A new fiber functionalization architecture for single‐fiber imaging and sensing is presented. 5(6)Carboxy‐seminaphthorhodafluor‐2 (a fluorescent pH sensor) is attached to a silk‐binding peptide and the complex added to aqueous silk fibroin protein. These bind with a Kd of 36 µM as determined by a fluorescence polarization assay. The fiber is dip‐coated into the silk and peptide mixture, and scanning electron microscopy images reveal a uniform silk coating on the fiber tip. The coating is stable to repeated washes and does not affect the imaging light emitted from the fiber, which allows concurrent optical coherence tomography (OCT) imaging and pH sensing. Oocytes are metabolically stimulated with CoCl2 to produce lactic acid, and a pH reduction of 0.04 is measured using the probe. The distance between fiber tip and oocyte is monitored by simultaneous OCT acquisitions to precisely position the probe. Lastly, OCT imaging of an ovary revealed the presence/absence of an oocyte within a follicle, an important step toward improving patient outcomes during in vitro fertilization, by limiting the number of invasive follicle punctures required. These results demonstrate the utility of this new coating to enable simultaneous OCT imaging and sensing, which provides significant insight into complex biological systems.
Purpose Intracytoplasmic sperm injection (ICSI) addresses male sub-fertility by injecting a spermatozoon into the oocyte. This challenging procedure requires the use of dual micromanipulators, with success influenced by inter-operator expertise. We hypothesized that minimizing oocyte handling during ICSI will simplify the procedure. To address this, we designed and fabricated a micrometer scale device that houses the oocyte and requires only one micromanipulator for microinjection. Methods The device consisted of 2 components, each of sub-cubic millimeter volume: a Pod and a Garage. These were fabricated using 2-photon polymerization. Toxicity was evaluated by culturing single-mouse presumptive zygotes (PZs) to the blastocyst stage within a Pod, with several Pods (and embryos) docked in a Garage. The development was compared to standard culture. The level of DNA damage/repair in resultant blastocysts was quantified (γH2A.X immunohistochemistry). To demonstrate the capability to carry out ICSI within the device, PZs were microinjected with 4-μm fluorescent microspheres and cultured to the blastocyst stage. Finally, the device was assessed for oocyte traceability and high-throughput microinjection capabilities and compared to standard microinjection practice using key parameters (pipette setup, holding then injecting oocytes). Results Compared to standard culture, embryo culture within Pods and a Garage showed no differences in development to the blastocyst stage or levels of DNA damage in resultant blastocysts. Furthermore, microinjection within our device removes the need for a holding pipette, improves traceability, and facilitates high-throughput microinjection. Conclusion This novel device could improve embryo production following ICSI by simplifying the procedure and thus decreasing inter-operator variability.
Purpose Vitrification permits long-term banking of oocytes and embryos. It is a technically challenging procedure requiring direct handling and movement of cells between potentially cytotoxic cryoprotectant solutions. Variation in adherence to timing, and ability to trace cells during the procedure, affects survival post-warming. We hypothesized that minimizing direct handling will simplify the procedure and improve traceability. To address this, we present a novel photopolymerized device that houses the sample during vitrification. Methods The fabricated device consisted of two components: the Pod and Garage. Single mouse oocytes or embryos were housed in a Pod, with multiple Pods docked into a Garage. The suitability of the device for cryogenic application was assessed by repeated vitrification and warming cycles. Oocytes or early blastocyst-stage embryos were vitrified either using standard practice or within Pods and a Garage and compared to non-vitrified control groups. Post-warming, we assessed survival rate, oocyte developmental potential (fertilization and subsequent development) and metabolism (autofluorescence). Results Vitrification within the device occurred within ~ 3 nL of cryoprotectant: this volume being ~ 1000-fold lower than standard vitrification. Compared to standard practice, vitrification and warming within our device showed no differences in viability, developmental competency, or metabolism for oocytes and embryos. The device housed the sample during processing, which improved traceability and minimized handling. Interestingly, vitrification-warming itself, altered oocyte and embryo metabolism. Conclusion The Pod and Garage system minimized the volume of cryoprotectant at vitrification—by ~ 1000-fold—improved traceability and reduced direct handling of the sample. This is a major step in simplifying the procedure.
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