Biophotonics Solutions for Quantitative Live Cell Near Infrared Raman Spectral Analysis of DNA Damage in Spermatozoa

在英国，至少有六分之一的夫妇会受到不孕不育的影响。最常见的原因是男性不育。男性因素不育症的特征通常是精子太弱，不能游到卵子里，或者根本不游。这些不健康的精子中保存的遗传物质(DNA)极有可能受到损害。在这些情况下，一种治疗方法是在实验室将精子注射到卵子中。然而，目前还没有办法确保在这一过程中转移的DNA质量良好。医学界担心，这可能会导致高发病率，如通过精子注射怀孕的儿童患上癌症。目前所有可用的DNA损伤测试都需要将精子打碎，并对提取的DNA进行化学分析。这项拟议的工作将开发一种方法来捕获单个精子，并在不伤害精子的情况下测量其DNA完整性。然后，可以选择一个好的精子来使卵子受精。开发的这项技术应该会极大地降低通过精子注射受孕的儿童的异常风险。这也可能提高辅助受精的成功率。我们建议结合使用激光的两种方法：光镊法和拉曼光谱法。如果一束激光被聚焦得非常紧，它可以产生足够的力来支撑一个非常小的物体--这被称为光学陷阱或光学镊子。可能试图游走的精子将通过光学捕获被保存在我们想要的地方。现在我们已经捕获了我们非常小的蠕动细胞，我们必须描述它的DNA。这将通过使用不同颜色的激光来振动细胞的分子来完成。然后我们可以看看振动是如何影响光的。通过使用强大的统计分析，我们可以建立与好的DNA相比，DNA损伤是如何振动的知识。这项工作将涉及大量的光学设计，以创建完成这项工作所需的仪器。然后我们必须证明我们的激光本身没有对精子造成任何额外的损害。镊子和拉曼以前都曾在细胞上使用过，但没有影响它们，但我们需要绝对确定精子也是如此。接下来，我们将通过获取健康的精子并用化学物质产生已知程度的损伤来建立统计模型，并告诉我们的拉曼软件这些精子是什么样子的。最后，我们将从健康程度不同的不育男性身上提取精子，看看我们的仪器是否能区分他们之间的区别。这种仪器很有可能能够用于世界各地治疗不育症患者或研究生殖的实验室。

Infertility affects at least one in six couples in the UK. The most common cause man infertility. Male factor infertility is usually characterised by sperm which are too weak to swim to the egg or don't swim at all. The genetic material (DNA) held in these unhealthy sperm is highly likely to be damaged.A treatment which is used in these cases is to inject the sperm into the egg in the laboratory. However, there is at present no way to ensure that the DNA which is transferred in this process is of good quality. There is concern in the medical community that this may lead to high rates of disease such as childhood cancer for children conceived by sperm injection.All the currently available tests for DNA damage require the sperm to be broken open and the DNA extracted to be chemically analysed. The proposed work will develop a method to trap a single sperm and measure its DNA integrity without hurting the sperm. A good sperm can then be selected to use to fertilise the egg. The technique developed should greatly reduce the risk of abnormalities in the children conceived by sperm injection. It may also increase the success rates for assisted fertilisation.We propose to combine two methods which use laser light: optical tweezers and Raman spectroscopy. If a beam of laser light is focused very tightly it can create enough force to hold a very small object - this is called optical trapping or optical tweezers. The sperm which may be trying to swim away will be held where we want it by optical trapping. Now we have trapped our very small and wriggling cell we have to characterise its DNA. This will be done by using a different colour of laser to vibrate the molecules of the cell. We can then look at how the vibrations affect the light. By using powerful statistical analysis we can build up knowledge of how damage DNA vibrates compared to good DNA.The work will involve a lot of optical design to create the instrument needed to do this work. We will then have to prove that our lasers themselves are not causing any additional damage to, the sperm. Tweezers and Raman have both been used before on cells without affecting them but we need to be absolutely sure that this is the case for sperm too. Next we'll build statistical models by taking healthy sperm and producing a known level of damage in them with chemicals and teaching our Raman software what that sperm 'looks' like. Finally, we'll take sperm from infertile men which have been sorted into different degrees of healthiness and we'll see if our instrument can tell the difference between them.There is a good chance that this instrument will be able to be used in laboratories around the world which are treating people with infertility or doing research into reproduction.