New publication

Quantum dots for Immunofluorescence

Guest post by Dave Mason

In modern cell biology and light microscopy, immunofluorescence is a workhorse experiment. The same way antibodies can recognise foreign pathogens in an animal, so the specificity of antibodies can be used to label specific targets within the cell. When antibodies are bound to a fluorophore of your choice, and in combination with light microscopy, this makes for a versatile platform for research and diagnostics.

Most small-dye based fluorophores that are used in combination with antibodies suffer from a limitation; hit them with enough light and you irreversibly damage the fluorochrome, rendering the dye ‘invisible’ or photobleached. This property is the basis of several biophysical techniques such as Fluorescence Recovery After Photobleaching (FRAP) but for routine imaging it is largely an unwanted property.

Over 20 years ago, a new class of fluorescent conjugate was introduced in the form of Quantum Dots (QDots); semiconductor nanocrystals that promised increased brightness, a broad excitation and narrow emission band (good when using multi-channel imaging) and most importantly: no photobleaching. They were hailed as a game changer: “When the methods are worked out, they’ll be used instantly” (ref). With the expectation that they would “…soon be a standard biological tool” (ref).

So what happened? Check the published literature or walk into any imaging lab today and you’ll find antibodies conjugated to all manner of small dyes from FITC and rhodamine to Cyanine and Alexa dyes. Rarely will you find QDot-conjugated antibodies used despite them being commercially available. Why would people shun a technology that seemingly provides so many advantages?

Based on some strange observations, when trying to use QDot-conjugated antibodies, Jen Francis, investigated this phenomenon more closely, systematically labelling different cellular targets with Quantum dots and traditional small molecule dyes.

Francis_et_alFig3_GM

Figure 3 from doi:10.3762/bjnano.8.125 shows Tubulin simultaneously labelled with small fluorescent dye (A) and QDots (B). Overlay shows Qdot in green and A488 in Magenta. See paper for more details. See UPDATE below.

The work published in the Beilstein Journal of Nanotechnology (doi: 10.3762/bjnano.8.125) demonstrates a surprising finding. Some targets in the cell such as tubulin (the ‘gold standard’ for QDot labelling) label just as well with the QDot as with the dye (see above). Others however, including nuclear and some focal adhesion targets would only label with the organic dye.

2190-4286-8-125-graphical-abstract.png

The important question of course is: why the difference in labelling when using Quantum Dots or dyes? This is discussed in more detail in the paper but one explanation the evidence supports is that it is the size of the QDots that hinder their ability to access targets in the nucleus or large protein complexes. This explanation further highlights how little we really know about the 3D structure of protein complexes in the cell and the effect of fixation and permeabilisation upon them. Why for example, can tubulin be labelled with QDots but F-actin cannot, despite them both being abundant filamentous cytosolic structures? At this point we can’t say.

So why is this study important? Publication bias (the preferential publication of ‘positive’ results) has largely hidden the complications of using QDots for immunofluorescence. We and others have spent time and money, trying to optimise and troubleshoot experiments that upon closer study, have no chance of working. We therefore hope that by undertaking and publishing this study, other researchers can be better informed and understand when (or whether) it might be appropriate to use Quantum Dots before embarking on a project.

This paper was published in the Beilstein Journal of Nanotechnology, an Open Access, peer-reviewed journal funded entirely by the Beilstein-Institut.

UPDATE [2017-06-13]: in response to a comment below, I’ve updated the overlay figure to use green/magenta instead of green/red. The original figure can be seen in the paper or here.

How to Characterize Gold Nanoparticles’ Surface?

Guest post by Elena Colangelo

Our Topical Review on the characterization of gold nanoparticles (GNPs) has just been published in the Bionconjugate Chemistry Special Issue “Interfacing Inorganic Nanoparticles with Biology”.

The literature is abounding in works on GNPs for applications in biology, catalysis and sensing, among others. GNPs’ appeal resides in their optical properties, together with the well-developed methods of synthesis available and the possibility of functionalizing their surface with small molecules of interest, which can readily self-assemble on the GNPs’ surface forming a monolayer.

However, allegedly the structure and organization of self-assembled monolayers (SAMs) at the GNPs’ surface are in fact aspects too often neglected [though surely not on this blog; RL]. Such elucidation is challenging experimentally, but it is crucial not only to ensure reproducibility, but also to design nanosystems with defined (bio)physicochemical and structural properties, which could then be envisioned to assemble in more complex systems from a “bottom-up” approach.

