講演情報
[10p-N305-10]Label-Free Characterization of Plant Protoplasts Using Multifrequency Impedance Flow Cytometry
〇(DC)Trisna Julian1, Muhammad Ridho Priyo1, Tao Tang2, Naomi Tanga1, Yang Yang3, Yoichiroh Hosokawa1, Yaxiaer Yalikun1 (1.NAIST, 2.Chongqing Gen. Hosp., 3.Chinese Aca. of Sci.)
キーワード：
cell characterization、impedance measurement、microfluidics device
Genetically modified (GM) plants provide a promising approach for developing climate-resilient crops. A key method in plant genetic engineering involves protoplasts—plant cells without cell walls—which offer direct access to intracellular machinery and can regenerate into full plants. However, characterizing individual protoplasts remains difficult because conventional methods like flow cytometry depend on fluorescent labeling, which can compromise viability, limit marker compatibility, and hinder long-term observation. To address this, we developed a label-free, multifrequency impedance flow cytometry system for single-cell characterization of plant protoplasts.
Our microfluidic device incorporates three electrodes: the central electrode is stimulated with an AC signal containing three frequencies targeting different cellular components. The current across the side electrodes is differentially converted to voltage and demodulated using a lock-in amplifier, yielding frequency-resolved impedance data.
To validate the system, we measured tobacco BY-2 protoplasts at 0, 12, and 24 hours after enzymatic cell wall removal. As the cell wall regenerates, changes in membrane and cytoplasmic properties are reflected in the impedance signal. Electrical diameter and low-frequency phase shift correlated with increasing cell size and wall formation. Frequency-dependent opacity, reflecting membrane shielding, increased with maturation. We also assessed mechanical properties by introducing a channel constriction. Increased transit time at lower channel height indicated rising stiffness as regeneration progressed.
These results demonstrate that label-free, multifrequency impedance cytometry enables non-invasive, scalable evaluation of plant protoplasts' electrical and mechanical properties during regeneration.
Our microfluidic device incorporates three electrodes: the central electrode is stimulated with an AC signal containing three frequencies targeting different cellular components. The current across the side electrodes is differentially converted to voltage and demodulated using a lock-in amplifier, yielding frequency-resolved impedance data.
To validate the system, we measured tobacco BY-2 protoplasts at 0, 12, and 24 hours after enzymatic cell wall removal. As the cell wall regenerates, changes in membrane and cytoplasmic properties are reflected in the impedance signal. Electrical diameter and low-frequency phase shift correlated with increasing cell size and wall formation. Frequency-dependent opacity, reflecting membrane shielding, increased with maturation. We also assessed mechanical properties by introducing a channel constriction. Increased transit time at lower channel height indicated rising stiffness as regeneration progressed.
These results demonstrate that label-free, multifrequency impedance cytometry enables non-invasive, scalable evaluation of plant protoplasts' electrical and mechanical properties during regeneration.