Microfluidic cryo-fixation of biomass, e.g., cells, flagella/cilia, or proteins for (in particular) correlative light-electron microscopy systems

Here, a method was invented to enable the culturing, stimulation, and in-situ cryo-fixation of cells on a microfluidic chip without the need for chemical cryo-protectants.

The enabling concept is to suspend cells in a freestanding microfluidic channel that has a low volume and a very short thermal time constant. If the channel is connected to a cold reservoir through at least one heated support, cells and nutrients can be delivered while the heater keeps the fluid at room temperature. When the heater is switched off, the channel equilibrates rapidly with the cold reservoir. This results in vitrification of the sample if the thermal time constant is sufficiently short (Fig. 1).

• Ultra-rapid cooling below critical water crystallization temperature in the rage of 10 to 100µs
• Works at atmospheric pressures
• Uninterrupted visual sample access prior and during freezing

• Post-fixation optical, Electron, or X-ray microscopy
• Organic single cells as samples (no cryo-protectants needed)
• FIB-SEM systems (cf. [2,3])

Cryofixation yields outstanding ultrastructural preservation of cells for electron microscopy, but current methods disrupt live cell imaging. Here a microfluidic approach is demonstrated that enables cryofixation to be performed directly in the light microscope with millisecond time resolution and at atmospheric pressure without using chemical cryo-protectants. This provides a link between imaging/stimulation of live cells and post-fixation optical, electron, or X-ray microscopy.

Microfluidic technology provides an excellent platform for applications that require localized heating and cooling with short time constants. Some examples are briefly outlined within the electronic supporting information of Ref. [1]. Compared with these studies, the work described here overcomes three key challenges. The first is to approach initial cooling rates of at least ~10⁴ °C s⁻¹ to suppress ice crystallization. Secondly, the final temperature must be below −140 °C to prevent de-vitrification; and third, continuous flow of fresh media is required during live imaging prior to freezing. These points are addressed in the design by

1. minimizing the thermal mass of the channel containing the sample,
2. minimizing the distance between the cold surface and the sample, and
3. maintaining uninterrupted, room-temperature connections into and out of the heated microchannel.

In particular, it was found that microfluidic perfusion devices of extremely low thermal mass could be made using microfluidic polymer foils in combination with silicon micromachined components.

[1] Mejia, Y. X., Feindt, H., Zhang, D., Steltenkamp, S., & Burg, T. P. (2014). Microfluidic cryofixation for correlative microscopy. Lab on a Chip, 14(17), 3281–3284. https://doi.org/10.1039/C4LC00333K

[2] Fuest, M., Nocera, G. M., Modena, M. M., Riedel, D., Mejia, Y. X., Burg, T. P. (2018). Cryofixation during live-imaging enables millisecond time-correlated light and electron microscopy. Journal of Microscopy, 272 (2), 87-95. https://doi.org/10.1111/jmi.12747

[3] Fuest, M., Schaffer, M., Nocera, G. M., Galilea-Kleinsteuber, R. I., Messling, J.-E., Heymann, M., Plitzko, J. M., Burg, T. P. (2019). In situ Microfluidic Cryofixation for Cryo Focused Ion Beam Milling and Cryo Electron Tomography. Scientific Reports, 9, 19133. https://doi.org/10.1038/s41598-019-55413-2

[4] PCT (WO2009065585A3), EP, US, JP