Our Topical Review gives an overview of the current knowledge and methods available to characterize the GNPs’ surface with different molecular details.

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Cartoon illustrating the different levels of GNPs’ surface characterization discussed in the Topical Review.

First, the experimental methods commonly used to provide the basic characterization of functionalized GNPs, such as identification and quantification of the ligands within the monolayer, are detailed with the aid of some examples.

Second, the experimental methods providing information on the monolayer thickness and compactness are reviewed.

Third, considering that the SAM’s thickness and compactness do not only depend on the amount of ligands within the monolayer, but also on their conformation, the experimental methods that can provide such insights are recapitulated. However, we also stressed on the limitations intrinsic to these methods and on the challenges associated to the determination of the structure of SAMs on GNPs.

Fourth, we summarized some of the approaches used to give insights into the organization of different ligands within a SAM. Indeed, mixed SAMs on GNPs are useful since they can impart to the nanoparticles different functionalities and offer a way to tune their stability.

Fifth, highlighting again the limited insights into the SAM’s structure and organization that can be gathered with experimental techniques, we detailed some examples where a combination of experimental and computational approaches was able to provide a compelling description of the system and to assess molecular details that could not have been revealed experimentally.

Overall, this Topical Review gives emphasis on the importance of GNPs’ surface characterization and on fact that even though a number of experimental techniques are available, they are intrinsically limited and they cannot provide a fully detailed picture. Hence, it is advantageous to combine experimental and theoretical approaches to design nanoparticles with desired (bio)physicochemical properties [such as, e.g., our paper under review, currently available as a preprint; RL].

How to Elucidate the Structure of Peptide Monolayers on Gold Nanoparticles?

I have recently submitted my PhD thesis and we have now pre-printed on bioRxiv the work constituting its major chapter. Together with the pre-print, the data have been made publicly available in an online repository of the University of Liverpool. Well isn’t it perfect timing that this week is open access week? 😉

This work has been conducted nearly entirely during the 2 years of my PhD spent at the A*STAR Institute of Materials Research and Engineering (IMRE) and at the A*STAR Institute of High Performance Computing (IHPC) in Singapore.

In this study, peptide-capped gold nanoparticles are considered, which offer the possibility of combining the optical properties of the gold core and the biochemical properties of the peptides.

In the past, short peptides have been specifically designed to form self-assembled monolayers on gold nanoparticles. Thus, such approach was described as constituting a potential route towards the preparation of protein-like nanosystems. In other words, peptide-capped gold nanoparticles can be depicted as building-blocks which could potentially be assembled to form artificial protein-like objects using a “bottom-up” approach.

However, the structural characterization of the peptide monolayer at the gold nanoparticles’ surface, essential to envision the design of building-blocks with well-defined secondary structure motifs and properties, is poorly investigated and remains challenging to assess experimentally.

In the pre-printed manuscript, we present a molecular dynamics computational model for peptide-capped gold nanoparticles, which was developed using systems characterized by mean of IR spectroscopy as a benchmark. In particular, we investigated the effect of the peptide capping density and the gold nanoparticle size on the structure of self-assembled monolayers constituted of either CALNN or CFGAILSS peptide.

The computational results were found not only to well-reproduce the experimental ones, but also to provide insights at the molecular level into the monolayer’s structure and organization, e.g., the peptides’ arrangement within secondary structure domains on the gold nanoparticle, which could not have been assessed with experimental techniques.

Overall, we believe that the proposed computational model will not only be used to predict the structure of peptide monolayers on gold nanoparticles, thus helping in the design of bio-nanomaterials with well-defined structural properties, but will also be combined to experimental findings, in order to obtain a compelling understanding of the monolayer’s structure and organization.

In this sense, we would like to stress that, in order to improve data reproducibility, enable further analysis and the use of the proposed computational model for peptide-capped gold nanoparticles, we are making the data and the custom-written software to assemble and analyse the systems publicly available.

picture1

Snapshots of the final structure of the simulated 5 (left) and 10 (right) nm CFGAILSS-capped gold nanoparticle, illustrating different content and organization of secondary structure motifs.

Gold nanorods to shine light on the fate of implanted stem cells

people_JC

Joan Comenge

This is a guest post by Joan Comenge

Our work regarding the use of gold nanorods as contrast agents for photoacoustic tracking of stem cells has been just published (or here*). You can find all the technical details of the work there, so I will try to explain here the work for the readers who are not very familiar with our field.

