Microfluidic chip features a microstructure with cavity, which is formed by a piece of Polydimethylsiloxane (PDMS) with microstructure etched onto its surface bonded to a piece of glass plate of certain thickness. PDMS is transparent, physiologically inert, chemically stable, hydrophobic and resilient, making it suitable for building microfluidic chips.

The microstructures include the “cell trap”, the “channel”, the heat exchanger, the pores and encoding matrix.

The cell trap is a channel of certain size, featuring a “neck” of certain size, which fixes yeast cells (mother cells) of certain range of sizes while allowing the spores (daughter cells) of the mother cells to detach under static pressure (Figure). This allows the fixation/anchoring of the targeted cells, constant supplement of fresh culturing media, and separation of daughter cells, enabling the robust real-time tracking of fluorescent intensity in single cell resolution and RLS measurement of yeast cells.

The “channel” is responsible for distributing cells and culturing media into “cell traps”, while maintaining a dynamic pressure caused by different flow rates.

The heat exchanger is a twisting section where fluid inside slows down under larger resistance and exchange heat with the environment of incubator, which provides normal temperature for yeast growth and survival.

The pores include holes punctured all the way through the PDMS material and etched areas enabling continuity of the holes to the rest of the microstructures.

The encoding matrix are made of several submatrix, each of which include one positioning dot and five encoding dots. Positioning dot confirms the existence of the submatrix and provides absolute positioning information relative to adjacent “cell traps”. Encoding dots in each submatrix occupy five positions, corresponding to five digits in binary. Etched holes indicate “one”, while unetched places indicate “zero”, hence the 32 differentiated submatrix for the positioning of “cell traps”.

The microstructures are etched on the surface of the molds using lithography, which are then formed on PDMS materials. There are two etching depths to the microstructures, 4.5 micron meter and 15 micron meter, requiring overlay process, which involves two sets of masks and positioning errors of patterns of two etching depths. Considering the required preciseness of relative position between positioning dots in dot submatrix and “cell traps”, the dot matrix and “cell traps” are designed with identical etching depth 4.5 micron meter and laid out on the same set of mask, ensuring robustness pf positioning assistance.

The PDMS material with microstructure is bonded to glass plate with certain thickness, which is primarily determined considering the lens used for measurement. Quantification of the intensity of mcherry requires using 60x lens to observe the nucleus, which has a limited working distance below 0.2mm. Therefore, cover slips that are 0.17mm in thickness are bond to the PDMS material.

Cell loading and constant supply of culturing media are achieved through the plumbing system, which includes 50ml syringe as reservoir, Teflon tubing, syringe needle with flat top as adapter between syringe and Teflon tubes, 90-degree bended stainless-steel tubes as adapter between Teflon tubes and the microfluidic chip.

The whole system is gravity driven. Syringe containing culturing media is secured on certain height relative to the chip for constant working pressure and fluid supply. A tripod-based stand is used to secure and record all equipment with height differences. An outlet tube is connected to the outlet pore and the outlet of tube is kept on the same height as the outlet pore to avoid excessive positive or negative pressure.

Choosing thin bed plates bring about less structural integrity and robustness. The 60x lens in measurement have shallow depth of field, which means that subtle deformation caused by small forces are significant enough to bring trouble to observation and quantification. Strains from the pipes are also transduced to the chip through the stainless-steel tubes, causing deformation to the whole chip. Further, the strains are higher around the pores to the extent that the pores tore, reducing concealment under high hydraulic pressure.

A jig was designed and fabricated, containing two pieces, the bottom plate and the midframe-cover assembly.

The bottom plate is a thin plate which is not the main contributor to increasing structural integrity. There is a hole designed in the middle, where the lens approaches the chip, to avoid interference of the lens with the bottom plate.

The midframe is the main load-carrying structure of the jig. There is a square hole in the middle to accommodate the PDMS part of the chip. On edges of the short sides, two sinking structures are designed for compatibility to the clamp in the incubation chamber of the microscope. On the top surface, several screw holes are designed to attach the cover to the midframe with M2 screws.

Both bottom plate and midframe are fabricated through 3D printing using light curing resin for ease of manufacturing.

The cover is made of acrylic for transparency. Several screw holes are reserved for attachment to midframe. Two holes of slightly larger diameter to stainless-steel tubes are hand drilled to allow passage of tubes while reducing stress on the PDMS material from pipes. The cover plate screwed to midframe adds to the rigidity of the assembly.

The jig and chip are assembled with paper tapes securing them from the vertical sides.