Hyperspectral multiplex single-particle tracking of different receptor subtypes labeled with quantum dots in live neurons

ABSTRACT

The efficacy of existing therapies and the discovery of innovative treatments for central nervous system (CNS) diseases have been limited by the lack of appropriate methods to investigate complex molecular processes at the synaptic level. To improve our capability to investigate complex mechanisms of synaptic signaling and remodeling, we designed a fluorescence hyperspectral imaging platform to simultaneously track different subtypes of individual neurotransmitter receptors trafficking in and out of synapses. This imaging platform allows simultaneous image acquisition of at least five fluorescent markers in living neurons with a high-spatial resolution. We used quantum dots emitting at different wavelengths and functionalized to specifically bind to single receptors on the membrane of living neurons. The hyperspectral imaging platform enabled the simultaneous optical tracking of five different synaptic proteins, including subtypes of glutamate receptors (mGluR and AMPAR) and postsynaptic signaling proteins. It also permitted the quantification of their mobility after treatments with various pharmacological agents. This technique provides an efficient method to monitor several synaptic proteins at the same time, which could accelerate the screening of effective compounds for treatment of CNS disorders

1. INTRODUCTION

Synaptic transmission and plasticity involve a plethora of different molecules and proteins. Understanding synaptic function at the molecular level is a crucial step in the discovery and development of compounds for treating neurological, neurodegenerative, and psychiatric diseases. To accelerate such discoveries, the capacity to monitor several molecules in parallel would be highly valuable. Imaging studies have demonstrated that synaptic proteins are extremely mobile between extrasynaptic and synaptic domains.1 Thus, being able to track the spatial dynamics of several proteins on the same sample becomes essential. Optical methods based on fluorescence microscopy enable the investigation of specific molecules at low concentration with a good signal-to-noise ratio, either in fixed or living specimen. However, due to spectral overlap, conventional fluorescence imaging techniques are usually limited to a maximum of three fluorophores.

Hyperspectral fluorescence imaging can increase the capability to discriminate additional fluorophores simultaneously since it detects photons emitted over many narrow spectral bands.2 Important factors for spectral filtering systems include the degree of transmission, wavelength tuning range, bandwidth, tuning speed, and out-of-band blocking power.3 Several hyperspectral filtering systems covering the visible spectrum are already available for microscopy systems, such as acousto-optic tunable filters (AOTF),4 liquid crystal tunable filters (LCTF),5 or thin-film tunable filters (TFTF).3 Both AOTF and LCTF crystals are polarization-dependent and have a low light-transmission rate (25% AOTF, 5% to 35% LCTF). They have an out-of-band blocking power that is less than 3 to 4 OD in the case of AOTF and 2 OD in that of LCTF. TFTF applications in hyperspectral imaging are just beginning; they show a high transmission rate (90%), a full visible spectrum acquisition, a bandwidth of 13 to 16 nm, a high out-of-band rejection (5 OD), but they have a slow acquisition speed.3 Several of these systems have been used in live cell microscopy, for instance, in Förster resonance energy transfer (FRET),6 in discriminating between autofluorescence and GFP fluorescence7 and in bulk quantum dot (QDs) tracking in non-neuronal cells.8 However, no hyperspectral system has been exploited for single-molecule imaging in live neurons.

Here, we present an application for a hyperspectral tunable filter originally designed for astrophysics.9 It is applied to live neuron optical imaging and allows single-molecule detection and tracking. The hyperspectral microscopy system that we (Photon etc.) developed uses a resonant Bragg tunable filter (BTF) imaging spectrometer mounted in front of an EMCCD camera. The BTF shows a high light-transmission rate, broad wavelength tuning range, and a fast spectrum acquisition rate. Furthermore, spectral properties of the tunable filters are almost identical for both s and p polarizations of light.

Для продолжения чтения вы можете скачать полную версию материала по ссылке ниже