It is important to have the appropriate tools to evaluate safety and efficacy of regenerative medicine therapies in preclinical models before they can be translated to the clinics. This is why there is an interest in developing new imaging technologies that enable real time cell tracking with improved sensitivity and/or resolution. This work is our contribution to this field.

To distinguish therapeutic cells from the patient’s own cells (or here from the mouse’s own cell),  the therapeutic cells have to be labelled before they are implanted. It is well known, that biological tissue is more transparent to some regions of the light spectrum than others. This fact is very easy to try at home (or at your favourite club): if you put your hand under a green light, no light will go through it, whilst doing the same under a red light the result will be very different. That means that red light is less absorbed by our body. Near infrared light is even less absorbed and this is why this region of the spectrum is ideal for in vivo imaging. Therefore, we made our cells to absorb strongly in the near infrared so we can easily distinguish them.

Gold nanoparticles of different sizes and shapes (synthesis and picture by Joan Comenge).

Gold nanoparticles of different sizes and shapes (synthesis and picture by Joan Comenge).

To do this, we labelled cells with gold nanoparticles. Interestingly, the way gold nanoparticles interact with light depends on how their electrons oscillate. That means that size and shape of the nanoparticles determine their optical properties, and this is one of the reasons why we love to make different shapes of nanoparticles. In particular, gold nanorods strongly absorb in the near infrared and they are ideal contrast agents for in vivo imaging.

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Figure reproduced from: The production of sound by radiant energy; Science 28 May 1881; DOI: 10.1126/science.os-2.49.242

We have now cells that interact with light in a different way than the tissue. The problem is that light is scattered by tissue, so resolution is rapidly lost as soon as you try to image depths beyond 1 mm. Obviously, this is not the best for in vivo imaging. Luckily for us, Alexander Graham Bell realised 130 years ago that matter emits sounds when is irradiated by a pulsed light. This is known as the photoacoustic effect and it has been exploited recently for bioimaging. Photoacoustic imaging combines the advantages of optical imaging (sensitivity, real-time acquisition, molecular imaging) and the good resolution of ultrasound imaging because ultrasounds (or phonons), contrarily to photons, are not scattered by biological tissue.
GNR-35.2Si3 in cells_16

Silica-coated gold nanorods inside cells

To optimise the performance of our gold nanorods, we coated them with silica. Silica is glass and therefore it protects the gold core without interfering with its optical properties. This protection is required to maintain gold nanorods isolated inside cells since nanorods are entrapped in intracellular vesicles, where they are very packed. The absence of a protective coating ultimately would result in a broader and less intense absorbance band, which would be translated to a less intense photoacoustic signal and consequently a lower sensitivity in cell detection. This of special importance in our system, a photoacoustic imaging system developed by iThera Medical which uses a  multiwavelength excitation to later deconvolute the spectral information of the image to find your components of interest. Thus, narrow absorption bands helps to improve the detection sensitivity even further. With this we demonstrated that we were able to monitor a few thousand nanorods labelled-cells with a very good 3D spatial resolution for 15 days. This allowed for example to see how a cell cluster changed with time, see how it grows or which regions of the cell cluster shows the highest cell density. In addition, this work opens the door to new opportunities such as  multilabelling using gold nanorods of different sizes and consequently different optical properties to observe simultaneously different type of cells. We also believe that not only stem cell therapies, but also other fields that are interested in monitoring cells such as cancer biology or immunology can benefit from the advances described in our work.

You can find the original publication here (or here*).
All the datasets are available via Figshare.

This work was supported by the UK Regenerative Medicine Platform Safety and efficacy, focusing on imaging technologies. Joan Comenge was funded by the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme. The in vivo imaging was done in the Centre for Preclinical Imaging, the Electron Microscopy in the Biomedical EM unit and the Optical Microscopy in the Centre for Cell Imaging.

* the alternative link is to 50 free e-prints; the link will be removed once the paper is fully open access (in a couple of days).

day3_200k

Cluster of gold nanorod-labelled cells imaged by photoacoustic imaging three days after implantation in mice.

The Spherical Nucleic Acids mRNA Detection Paradox

We are publishing today “The Spherical Nucleic Acids mRNA Detection Paradox“, the outcome of an open science project which started with an Hns student last year. In the last 12 months, we have reported in quasi real-time our experiments, protocols and analyses in an open science notebook and shared the data on our repository. The data are also stored at FigShare (e.g. Electron Microscopy results).

