based on micro-hotplates counter-cooled with liquid nitrogen. While the power is on, the sample inside a foil-embedded microchannel on top of the micro-hotplate is kept warm. When the heater is turned off, the thermal energy is drained rapidly and the sample freezes. While this principle has been demonstrated experimentally with small samples (<0.5 mm), there is an important trade-off between the attainable cooling rate, sample size, and heater power. Here, we elucidate these connections by theoretical modeling and by measurements. Our findings show that cooling rates of 10 K s, which are required for the vitrification of pure water, can theoretically be attained in samples up to ∼1 mm wide and 5 μm thick by using diamond substrates. If a heat sink made of silicon or copper is used, the maximum thickness for the same cooling rate is reduced to ∼3 μm. Importantly, cooling rates of 10 K s to 10 K s can theoretically be attained for samples of arbitrary area. Such rates are sufficient for many real biological samples due to the natural cryoprotective effect of the cytosol. Thus, we expect that the vitrification of millimeter-scale specimens with thicknesses in the 10 μm range should be possible using micro-hotplate-based microfluidic cryofixation technology.
超快速冷冻的低温固定被广泛认为是用于电子显微镜下无伪像保存细胞结构的金标准。然而，传统的低温固定技术与实时成像不兼容，使得难以在精确时间捕捉动态细胞过程。为克服这一限制，我们最近引入了一种名为微流控低温固定的新技术。其原理基于用液氮反向冷却的微型热板。当电源开启时，微型热板顶部箔片嵌入微通道内的样品保持温热。当加热器关闭时，热能迅速排出，样品冻结。虽然这一原理已在小样品（<0.5毫米）上通过实验得到证明，但在可实现的冷却速率、样品尺寸和加热器功率之间存在重要的权衡。在此，我们通过理论建模和测量来阐明这些关系。我们的研究结果表明，通过使用金刚石基板，理论上在宽度达约1毫米、厚度为5微米的样品中可实现纯水玻璃化所需的10 K/s的冷却速率。如果使用由硅或铜制成的散热器，对于相同的冷却速率，最大厚度会减小到约3微米。重要的是，对于任意面积的样品，理论上可实现10 K/s至10 K/s的冷却速率。由于细胞质溶胶的天然冷冻保护作用，这样的速率对于许多实际生物样品来说是足够的。因此，我们预计使用基于微型热板的微流控低温固定技术，应该能够实现厚度在10微米范围内的毫米级标本的玻璃化。
