■为了在不使用破坏性探针的情况下对完整肿瘤组织中的药物进行非破坏性纵向评估,我们设计了一种无标记方法,使用多光子荧光寿命成像显微镜(MP-FLIM)对切除的肿瘤组织中单个肿瘤细胞的健康状况进行定量.
■使用保留天然肿瘤微环境的鼠肿瘤片段,我们试图证明固有荧光代谢辅因子烟酰胺腺嘌呤二核苷酸磷酸[NAD(P)H]和黄素腺嘌呤二核苷酸(FAD)产生的信号与导致细胞死亡的不可逆级联反应相关。
■我们在组织上使用NAD(P)H和FAD的MP-FLIM,并使用标准凋亡和活/死(Caspase3/7和碘化丙啶,分别)测定。
■通过统计方法,FLIM数据的可重复变化,通过相量分析确定,显示与细胞活力的丧失相关。有了这个,我们证明可以区分通过凋亡/坏死或坏死性凋亡实现的细胞死亡。此外,检测到对常见化疗治疗诱导细胞死亡的特异性反应。
■这些数据表明,MP-FLIM可以在不使用潜在毒性染料的情况下检测和定量细胞活力,因此,能够进行为期多天的纵向研究,评估治疗药物对肿瘤碎片的影响。
UNASSIGNED: To enable non-destructive longitudinal assessment of drug agents in intact tumor tissue without the use of disruptive probes, we have designed a label-free method to quantify the health of individual tumor cells in excised tumor tissue using multiphoton fluorescence lifetime imaging microscopy (MP-FLIM).
UNASSIGNED: Using murine tumor fragments which preserve the native tumor microenvironment, we seek to demonstrate signals generated by the intrinsically fluorescent metabolic co-factors nicotinamide adenine dinucleotide phosphate [NAD(P)H] and flavin adenine dinucleotide (FAD) correlate with irreversible cascades leading to cell death.
UNASSIGNED: We use MP-FLIM of NAD(P)H and FAD on tissues and confirm viability using standard apoptosis and live/dead (Caspase 3/7 and propidium iodide, respectively) assays.
UNASSIGNED: Through a statistical approach, reproducible shifts in FLIM data, determined through phasor analysis, are shown to correlate with loss of cell viability. With this, we demonstrate that cell death achieved through either apoptosis/necrosis or necroptosis can be discriminated. In addition, specific responses to common chemotherapeutic treatment inducing cell death were detected.
UNASSIGNED: These data demonstrate that MP-FLIM can detect and quantify cell viability without the use of potentially toxic dyes, thus enabling longitudinal multi-day studies assessing the effects of therapeutic agents on tumor fragments.