König K, Tadir Y, Patrizio P, Berns M W, Tromberg B J
Institute of Anatomy II, Friedrich Schiller University, Jena, Germany.
Hum Reprod. 1996 Oct;11(10):2162-4. doi: 10.1093/oxfordjournals.humrep.a019069.
Photostress has to be considered during optical micromanipulation of gametes. Ultraviolet light, including low-energy UVA (32-400 nm) radiation, as well as high-intensity near infrared (NIR) laser radiation may induce cell damage. A total number of 580 light-exposed sperm cells were studied in single-cell photostress experiments. Low-power (1.5 mW, 5.3 W/cm2) UVA exposure with 365 nm radiation of a standard mercury microscopy lamp to human spermatozoa resulted within 109 +/- 30 s in paralysis and within 310 +/- 110 s in cell death. Cytotoxic effects during cell manipulation with laser microbeams were found to be partly based on non-linear excitation phenomena, in particular two-photon absorption by endogenous cell chromophores. Two-photon absorption will be more intense in the case of pulsed laser microradiation, but occur also during micromanipulation with highly focused continuous wave (cw) microbeams used as laser tweezers ('optical traps'). In particular, short-wavelength NIR traps < 800 nm induce UVA-like biological effects (oxidative stress). For example, sperm trapping with 760 nm microbeams resulted in UVA-like autofluorescence modifications, paralysis within 35 +/- 20 s and cell death within 65 +/- 20 s. In contrast, laser microbeams at 800-1064 nm may act as relatively safe micromanipulation tools. In most optical traps multifrequency cw lasers are employed. Radiation of these lasers can magnify cytotoxic effects. Therefore, single-frequency laser operation should be preferred. In general, laser assisted cell micromanipulation requires a new understanding of microbeam-cell interaction, including aspects of non-linear optics.
在对配子进行光学显微操作时必须考虑光应激。紫外线，包括低能量的UVA（32 - 400纳米）辐射以及高强度近红外（NIR）激光辐射，都可能诱导细胞损伤。在单细胞光应激实验中研究了总共580个受光照射的精子细胞。用标准汞显微镜灯的365纳米辐射对人类精子进行低功率（1.5毫瓦，5.3瓦/平方厘米）UVA照射，在109±30秒内导致精子麻痹，在310±110秒内导致细胞死亡。发现用激光微束进行细胞操作时的细胞毒性作用部分基于非线性激发现象，特别是内源性细胞发色团的双光子吸收。在脉冲激光微辐射情况下双光子吸收会更强，但在用用作激光镊子（“光阱”）的高度聚焦连续波（cw）微束进行显微操作时也会发生。特别是，波长小于800纳米的短波近红外光阱会诱导类似UVA的生物学效应（氧化应激）。例如，用760纳米微束捕获精子会导致类似UVA的自发荧光改变，在35±20秒内导致麻痹，在65±20秒内导致细胞死亡。相比之下，800 - 1064纳米的激光微束可能是相对安全的显微操作工具。在大多数光阱中使用多频cw激光器。这些激光器的辐射会放大细胞毒性作用。因此，应优先选择单频激光操作。一般来说，激光辅助细胞显微操作需要对微束 - 细胞相互作用有新的认识，包括非线性光学方面。