In addition to being exciting scientifically (he says!), this has been an experiment in how do science in the open using the tools of the 21st century to share information and solicit feedback. It is therefore fitting to publish it on a platform that challenges conventional modes of peer review.

We have chosen ScienceOpen where publication happens immediately (a couple of hours from submission to publication), followed by open peer review. In the coming weeks and months, I hope that many scientists will provide their expert evaluation of our article. In particular, Chad Mirkin will be invited to provide a review.

This article is important to all scientists who are using nanoparticles for imaging and sensing inside living cells. It should also be particularly relevant to past, current and prospective customers of the SmartFlares. Here is the abstract:

From the 1950s onwards, our understanding of the formation and intracellular trafficking of membrane vesicles was informed by experiments in which cells were exposed to gold nanoparticles and their uptake and localisation, studied by electron microscopy.  In the last decade, building on progress in the synthesis of gold nanoparticles and their controlled functionalisation with a large variety of biomolecules (DNA, peptides, polysaccharides), new applications have been proposed, including the imaging and sensing of intracellular events. Yet, as already demonstrated in the 1950s, uptake of nanoparticles results in confinement within an intracellular vesicle which in principle should preclude sensing of cytosolic events. To study this apparent paradox, we focus on a commercially available nanoparticle probe that detects mRNA through the release of a fluorescently-labelled oligonucleotide (unquenching the fluorescence) in the presence of the target mRNA. Using electron, fluorescence and photothermal microscopy, we show that the probes remain in endocytic compartments and that they do not report on mRNA level. We suggest that the validation of any nanoparticle-based probes for intracellular sensing should include a quantitative and thorough demonstration that the probes can reach the cytosolic compartment.

The paper will be typeset in the next few days and open peer review will be open from that point. Comments are already possible. Thank you to Dave Mason, Gemma Carolan, Joan Comenge and Marie Held for their contributions to this work.

A good day for science; respect to the Editor…

Earlier, I reported on the publication of our article on the internalisation of peptide-capped nanoparticles in cells. Today, I want to share with you the publication process as it happened at PloS One. The paper was submitted on the 20th of November 2014. The academic editor sent his decision, major revision, along with two referees reports on the 22nd of December, i.e. one month after submission [great turn around time!].

Reviewer 2 was very supportive but reviewer 1 much less so: there appeared to be a real difference of interpretation regarding the impact of cell-penetrating peptides on the intracellular localisation of ingested nanoparticles. The reviewer also requested additional experiments that we could not easily do at this time and that we felt were unnecessary to support our main conclusions. The academic editor himself, Dr Pedro V. Baptista [more on PloS One editorial process here], was author on a paper which in some ways could be seen as conflicting with our results and interpretation. The response to the referees and editors took me a long time to write. It was submitted on the 29th of January. I share it below.

The paper was accepted on the 6th of February. I welcome this decision, not just because our paper gets published -this is of course also great news!-, but because it demonstrates that there is space for open scientific debate in the peer reviewed literature. For this, I am immensely grateful to Dr Baptista.


Response to the referees.

Dear Dr Pedro V. Baptista

On behalf of my co-authors, I would like to thank you for handling our article and to thank the reviewers for their careful reading and for their comments.

Reviewer 2 notes that the context of our ms is the existence of conflicting reports on the effect of TAT and HA2 on intracellular fate of nanoparticles. Indeed, some articles have reported efficient access to the cytosol, while other studies indicate that most particles remain confined in endosomal compartments. Our own experiments are in line with this second group of articles. Reviewer 2 notes that “the study is well designed and executed and the results are interpreted appropriately”. Reviewer 2 supports publication in its current form.

Reviewer 1 has concerns about novelty. Reviewer 1 also suggests that we should add three references. These fall in the first category mentioned above, i.e. articles that support the notion that TAT enables access to the cytosol. It is of course appropriate that we should cite studies from both groups of articles. One of the three, […], was in fact already cited. We have now added the other two, i.e.: […]

Experiments related to this topic have led to many articles in the past 10 years. However, the persistence of conflicting reports and the importance of the topic for many envisioned applications require new insights. This we have provided through the use of imaging modalities that provide information across different scales: electron microscopy measures what occurs to a few nanoparticles in a very small part of the cell; photothermal microscopy measures what happens to the bulk of nanoparticles across a large part of the cell. This combination is thus uniquely able to address, in at least one cell type and a particular formulation of nanoparticle, the fate of TAT-functionalised nanoparticles after they bind to the cell surface.

