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Monitoring reactivation of latent HIV by label-free gradient light interference microscopy.

ABSTRACT: Human immunodeficiency virus (HIV) can infect cells and take a quiescent and nonexpressive state called latency. In this study, we report insights provided by label-free, gradient light interference microscopy (GLIM) about the changes in dry mass, diameter, and dry mass density associated with infected cells that occur upon reactivation. We discovered that the mean cell dry mass and mean diameter of latently infected cells treated with reactivating drug, TNF-α, are higher for latent cells that reactivate than those of the cells that did not reactivate. Cells with mean dry mass and diameter less than approximately 10 pg and 8 μm, respectively, remain exclusively in the latent state. Also, cells with mean dry mass greater than approximately 28-30 pg and mean diameter greater than 11-12 μm have a higher probability of reactivating. This study is significant as it presents a new label-free approach to quantify latent reactivation of a virus in single cells.

SUBMITTER: Goswami N

PROVIDER: S-EPMC8367845 | biostudies-literature | 2021 Aug

REPOSITORIES: biostudies-literature

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Monitoring reactivation of latent HIV by label-free gradient light interference microscopy.

Goswami Neha N Lu Yiyang Y Kandel Mikhail E ME Fanous Michael J MJ Bohn-Wippert Kathrin K Tevonian Erin N EN Dar Roy D RD Popescu Gabriel G

iScience 20210804 8

Human immunodeficiency virus (HIV) can infect cells and take a quiescent and nonexpressive state called latency. In this study, we report insights provided by label-free, gradient light interference microscopy (GLIM) about the changes in dry mass, diameter, and dry mass density associated with infected cells that occur upon reactivation. We discovered that the mean cell dry mass and mean diameter of latently infected cells treated with reactivating drug, TNF-α, are higher for latent cells that r ...[more]

PMID: 34430819

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Project description:When studying microtubules in vitro, label free imaging of single microtubules is necessary when the quantity of purified tubulin is too low for efficient fluorescent labelling or there is concern that labelling will disrupt function. Commonly used techniques for observing unlabelled microtubules, such as video enhanced differential interference contrast, dark-field and more recently laser-based interferometric scattering microscopy, suffer from a number of drawbacks. The contrast of differential interference contrast images depends on the orientation of the microtubules, dark-field is highly sensitive to impurities and optical misalignments. In addition, all of these techniques require costly optical components such as Nomarski prisms, dark-field condensers, lasers and laser scanners. Here we show that single microtubules can be imaged at high speed and with high contrast using interference reflection microscopy without the aforementioned drawbacks. Interference reflection microscopy is simple to implement, requiring only the incorporation of a 50/50 mirror instead of a dichroic in a fluorescence microscope, and with appropriate microscope settings has a similar signal-to-noise ratio to differential interference contrast and fluorescence. We demonstrated the utility of interference reflection microscopy by high-speed imaging and tracking of dynamic microtubules at 100 frames per second. In conclusion, the optical quality of interference reflection microscopy falls within the range of other microscope techniques, being inferior to some and superior to others, depending on the metric used and, with minimal microscope modification, can be used to study the dynamics of unlabelled microtubules.Lay descriptionThe cytoskeleton gives a cell its shape and plays a major role in its movement and division. It's also helps organise the content of cells and is the base for intracellular transport. Important components of the cytoskeleton are microtubules, which are hollow cylindrical beams (25 nm in diameter) that assemble from protein building blocks called tubulin. Deficiencies in microtubules are related to many diseases including cancer and Alzheimer. Given their important role, microtubules are heavily investigated in many laboratories. One way to study microtubules is to isolate them from cells and image them using light microscopy. Over the years a number of imaging techniques have been used. These techniques have a number of drawbacks which are addressed by ongoing efforts which this work is a part of. Here, we present a method based on light interference that produce high quality images of microtubules. The technique is cheap and easy to implement making it accessible to a wide base of researchers.

Dataset Information

Monitoring reactivation of latent HIV by label-free gradient light interference microscopy.

Publications

Monitoring reactivation of latent HIV by label-free gradient light interference microscopy.

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