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Related Experiment Video

Updated: Nov 15, 2025

Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body
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Imaging Cryptococcus spp. Capsule by Differential Interference Contrast Microscopy Using Percoll®.

Hugo Costa Paes1, Stefânia de Oliveira Frazão2, Maria Sueli Soares Felipe2,3

  • 1Faculty of Medicine, University of Brasília, Brasília, Brazil.

Bio-Protocol
|March 3, 2021
PubMed
Summary
This summary is machine-generated.

Researchers developed a new, non-toxic method using Percoll® and DIC microscopy to image the Cryptococcus polysaccharide capsule. This technique allows clear, real-time observation of capsule growth dynamics without interference from bound proteins.

Keywords:
CapsuleCryptococcus neoformansDifferential interference microscopyLive-cell imagingTime-lapse microscopy

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Area of Science:

  • Mycology
  • Microscopy
  • Infectious Diseases

Background:

  • The polysaccharide capsule is a key virulence factor in Cryptococcus species, which cause invasive fungal infections.
  • Current methods for imaging the capsule, like India ink, have limitations and can be affected by capsule-binding proteins.

Purpose of the Study:

  • To develop and validate a novel, non-invasive method for static and time-lapse imaging of the Cryptococcus polysaccharide capsule.
  • To overcome the limitations of India ink staining and protein interference in capsule growth studies.

Main Methods:

  • Utilized Percoll®, a suspension of non-penetrating nanometric spheres with a high refractive index.
  • Employed differential interference contrast (DIC) microscopy for imaging the capsule based on Percoll® exclusion halos.
  • Integrated capsule-inducing media, a glass-bottom incubation chamber, and live-imaging for time-lapse studies.

Main Results:

  • Percoll® and DIC microscopy provided static capsule thickness measurements comparable to India ink.
  • The method enabled crisp, time-lapse imaging of capsule growth dynamics.
  • Observations were free from interference by bound proteins, unlike methods using opsonins.

Conclusions:

  • Percoll® with DIC microscopy offers a superior, non-toxic alternative for visualizing Cryptococcus capsule morphology and growth.
  • This technique provides crucial insights into capsule dynamics without altering capsule physiology.
  • The developed method enhances the study of fungal virulence factors and infection mechanisms.