Single molecule localization imaging of telomeres and centromeres using fluorescence in situ hybridization and semiconductor quantum dots

Abstract

Single molecule localization microscopy (SMLM) is a powerful tool for imaging biological targets at the nanoscale. In this report, we present SMLM imaging of telomeres and centromeres using fluorescence in situ hybridization (FISH). The FISH probes were fabricated by decorating CdSSe/ZnS quantum dots (QDs) with telomere or centromere complementary DNA strands. SMLM imaging experiments using commercially available peptide nucleic acid (PNA) probes labeled with organic fluorophores were also conducted to demonstrate the advantages of using QDs FISH probes. Compared with the PNA probes, the QDs probes have the following merits. First, the fluorescence blinking of QDs can be realized in aqueous solution or PBS buffer without thiol, which is a key buffer component for organic fluorophores' blinking. Second, fluorescence blinking of the QDs probe needs only one excitation light (i.e. 405 nm). While fluorescence blinking of the organic fluorophores usually requires two illumination lights, that is, the activation light (i.e. 405 nm) and the imaging light. Third, the high quantum yield, multiple switching times and a good optical stability make the QDs more suitable for long-term imaging. The localization precision achieved in telomeres and centromeres imaging experiments is about 30 nm, which is far beyond the diffraction limit. SMLM has enabled new insights into telomeres or centromeres on the molecular level, and it is even possible to determine the length of telomere and become a potential technique for telomere-related investigation.

Enhanced mRNA FISH with compact quantum dots

“…This is significant because tens of seconds are needed to acquire a full z-stack for 3D cell imaging. In comparison, QDs exhibit long-term stability with no significant change in GAPDH mRNA counts ( p > 0.05; Student’s t -test) after 12 min of excitation, which is consistent with previous results for QD-based stains measured by net intensity 16 . QD-FISH yielded a significantly higher 3D count number compared with Dye-FISH (by 15–30%) for cells with the same 2D counts at a single nuclear focal plane (Fig.…”

Telomerase detection using a DNA-PAINT strategy

“…Figure 2(a) is the time-dependent fluorescence intensity profile of an individual anchor probe, where obvious photoswitching was observed. This is in consistence with our observation reported previously, indicating that super resolution imaging of the anchor probes can be achieved by SMLM [30]. The localization precision of anchor probes analyzed using the Zeiss Zen 2012 software is shown in figure 2(b), which is about 27 nm.…”

Meiotic nondisjunction and sperm aneuploidy in humans

“…Future developments may leverage existing technology to further facilitate the rapid detection of more chromosomes simultaneously in a single cell. Examples include the possible development of novel fluorescent dyes to replace traditional organic fluorochromes, such as quantum dots, which has already been applied to FISH with mixed success (Ioannou et al 2009, Yusuf et al 2011, Hwang et al 2015, Wang et al 2018. Quantum dots are inorganic fluorochromes that are resistant to photobleaching and excite at a wide range of wavelengths but emit light in a very narrow band (controlled by particle size) (Ioannou et al 2009).…”