A novel method for determining murine skeletal muscle fiber type using autofluorescence lifetimes.
A novel method for determining murine skeletal muscle fiber type using autofluorescence lifetimes.
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DOI：
10.1085/jgp.202213143
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发表时间：
2022-09-05
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Manno等人。展示了一种简单、快速和非侵入性的程序，通过在活体骨骼肌上使用可见光激发并通过共聚焦光子计数测量的自体荧光的寿命来区分I型和II型肌纤维。这项工作描述了一种通过荧光寿命成像显微镜(FLIM)识别活体肌肉中纤维类型的简单方法。我们量化了新鲜解剖的小鼠指短屈肌和比目鱼肌的寿命平均值τ1和τ2，这是由两个指数拟合得出的。虽然τ1的值随肌肉之间的双峰行为而变化，但τ2的分布在FDB中向更高的值移动。为了了解这种差异的来源，我们在冰冻切片中获得了黄素单核苷酸和二核苷酸(FMN/FAD)的自发荧光寿命图，其中激发波长设置为440 nm，发射带宽设置在500到570 nm之间，并将它们与肌球蛋白重链亚型的免疫荧光图像配对，从而可以识别纤维类型。比目鱼肌τ为3.16 ns(SD 0.11，97个纤维)，IIA为3.45 ns(0.10，69)，IIX为3.46 ns(0.12，65)。τ-2分别为3.17 ns(0.08，22)、3.46 ns(0.16，48)和3.66 ns(0.15，43)。从τ2的分布可以推断，τ2为3.3 ns的FDB光纤应为类型II，否则为类型I。这种简单的分类方法的第一类和第二类误差估计为0.02和0.10，可以通过降低I型识别的阈值和增加II型的识别阈值来降低。因此，自体荧光寿命图构成了一种识别纤维类型的工具，因为它实用、快速和非侵入性，可以在不影响其他实验干预的情况下应用于活组织。
Manno et al. demonstrate a simple, fast, and noninvasive procedure, whereby the lifetimes of autofluorescence excited with visible light and measured by confocal photon counting are used on live skeletal muscle to distinguish type I from type II myofibers. This work describes a simple way to identify fiber types in living muscles by fluorescence lifetime imaging microscopy (FLIM). We quantified the mean values of lifetimes τ1 and τ2 derived from a two-exponential fit in freshly dissected mouse flexor digitorum brevis (FDB) and soleus muscles. While τ1 values changed following a bimodal behavior between muscles, the distribution of τ2 is shifted to higher values in FDB. To understand the origin of this difference, we obtained maps of autofluorescence lifetimes of flavin mononucleotide and dinucleotide (FMN/FAD) in cryosections, where excitation was set at 440 nm and emission at a bandwidth of between 500 and 570 nm, and paired them with immunofluorescence images of myosin heavy chain isoforms, which allowed identification of fiber types. In soleus, τ2 was 3.16 ns for type I (SD 0.11, 97 fibers), 3.45 ns for IIA (0.10, 69), and 3.46 ns for IIX (0.12, 65). In FDB muscle, τ2 was 3.17 ns for type I (0.08, 22), 3.46 ns for IIA (0.16, 48), and 3.66 ns for IIX (0.15, 43). From τ2 distributions, it follows that an FDB fiber with τ2 > 3.3 ns is expected to be of type II, and of type I otherwise. This simple classification method has first and second kind errors estimated at 0.02 and 0.10, which can be lowered by reducing the threshold for identification of type I and increasing it for type II. Lifetime maps of autofluorescence, therefore, constitute a tool to identify fiber types that, for being practical, fast, and noninvasive, can be applied in living tissue without compromising other experimental interventions.
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