Imaging Cryptococcus spp. Capsule by Differential Interference Contrast Microscopy Using Percoll®
利用 Percoll®在微分干涉显微镜下对隐球菌荚膜成像
发布: 2019年11月20日第9卷第22期 DOI: 10.21769/BioProtoc.3423 浏览次数: 3534
评审: Samantha E. R. DundonJulie WeidnerAnonymous reviewer(s)
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The authors used this protocol in:
Sep 2018
Abstract
The most important virulence factor in the Cryptococcus 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.
Keywords: Cryptococcus neoformans (新生隐球菌)
Capsule (荚膜)
Differential interference microscopy (微分干涉显微镜)
Time-lapse microscopy (时间间隔显微镜)
Live-cell imaging (活细胞成像)
Background
Cryptococcosis affects more than two hundred thousand people each year, of which approximately 180 thousand die (Rajasingham et al., 2017). It presents most commonly as a severe meningoencephalitis in patients with AIDS, but the fungi can affect other immunocompromised and immunocompetent people and cause damage in other organs such as lungs and skin (Perfect et al., 2010). A hallmark characteristic of the Cryptococcus yeast cell is a polysaccharide capsule that surrounds the cell, which helps in evasion from the host’s immune responses (Agustinho et al., 2018).
The transparent capsule is most frequently observed using India ink, a suspension of dark particles that get excluded from the capsule and generate negative staining. It can also be observed using the Quellung reaction, in which the capsule becomes visible by differential interference contrast microscopy due to the increase in refractive index that results from antibody binding to it (Mukherjee et al., 1995; MacGill et al., 2000). In addition to observing the capsule statically, the binding of antibodies-as well as complement-has also been exploited to understand the mechanism used by C. neoformans to increase capsule thickness (Cordero et al., 2013a; García-Rodas et al., 2014). These methods brought a lot of information about the structure and formation of the most important Cryptococcus virulence factor, but they have intrinsic limitations. For one, we found India ink to be toxic to cryptococcal cells and thus not useful for time-lapse imaging of capsule growth. Binding of antibodies to the C. neoformans capsule was also found to change the capsular rigidity (Cordero et al., 2013b) and cellular metabolism (McClelland et al., 2010), so methods that rely on binding of an antibody or complement to observe capsule growth can give misleading results.
Here we provide additional details for a new method to image the capsule that we developed recently (Paes et al., 2018), which is able to overcome these limitations and allows imaging of the capsule in the absence of any protein binding to it. It relies on the same principle as India ink, that of negative staining using particles that are large enough to be excluded from the capsule. Instead of dark particles that absorb light, however, we used nanoparticles with high refractive index, which generate contrast in DIC microscopy (Figure 1). We have successfully used it to do one-time observations of the capsule and also in time-lapse experiments showing how the capsule thickness increased during growth and reproduction.
Figure 1. Example of capsule imaging by DIC using Percoll®. C. neoformans strain H99 was incubated for five days in the capsule-inducing medium Sab-MOPS. The cells were then imaged as described below. Brightness and contrast were adjusted in the image to improve clarity.
Materials and Reagents
- Tweezers, autoclave-sterilized
We prefer smaller tweezers (approximately 10 cm length) with a fine point that is either flat or serrated - P200 micropipette
- Microscope slides
We prefer pre-cleaned frosted slides (e.g., Fisher Scientific, catalog number: 12-550-143) - Coverslips, #1.5 or preferentially 1.5H thickness
We use 18 x 18 mm square coverslips (Zeiss, 474030-9000-000) - Sterile 1 ml syringe and two compatible 30-gauge sterile needles (e.g., BD, catalog numbers 309628 and 305128, respectively)
- Polypropylene tubes in 1.5 ml and 2.0 ml volumes (USA Scientific, catalog number: 1615-5500 and 1620-2700, respectively)
- Wooden toothpicks, autoclave-sterilized
- C. neoformans and C. gattii species complex strains
- NaCl (Fisher Scientific, catalog number: S271-3)
- KCl (Fisher Scientific, catalog number: P217-3)
- Na2HPO4 (Fisher Scientific, catalog number: S374-500)
- KH2PO4 (Fisher Scientific, catalog number: P-285)
- Hydrochloric acid (Fisher Scientific, catalog number: A144-212)
- Percoll® (GE Life Sciences, catalog number: 17089102), stored at 2-8 °C, shelf-life of two years
- 3-(N-Morpholino) propanesulfonic acid-MOPS (Sigma-Aldrich, catalog number: M3183)
- Peptone (BD, catalog number: 211677)
- Dextrose (BD, catalog number: 238230) or Dextrose (Sigma, catalog number: G7021)
- Yeast extract
- Agar (BD, catalog number: 242720)
- Ampicillin (Sigma-Aldrich, catalog number: A5354)
- Percoll®/Sab-MOPS suspensions (see Recipes)
- YPD broth and agar (see Recipes)
- Sabouraud dextrose broth (see Recipes)
- Phosphate buffer saline (see Recipes)
- Ampicillin solution (100 mg/ml) (see Recipes)
Equipment
- Microscope equipped for DIC microscopy
We use a Zeiss AxioObserver Z1 temperature-controlled inverted microscope (Carl Zeiss GmbH), fitted with a 63x NA 1.4 oil immersion objective, DIC polarizers and prisms, motorized stage and focus and a cooled monochromatic CCD camera - Incubation chamber for live-cell imaging
We use the POCmini-2 Cell Cultivation System (PeCon GmbH) in its closed cultivation mode (Figure 2) - Microcentrifuge with fixed-angle rotor to fit 1.5-2.0 ml tubes
- Orbital shaker incubator
We generally use heating incubated shakers with orbits between 25 and 28 mm, but most shakers used for microbiological cultures should work - Hemocytometer
- Biosafety cabinet
- Autoclave
Figure 2. Assembly of the POCmini-2 chamber. A. The separate components from left to right: POCmini2 system specific screwdriver; screw-on gasket; spacer; metal frame with 170 µm glass coverslips on top of it. B. Glass coverslips mounted into the frame with the spacer between them, done with the tweezers shown. C. Screw-on gasket partially fitted into the frame. The system can be autoclaved like this. D. Screwdriver in position for tightening the screw-on gasket onto the coverslips. E. mounted system with the syringe and needle on opposite orifices, for injecting the cell suspension while expelling the air between the spacers.
Software
- Image collection: Zen 2 (Zeiss GmbH)
https://www.zeiss.com/microscopy/us/products/microscope-software/zen.html - Image processing: ImageJ (NIH) (Schindelin et al., 2012; Schneider et al., 2012)
https://imagej.nih.gov/ij/
Procedure
文章信息
版权信息
© 2019 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Paes, H. C., Frazão, S. D. O., Felipe, M. S. S., Casadevall, A. and Nicola, A. M. (2019). Imaging Cryptococcus spp. Capsule by Differential Interference Contrast Microscopy Using Percoll®. Bio-protocol 9(22): e3423. DOI: 10.21769/BioProtoc.3423.
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分类
微生物学 > 微生物细胞生物学 > 细胞成像
微生物学 > 微生物生理学 > 细胞壁
细胞生物学 > 细胞成像 > 活细胞成像
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