Category Archives: red dye

Eliminating mutated mitochondria during in-vitro fertilization

There are several genetic diseases which originate not from mutations in the nuclear genome but mutations in the mitochondrial genome. In humans, the threshold for disease occurrence is if 60% of the mitochondria has mutated mitochondrial DNA (mtDNA) (a mixed mitochondrial origin is called heteroplasmy). There is currently no cure, and no good way to prevent these genetic diseases.

Since the source of the mitochondria is solely from the oocyte, a lot of effort is invested in trying to get rid of mutated mitochondria by in-vitro fertilization (IVF) procedures – thus preventing the disease in the offspring. Two methods have been tried so far – pronuclear transfer (PNT) and spindle-chromosome transfer (ST).  The idea is to extract the nuclear genetic material of the oocyte or zygote and transfer it to an enucleated oocyte or zygote with healthy mitochondria. However, in both cases, there is still carry-over of some mutated mitochondria to levels that can be as high as 44%.

In this new paper published in Cell, a group of researchers from China suggest a different approach. Continue reading

Silicon nanocrystals – the next generation of fluorescent dyes?

Fluorescent microscopy is the only current method to follow biological structures and molecules in real-time in live specimens. Many advances were made but there are still a few problems with present fluorescent probes. Photobleaching (permanent disappearance of the fluorescent signal due to destruction of the fluorophore) is a major problem for all current fluorescent probes, be it chemical or biological. Photobleaching prevents prolong imaging.

A second problem is blinking of the fluorophore. Although blinking is actually useful in some super resolution techniques, it may still pose a problem for some applications.

Another problem is the size of some fluorescent tags such as fluorescent proteins and quantum dots. FP fusion to the studied protein can affect to protein function, simply by spatial interference.

Now, a new type of fluorophore was developed by the lab of Akihiro Kusumi from Kyoto university. Silicon nanocrystals (SiNCs) were apparently known for two decades as fluorescent, that are stable and can be used for biological applications. However, no serious effort was made to make SiNCs applicable in cell biology studies. This, he says, is due to the difficulty in producing SiNCs, manufacturing them in uniform size (and the size determines the fluorescent properties) and properties and conjugating them to biomolecules.

Kusumi developed a simplified protocol to produce mercaptosilane coated SiNC (mSiNC) that has a hydrodynamic diameter of 4.1 nm.  This was confirmed by three independent methods: gel filtration, FCS and dynamic light scattering (DLS). The hydrodynamic diameter of GFP is ~5.5nm and of Qdot655 – 19.5nm. They further measured by electron microscopy the actual size of the mSiNC core to be 3.2nm.

A & B - Elution traces of gel filtration experiments. The longer the molecule stays in the gel, the smaller it is. C- DLS analysis of mSiNC. D - FCS curves of the indicated molecules. E - image of mSiNCs obtained by electron microscopy (EM). F - distribution of measured diameter based on EM imaging.

A & B – Elution traces of gel filtration experiments. The longer the molecule stays in the gel, the smaller it is. C- DLS analysis of mSiNC. D – FCS curves of the indicated molecules. E – image of mSiNCs obtained by electron microscopy (EM). F – distribution of measured diameter based on EM imaging.

Characterization of the fluorescent properties showed that  the mSiNC fluorescent emission peak is at 655nm with a broad spectrum. Storage of dehydrated mSiNC did not affect the properties for over two months. However, fluorescence intensity decrease if stored in aqueous buffer at 37C (exponential decay time of 42 hours). Exposure of mSiNCs to excitation under fluorescent microscopy conditions showed that they are not blinking of photobleacing for over 300 minutes! Under similar conditions, GFP and the Cy3 dye photobleach within 3 seconds. the Qdot exhibited frequent blinking and fluctuations in fluorescent intensity compared to the mSiNC. mSiNCs fluorescence intensity is lower than Qdots. However, due to the instability of Qdot fluorescence, this difference diminishes over time.

After characterization came the actual test: they conjugated mSiNC to the transferrin protein (mSiNC-Tf) and applied it to cells, to follow the dynamics of cell surface transferrin receptors (TfR). whereas GFP-Tf allowed tracking for only 20 frames (0.67 sec), and Cy3-Tf allowed tracking of 150 frames (5sec), mSiNC-Tf allowed tracking for 3600 frames (120 seconds). This is quite an improvement.

A - a typical image of immobilized single mSiNC molecules. B - Distribution of fluorescence intensities of of single mSiNC spots (top) and single Qdot655 (bottom). C - Typical time-dependent changes of the fluorescence intensities of an mSiNC spot (top) and of a Qdot655 spot (bottom).

A – a typical image of immobilized single mSiNC molecules. B – Distribution of fluorescence intensities of of single mSiNC spots (top) and single Qdot655 (bottom). C – Typical time-dependent changes of the fluorescence intensities of an mSiNC spot (top) and of a Qdot655 spot (bottom).

Usually, due to blinking, single-molecule trajectories have gaps in them. These trajectories are later reconstructed based on the assumption that these gaps are due to blinking. With mSiNC-Tf, no gaps were observed (even of a single frame) for the whole 120 second (3600 frames) tracking. Thus, using mSiNC-Tf, they could follow the dynamics of TfR for long periods of time.

So, the advantages of mSiNC are very clear – they are extremely photostable, which allows for prolong imaging without loss of fluorescence. They are also small, allowing single molecule imaging with minimal perturbation. They can be conjugated to proteins.

Here are links to videos showing the lack of blinking; mSiNC-Tf on cell membrane (exhibiting Brownian motion) and what seems to be receptor internalization.

What are the disadvantages?

Well, first, the protocol to produce them, and verify their uniformity, though simplified, is yet not simple.

Second, the very wide emission spectra limits the use for multicolor imaging.

Last, similar to other dyes, it is excellent for live imaging of outer-membrane molecules, but may be more difficult for intra-cellular tagging for live imaging.

ResearchBlogging.orgNishimura H, Ritchie K, Kasai RS, Goto M, Morone N, Sugimura H, Tanaka K, Sase I, Yoshimura A, Nakano Y, Fujiwara TK, & Kusumi A (2013). Biocompatible fluorescent silicon nanocrystals for single-molecule tracking and fluorescence imaging. The Journal of cell biology, 202 (6), 967-83 PMID: 24043702