Readily Accessible Multiplane Microscopy: 3D Tracking the HIV‐1 Genome in Living Cells

Abstract

HIV-1 infection and the associated disease AIDS are a major cause of human death worldwide with no vaccine or cure available. The trafficking of HIV-1 RNAs from sites of synthesis in the nucleus, through the cytoplasm, to sites of assembly at the plasma membrane are critical steps in HIV-1 viral replication, but are not well characterized. Here we present a broadly accessible microscopy method that captures multiple focal planes simultaneously, which allows us to image the trafficking of HIV-1 genomic RNAs with high precision. This method utilizes a customization of a commercial multichannel emission splitter that enables high-resolution 3D imaging with single-macromolecule sensitivity. We show with high temporal and spatial resolution that HIV-1 genomic RNAs are most mobile in the cytosol, and undergo confined mobility at sites along the nuclear envelope and in the nucleus and nucleolus. These provide important insights regarding the mechanism by which the HIV-1 RNA genome is transported to the sites of assembly of nascent virions.

Recruitment of 7SL RNA to assembling HIV‐1 virus‐like particles

“…The cells were imaged under azimuthal TIR‐FM using a Cairn Opto‐Split III with a cube and appropriate filters and a Hamamatsu Flash 4.0 camera. Multiple colors were aligned by using calibration data obtained with 500 nm fluorescent beads (TetraSpeck Fluorescent Microspheres Size Kit, T14792, Invitrogen) mounted on a slide . The microscope was pre‐warmed to 37°C prior to imaging and temperature was maintained during imaging.…”

“…Microspheres Size Kit, T14792, Invitrogen) mounted on a slide. 34 The microscope was pre-warmed to 37 C prior to imaging and temperature was maintained during imaging.…”

Multifocal imaging for precise, label-free tracking of fast biological processes in 3D

“…(1) inserting an optical grating 13,18 , (2) using a deformable mirror 20 , (3) changing the optical path length 14,16 , and (4) by using lenses of different focal power 15,17,19 . Disadvantageously, optical gratings (approach (1)) are wavelength-dependent and have low optical efficiency.…”

“…MFI has been applied for 3D tracking 13,[15][16][17][18][19] , but only at low resolution or small sampled volume, and relying on fluorescent labels. The image of an object, i.e.…”

“…However, this approach constrains the field-of-view (FOV) 13 and lowers the signal-to-noise ratio (SNR) when increasing the number of focal planes. Thus, state-of-art 3D tracking based on MFI requires bright fluorescent labels and is limited to either lower speeds, small sampled volumes, or low precision 13,[15][16][17][18][19] . Here, we develop dark-field-microscopy-based MFI for label-free, long-term, and high-speed imaging at a high SNR and applicable over a large volume.…”