Excitation spectral microscopy for highly multiplexed fluorescence imaging and quantitative biosensing

Abstract

The multiplexing capability of fluorescence microscopy is severely limited by the broad fluorescence spectral width. Spectral imaging offers potential solutions, yet typical approaches to disperse the local emission spectra notably impede the attainable throughput. Here we show that using a single, fixed fluorescence emission detection band, through frame-synchronized fast scanning of the excitation wavelength from a white lamp via an acousto-optic tunable filter (AOTF), up to 6 subcellular targets, labeled by common fluorophores of substantial spectral overlap, can be simultaneously imaged in live cells with low (~1%) crosstalks and high temporal resolutions (down to ~10 ms). The demonstrated capability to quantify the abundances of different fluorophores in the same sample through unmixing the excitation spectra next enables us to devise novel, quantitative imaging schemes for both bi-state and FRET (Forster resonance energy transfer) fluorescent biosensors in live cells. We thus achieve high sensitivities and spatiotemporal resolutions in quantifying the mitochondrial matrix pH and intracellular macromolecular crowding, and further demonstrate, for the first time, the multiplexing of absolute pH imaging with three additional target organelles/proteins to elucidate the complex, Parkin-mediated mitophagy pathway. Together, excitation spectral microscopy provides exceptional opportunities for highly multiplexed fluorescence imaging. The prospect of acquiring fast spectral images without the need for fluorescence dispersion or care for the spectral response of the detector offers tremendous potential.

Single-molecule displacement mapping unveils sign-asymmetric protein charge effects on intraorganellar diffusion

“…We next examined diffusion in the mitochondrial matrix. Mito-Dendra2 of varied net charges were constructed by appending 20-AA Asp/Glu and Arg/Lys sequences at the C-terminus ( Table S1 ), after accounting for the basic (pH~7.9) 19,20 mitochondrial matrix environment.…”

“…We next examined diffusion in the mitochondrial matrix. Mito-Dendra2 of varied net charges were constructed by appending 20-AA Asp/Glu and Arg/Lys sequences at the C-terminus (Table S1), after accounting for the basic (pH~7.9) 19,20 mitochondrial matrix environment. Starting with the original Mito-Dendra2 of −3.9 net charges, SMdM indicated no strong directional preferences for its diffusion in the mitochondrial matrix (Figure 3b).…”

Unmixing for ultra-high-plex fluorescence imaging

“…One shortcoming of multiplexed imaging is the noise that is introduced into the readouts-fluorescence microscopy is limited by the broad fluorescence spectral width. A possible solution to this limitation could be by combining PICASSO with excitation unmixing which has been validated in other studies 14 .…”

Temporally multiplexed imaging of dynamic signaling networks in living cells

“…Microscopy of living cells expressing fully genetically encoded fluorescent reporters of molecular signals is important for measuring the dynamics of these signals in relation to cellular states and functions. Multiplexed fluorescence imaging enables the important ability to see more than one signal at once, in a single living cell, so that relationships between the signals can be derived (1)(2)(3)(4). Without this ability, it is hard to determine the relationships between different signals, key to understanding how they interact to yield cellular computations, and how such biological computations go wrong in disease states.…”