Direct Stochastic Optical Reconstruction Microscopy of Extracellular Vesicles in Three Dimensions

Meredith G. Chambers; Ryan P. McNamara; Dirk P. Dittmer

doi:10.3791/62845

JoVE Journal
Biology

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JoVE Journal Biology

Direct Stochastic Optical Reconstruction Microscopy of Extracellular Vesicles in Three Dimensions

3 차원에서 세포 외 소포의 직접 스토카스틱 광학 재건 현미경 검사

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09:36 min

August 26, 2021

DOI: 10.3791/62845-v

Meredith G. Chambers¹, Ryan P. McNamara¹, Dirk P. Dittmer¹

¹Department of Microbiology and Immunology, Lineberger Comprehensive Cancer Center,The University of North Carolina at Chapel Hill School of Medicine

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Overview

This study employs direct stochastic optical reconstruction microscopy (dSTORM) to visualize extracellular vesicles (EVs) and exosomes at a nanometer scale, surpassing the diffraction limits of traditional light microscopy. The protocol allows for precise imaging of EVs in three-dimensional space, facilitating the study of their biochemical nature and roles in disease, including viral infections such as SARS-CoV-2.

Key Study Components

Research Area

Microscopy and imaging techniques
Extracellular vesicles (EVs) and exosomes
Viral biology and disease progression

Background

Importance of EVs in cellular communication and disease biomarker identification
Challenges in visualizing small particles due to diffraction limits
Application of super-resolution techniques in biological imaging

Methods Used

Direct stochastic optical reconstruction microscopy (dSTORM)
Extracellular vesicles (EVs) sourced from affinity purification methods
Simulation of imaging conditions for enhanced resolution

Main Results

Successful visualization of individual EVs at nanometer resolution
Insights into the structural characteristics of EVs and their potential roles in viral infections
Demonstration of methodology as a reliable tool for characterizing biological samples

Conclusions

dSTORM proves to be an effective technique for studying EVs, enhancing our understanding of their role in health and disease
This methodology holds the potential for advancing biomarker discovery and understanding disease mechanisms

Frequently Asked Questions

What is dSTORM?

Direct stochastic optical reconstruction microscopy (dSTORM) is a super-resolution microscopy technique that allows visualization of biological samples at nanometer resolution.

How does dSTORM improve visualization compared to traditional microscopy?

dSTORM bypasses the diffraction limit of light, enabling the imaging of smaller structures with higher precision.

What biological entities can be studied using dSTORM?

dSTORM can be used to visualize extracellular vesicles, viruses, and other small cellular structures.

Why is understanding EVs important?

Understanding EVs is crucial for exploring their roles in intercellular communication and as potential biomarkers in diseases.

What are the key steps in preparing samples for dSTORM?

Key steps include adhering EVs to a surface, fixing them with paraformaldehyde, and preparing an appropriate imaging buffer.

Can dSTORM be used to study viral particles?

Yes, dSTORM can directly visualize viral particles such as SARS-CoV-2, providing insights into their structure and function.

What future applications does this methodology have?

This methodology has potential applications in fundamental biology research, diagnostics, and therapeutic developments targeting EVs.

직접 관면 광학 재구성 현미경 검사법 (dSTORM)은 빛 현미경 검사법의 전형적인 회절 한계를 우회하고 나노 미터 척도에서 엑소좀을 보는 데 사용됩니다. 엑소좀을 특성화하기 위해 2차원과 3차원에서 모두 사용할 수 있다.

이 프로토콜은 초해상도 현미경 검사법을 사용하며, 특히 dSTORM이라고도 하는 직접 관면 광학 재구성 현미경 검사법을 사용하여 회절 한계를 우회하고 3차원의 나노미터 정밀도로 전기를 시각화합니다. DSTORM의 한 가지 주목할 만한 장점은 EV의 생화학적 특성을 변화시키는 단계를 손상시키지 않으면서 빛의 회절 수준 아래에 입자를 직접 시각화하는 능력입니다. 많은 진화적으로 구별되는 바이러스는 EV 시그널링을 사용하므로 dSTORM을 사용하여 질병 바이오마커 및 진행을 위한 전기를 특성화하고 SARS-CoV2와 같은 개별 바이러스 입자를 시각화할 수 있습니다. 먼저 친화성 정제, 세포외 소포 또는 EV를 유리 바닥, 마이크로 슬라이드, 총 200 마이크로리터의 8웰 플레이트에 배치하고 섭씨 4도에서 밤새 표면에 부착할 수 있도록 합니다.

8웰 플레이트에서 기존 용액을 제거하지 않고, 1X PBS에 4%파라포름알데히드의 200마이크로리터를 각 웰의 EV 함유 용액에 추가하여 플레이트에 전기를 고정하고 플레이트가 실온에서 30분 동안 배양할 수 있도록 합니다. 조심스럽게 EV를 방해하지 않는 마이크로 파이펫파라포름알데히드와 과잉 용액을 제거합니다. 1X PBS로 EV를 세척하여 과도한 파라포름알데히드를 제거합니다.

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