Simultaneous multicolour imaging using quantum dot structured illumination microscopy

Abstract

Multicolour structured illumination microscopy (SIM) is a powerful tool used for the investigation of the dynamic interaction between subcellular structures. Nevertheless, most of the multicolour SIM schemes are currently limited by conventional fluorescent dyes and wavelength-dependent optical systems, and can only sequentially record images of different colour channels instead of obtaining multicolour datasets simultaneously. To address these issues, we present a novel multicolour SIM scheme referred to as quantum dot structured illumination microscopy (QD-SIM). QD-SIM enables simultaneously excitation and collection of multicolour fluorescent signals. We also propose a theoretical analysis of the image formation in two-dimensional multicolour SIM to help combine the optically sectioned and super-resolution attributes of SIM. Based on this theory, QD-SIM enables optically sectioned, super-resolution, multicolour simultaneous imaging at a single plane.

Fluorescent Nanoparticles for Super-Resolution Imaging

“…Using only a single excitation wavelength, QDs with different emission spectra could recorded simultaneously and differentiated by use of a color selective image splitter. Raw SIM images could thus be acquired simultaneously for each spectral channel, increasing acquisition speed …”

“…Raw SIM images could thus be acquired simultaneously for each spectral channel, increasing acquisition speed. 183 Despite of their favorable optical properties, such as superior photostability and brightness, some drawbacks prevail for the use of QDs in super-resolution imaging. For example, the broad absorption band and potential multiphoton absorption of QDs make them difficult to use for STED, similar to the problem discussed for CDs; blinking remains a major limitation; and the tendency of QDs to feature short off-state times compromises their use for single-molecule imaging.…”

Exploiting the UV excited size-dependent emission of PDMS-coated CdTe quantum dots for in vitro simultaneous multicolor imaging of HepG2 cellular organelles

“…There are reports available demonstrating the use of QDs for multicolor imaging applications. 2,7,16,17,20,35,36 However, very few reports are available for simultaneous imaging of biological moieties at a single excitation wavelength using QDs only. A study demonstrating the simultaneous imaging of tissue targets using streptavidin conjugated commercially available QDs at a single excitation wavelength was reported by Fountain et al 37 Emission fingerprinting was carried out to resolve the emission peaks of the QDs.…”

“…Multicolor bioimaging utilizes several fluorophores with different emission wavelengths to image various moieties of a given sample. [1][2][3][4] Fluorophores such as organic stains or dyes exhibit an excitation and emission pair for the visualization of biological moieties. The major limitation of organic dyes when used in multicolor imaging is their spectral overlap.…”

Combination of Structured Illumination Microscopy with Hyperspectral Imaging for Cell Analysis

“…However, when spectral detection is involved, many SIM methods require scanning process in either spectral (λ) or spatial ( x , y ) dimensions to capture the three-dimensional ( x , y , and λ) dataset (referred to as the datacube) of each SIM raw image, including sequentially switching the illumination wavelengths and filter sets, ,− or scanning the spectral detection slit over the field-of-view (FOV), , resulting in serious speed degradation. Although parallel detection for multicolor SIM raw images had been reported using an image splitter , or multiple detectors for each wavelength band, only few wavelength bands could be detected simultaneously; thus, these approaches lose abundant spectral information that might reveal the physicochemical properties of samples. There has always been a trade-off between dynamic performance and spectral detection capability in SIM.…”