A schematic of a microfluidic device to detect the oxygen consumption rates of a single developing zebrafish embryo by tracking the oxygen concentration of the medium over time via phase-based phosphorescence lifetime detection. The microfluidic device combines two components: an array of glass microwells deposited with PtOEP as an oxygen-sensitive luminescent layer and a microfluidic module with pneumatically actuated lids above the microwells to controllably seal the microwells of interest. The phase-based phosphorescence lifetime detection system utilizes a light modulation system, which is equipped with a UV LED as a light modulation source. The long-term cellular OCR measurement was repeated over time via a periodic three-stage operation for replenishing the microwell with fresh medium, sealing the microwell, and measuring the oxygen concentration.
(a) An expanded drawing and (b) an image of the microfluidic device, which was assembled using three layers (layers 1–3) of PDMS structures that served as a microfluidic module and one layer (layer 4) of a glass substrate with 2 × 2 microwells inside a PDMS microchamber. (c) Close-up of one microwell containing a single 24 hpf zebrafish embryo.
A schematic of the light modulation system equipped with a UV LED as the light modulation source. A programmable motorized X-Y stage was used to spatially direct the modulated excitation light toward the sealed microwell of interest to determine the OCR via phase-based phosphorescence lifetime detection. (CL: collimating lens; BS: beam splitter; RS: reference signal; DS: detection signal; APD: amplified photo-detector; and PMT: photo-multiplier tube).
Images of a single developing embryo inside a microwell taken at various time points of development from the blastula (3 hpf) to the hatching (48 hpf) stage to show the long-term development and survival.
(a) The time variation of the oxygen concentration [O2] and (b) the oxygen consumption rate of a single developing zebrafish embryo (OCR(t), −d[O2]/dt) at 3 hpf and 24 hpf inside microwells for 3 successive measurements. These measurements were performed by a periodic three-stage operation of replenishing the microwell with fresh medium (O-stage), sealing the microwell (S-stage), and measuring the oxygen concentration (M-stage). (Mean ± SEM; n > 8).
The total basal respiration (OCR, in pmol O2/min/embryo) during the development of a single zebrafish embryo in the blastula (3 hpf), gastrula (7 hpf), segmentation (12 hpf), segmentation/pharyngula transition (24 hpf), mid-pharyngula (36 hpf), and hatching (48 hpf) stages inside a microfluidic device. (Mean ± SEM; n = 4 from 2 × 2 microwells).
The metabolic profiles of a single developing zebrafish embryo at (a) 3 hpf and (b) 48 hpf, measured by sequentially adding pharmacological inhibitors of bioenergetic pathways. The time courses are annotated to show the relative contribution of non-mitochondrial respiration, respiration due to ATP turnover, respiration due to proton leak, maximal OCR after the addition of FCCP, and reserve capacity of a single developing zebrafish embryo. (oligomycin: inhibits ATP synthase; FCCP: an uncoupler that allows maximal respiration; and sodium azide inhibits total mitochondrial respiration) (Mean ± SEM; n > 8).
(a) The variation in the components of the total basal respiration and the mitochondrial reserve capacity in pmol O2/min/embryo with developmental stage at 3, 7, 12, 24, 36, and 48 hpf. The total basal respiration is composed of a non-mitochondrial fraction and a mitochondrial fraction, which is further composed of the parts of respiration associated with proton leak and with ATP turnover. (b) The change in the partitioning of the total basal respiration with zebrafish embryonic development, determined by calculating the percentage that each fraction of respiration contributes to the total basal respiration (100%). (Mean ± SEM; n = 4 from 2 × 2 microwells).
The developmental stages of a zebrafish embryo and the OCR measured at these stages.
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