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Utilizing a microfluidic chip with serpentine channels, we inoculated the chip with an agar plug with Neurospora crassa mycelium and successfully captured individual hyphae in channels. For the first time, we report the presence of an autonomous clock in hyphae. Fluorescence of a mCherry reporter gene driven by a clock-controlled gene-2 promoter (ccg-2p) was measured simultaneously along hyphae every half an hour for at least 6 days. We entrained single hyphae to light over a wide range of day lengths, including 6,12, 24, and 36 h days. Hyphae tracked in individual serpentine channels were highly synchronized (K = 0.60-0.78). Furthermore, hyphae also displayed temperature compensation properties, where the oscillation period was stable over a physiological range of temperatures from 24 °C to 30 °C (Q10 = 1.00-1.10). A Clock Tube Model developed could mimic hyphal growth observed in the serpentine chip and provides a mechanism for the stable banding patterns seen in race tubes at the macroscopic scale and synchronization through molecules riding the growth wave in the device. 

Abstract Utilizing a microfluidic chip with serpentine channels, we inoculated the chip with an agar plug withNeurospora crassamycelium and successfully captured individual hyphae in channels. For the first time, we report the presence of an autonomous clock in hyphae. Fluorescence of a mCherry reporter gene driven by aclock-controlled gene-2 promoter(ccg-2p) was measured simultaneously along hyphae every half an hour for at least 6 days. We entrained single hyphae to light over a wide range of day lengths, including 6,12, 24, and 36 h days. Hyphae tracked in individual serpentine channels were highly synchronized (K = 0.60-0.78). Furthermore, hyphae also displayed temperature compensation properties, where the oscillation period was stable over a physiological range of temperatures from 24 °C to 30 °C (Q₁₀ = 1.00-1.10). A Clock Tube Model developed could mimic hyphal growth observed in the serpentine chip and provides a mechanism for the stable banding patterns seen in race tubes at the macroscopic scale and synchronization through molecules riding the growth wave in the device. 

Abstract We determined the macroscopic limit for phase synchronization of cellular clocks in an artificial tissue created by a “big chamber” microfluidic device to be about 150,000 cells or less. The dimensions of the microfluidic chamber allowed us to calculate an upper limit on the radius of a hypothesized quorum sensing signal molecule of 13.05 nm using a diffusion approximation for signal travel within the device. The use of a second microwell microfluidic device allowed the refinement of the macroscopic limit to a cell density of 2166 cells per fixed area of the device for phase synchronization. The measurement of averages over single cell trajectories in the microwell device supported a deterministic quorum sensing model identified by ensemble methods for clock phase synchronization. A strong inference framework was used to test the communication mechanism in phase synchronization of quorum sensing versus cell-to-cell contact, suggesting support for quorum sensing. Further evidence came from showing phase synchronization was density-dependent. 

The use of a microwell microfluidic device allows separating single cells and tracking single cells data. The measurement of single cell fluorescent intensity trajectories in the microwell device supported a deterministic quorum sensing model identified by ensemble methods for clock phase synchronization. A strong inference framework was used to test the communication mechanism in phase synchronization of quorum sensing versus cell-to-cell contact, and the results lent support for quorum sensing. 

Most eukaryotes and cyanobacterial species have a biological clock that allows adaptation to the daily light/dark cycle of the planet. A central problem in the study of the biological clock is understanding the synchro-nization of the stochastic oscillators in different cells and tissues, but this problem is largely unstudied, particularly in the context of circadian rhythms. We developed a novel microfluidic platform to make high-throughput and high-precision measurements of biological clocks on a controlled number of Neurospora crassa (N. crassa) cells. Single cell measurements in this platform enabled us to test whether clocks of individual cells are able to communicate. 

We report a microfluidic device that mimics an artificial tissue to test the theory of quorum sensing as a method for synchronization of a model fungal system, Neurospora crassa (N. crassa). High synchronicity between cells were observed by calculating the Kuramoto order parameter (K) between different fields of view.The dimensions of the microfluidic chamber allows us to also calculate an upper limit of the radius of a hypothesized quorum sensing signal by using the diffusion approximation for signal travelling within the device.