Fluorescence lifetime imaging (FLIM) has been used in living cells to measure metabolic activity and demonstrate cell differentiation. The aim of this study was to investigate whether the FLIM technique could be able to demonstrate cell maturation during myelopoiesis and erythropoiesis in unlabeled routine bone marrow (BM) preparations. Air-dried, unstained smears of BM aspiration samples of 32 patients without BM disease and a normal morphology on May–Grünwald–Giemsa (MGG) stained smears entered the study. FLIM images were captured with a Zeiss LSM 780 NLO multiphoton microscope equipped with a Becker & Hickl SPC-830 TCSPC FLIM module and HPM-100-40 hybrid detector. The samples were irradiated by two-photon excitation at 800 nm with a titanium-sapphire laser of the LSM 780 NLO. FLIM images were compared with those obtained by autofluorescence high resolution imaging. FLIM images of unstained smears were highly contrasted. Different cell types could be easily recognized as they were similar to those seen in MGG stained preparations. Cytoplasm of cells from the erythroid lineage revealed relatively short fluorescence lifetimes due to the presence of hemoglobin, and therefore could easily be distinguished from granulocytic precursors. Nuclear fluorescence lifetimes of all cell types were higher than those of the corresponding cytoplasm. So, FLIM of unstained BM smears obtained under routine real-life conditions permits an easy identification of BM cells, by highlighting differences of their physicochemical properties.

中文翻译：

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荧光寿命成像能够识别未染色的常规骨髓膜中的不同造血前体

荧光寿命成像 (FLIM) 已在活细胞中用于测量代谢活动并证明细胞分化。本研究的目的是调查 FLIM 技术是否能够证明未标记的常规骨髓 (BM) 制剂中骨髓生成和红细胞生成过程中的细胞成熟。32 名无 BM 疾病且 May-Grünwald-Giemsa (MGG) 染色涂片形态正常的 BM 抽吸样本的风干、未染色涂片进入研究。FLIM 图像使用配备 Becker & Hickl SPC-830 TCSPC FLIM 模块和 HPM-100-40 混合探测器的 Zeiss LSM 780 NLO 多光子显微镜捕获。样品用 LSM 780 NLO 的钛蓝宝石激光器通过 800 nm 的双光子激发进行照射。将 FLIM 图像与通过自发荧光高分辨率成像获得的图像进行比较。未染色涂片的 FLIM 图像对比度很高。不同的细胞类型很容易被识别，因为它们与 MGG 染色制剂中看到的相似。由于血红蛋白的存在，红系细胞的细胞质显示出相对较短的荧光寿命，因此很容易与粒细胞前体区分开来。所有细胞类型的核荧光寿命均高于相应细胞质的核荧光寿命。因此，在常规现实生活条件下获得的未染色 BM 涂片的 FLIM 通过突出其理化特性的差异，可以轻松识别 BM 细胞。不同的细胞类型很容易被识别，因为它们与 MGG 染色制剂中看到的相似。由于血红蛋白的存在，红系细胞的细胞质显示出相对较短的荧光寿命，因此很容易与粒细胞前体区分开来。所有细胞类型的核荧光寿命均高于相应细胞质的核荧光寿命。因此，在常规现实生活条件下获得的未染色 BM 涂片的 FLIM 通过突出其理化特性的差异，可以轻松识别 BM 细胞。不同的细胞类型很容易被识别，因为它们与 MGG 染色制剂中看到的相似。由于血红蛋白的存在，红系细胞的细胞质显示出相对较短的荧光寿命，因此很容易与粒细胞前体区分开来。所有细胞类型的核荧光寿命均高于相应细胞质的核荧光寿命。因此，在常规现实生活条件下获得的未染色 BM 涂片的 FLIM 通过突出其理化特性的差异，可以轻松识别 BM 细胞。因此很容易与粒细胞前体区分开来。所有细胞类型的核荧光寿命均高于相应细胞质的核荧光寿命。因此，在常规现实生活条件下获得的未染色 BM 涂片的 FLIM 通过突出其理化特性的差异，可以轻松识别 BM 细胞。因此很容易与粒细胞前体区分开来。所有细胞类型的核荧光寿命均高于相应细胞质的核荧光寿命。因此，在常规现实生活条件下获得的未染色 BM 涂片的 FLIM 通过突出其理化特性的差异，可以轻松识别 BM 细胞。