Paes Hugo Costa, de Oliveira Frazão Stefânia, Felipe Maria Sueli Soares, Casadevall Arturo, Nicola André Moraes
Faculty of Medicine, University of Brasília, Brasília, Brazil.
Department of Cell Biology, University of Brasília, Brasília, Brazil.
Bio Protoc. 2019 Nov 20;9(22):e3423. doi: 10.21769/BioProtoc.3423.
The most important virulence factor in the genus is the polysaccharide capsule. This genus includes several species that cause life-threatening invasive disease. An increase in capsule thickness is important during fungal infection. The capsule is usually imaged using India ink, and crucial insights on the dynamics of its growth have been obtained using capsule-binding proteins such as specific antibodies or complement. We have developed an alternative method that allows both static and time-lapse imaging of the capsule using Percoll, a suspension of nanometric spheres that do not penetrate the capsule. Given that these particles have a higher refractive index than the capsule, the latter can be imaged by differential interference contrast (DIC) microscopy. Static observation of the capsule with DIC and Percoll results in capsule thickness measurements that match those made with India ink. Using capsule-inducing media, a glass-bottom incubation chamber and a live-imaging system equipped for DIC microscopy, this method allows time-lapse imaging of capsule growth. In contrast with India ink staining, DIC imaging of Percoll exclusion halos result in crisp images. The greatest advantage of this method, though, is that unlike India ink, the Percoll particles are non-toxic and unlike opsonins they do not bind the capsule, resulting in observations of capsule growth that are free from interference of bound proteins on capsule physiology.
该属中最重要的毒力因子是多糖荚膜。该属包括几种可引起危及生命的侵袭性疾病的物种。在真菌感染期间，荚膜厚度增加很重要。荚膜通常用印度墨汁成像，并且使用诸如特异性抗体或补体等荚膜结合蛋白获得了关于其生长动态的关键见解。我们开发了一种替代方法，该方法允许使用不穿透荚膜的纳米级球体悬浮液Percoll对荚膜进行静态和延时成像。鉴于这些颗粒的折射率高于荚膜，后者可以通过微分干涉对比（DIC）显微镜成像。用DIC和Percoll对荚膜进行静态观察可得到与用印度墨汁测量的荚膜厚度相匹配的结果。使用诱导荚膜形成的培养基、玻璃底培养室和配备DIC显微镜的实时成像系统，该方法允许对荚膜生长进行延时成像。与印度墨汁染色相比，Percoll排斥晕的DIC成像可得到清晰的图像。不过，该方法最大的优点是，与印度墨汁不同，Percoll颗粒无毒，并且与调理素不同，它们不结合荚膜，从而可以在不受结合蛋白对荚膜生理学干扰的情况下观察荚膜生长。