Below we respond to the detailed queries of reviewer 1 and trust that the manuscript now meets the standards required for publication in PLOS One.

Dr Raphaël Lévy, rapha@liverpool.ac.uk

Detailed response to reviewer 1 queries:
• Novelty. Our article is a significant piece of work that adds useful information towards understanding and clarifying the impact of cell penetrating peptides on intracellular localisation of nanoparticles. The work is novel because it builds on a new imaging methodology that directly images the nanoparticle cores (as opposed to an attached fluorescent molecule) and gives a better overview of an entire cell than just electron microscopy. It is also novel because our peptide self-assembled monolayer approach enables us to do systematic variations of the surface chemistry of the nanoconjugates.
• “To include as a new figure, the extinction spectra of all the nanoconjugates as well as all the scattering spectra […]”. The reviewer is right that extinction spectra are very useful to characterise functionalisation and colloidal stability. We have added the requested figure as Fig. S0. For the conjugates used in Fig. 1, the formation of the self-assembled monolayers results in a minimal shift of the plasmon band of ~1-3 nm. This shift is small compared to the width of the plasmon peak. Because photothermal microscopy relies on absorption at the wavelength of our heating laser which matches the position of the maximal absorbance, differences due to a 1-3 nm plasmon shift are negligible. Interestingly, particles presenting a higher percentage of TAT in their monolayer do show a larger plasmon shift indicative of aggregation. We have modified the paragraph on the formation of the SAMs as follows: “Formation of the monolayer was immediately visible because of the increased colloidal stability and of a small red shift of the nanoparticles plasmon band (Fig. S0). Higher proportions of TAT in the monolayer resulted in nanoparticle aggregation and therefore were not used for further studies (Fig. S0).”

• “To include the images and quantification in Figure 1 with cells only with naked gold nanoparticles and cells only with PEG-gold nanoparticles and compare intensities.” The images and quantification for “cells only” were already included (Fig. 1A and first column of Fig. 1F). We have not included “naked gold”. Instead, as a reference point, we have used PEG-gold particles that have a capping composition made of CALNN and CCALNN-PEG. “naked gold” does not remain naked: non-specific adsorption of proteins, e.g. serum albumin in the cell medium, very rapidly changes the properties of the surface [see for example, Time Evolution of the Nanoparticle Protein Corona, Casals et al., ACS Nano, 2010, 4, pp 3623–3632]. The CALNN and CCALNNPEG composition was optimised, as discussed p 7, line 213-220 and Fig. S2 “Gold nanoparticles uptake decreases with increasing percentages of CCALNN-PEG”. The selected composition leads to minimal uptake as shown in Fig. 1B and the second column in Fig. 1F. From this reference composition, we have made systematic variations where we include defined percentages of the two functional peptides (dHA2 and TAT). For all these conditions, exemplary images are shown in Fig. 1 A-E, additional images are shared via figshare (http://dx.doi.org/10.6084/m9.figshare.1088379) and the quantifications are shown in Fig. 1F.

• “To perform other technique to quantify the gold content […].To include more time points in the TEM studies […]. […] the efficacy results reported by the authors are premature without the additional data described above.” While we agree that the suggested experiments are interesting, they are not necessary to reach the conclusions arrived at in the ms. Those conclusions do not concern “efficacy”, but increased uptake and intracellular localisation. The increase in photothermal signal as well as in the counts of nanoparticles in EM images unambiguously demonstrate increased uptake. The non-homogenous distribution of signal observed in the photothermal images and the electron microscopy analyses unambiguously rule out cytosolic distribution of the nanoparticles. The time point of 3 hours used in our studies is a key point both from the perspective of applications and of cell entry mechanisms. We agree that a systematic analysis as a function of time after uptake would provide further insights into endocytotic mechanisms, but it is outside of the focus of this study. Furthermore, it has been done extensively by cell biologists since the 1950s using a variety of probes. Notably, one of the first applications of gold nanoparticles in biology precisely focused on the mechanisms by which cells probe their external environment (Electron microscopy of HeLa cells after ingestion of colloidal gold, Harford et al., J Biophys Biochem Cytol 1957 3:749-756; reference added into the ms).

The standards in the field have been to publish only one or two representative electron
microscopy images. The photothermal imaging provides a unique means for the reader to understand nanoparticle distribution over biologically representative scales. Importantly, we are sharing here 942 EM images and 37 photothermal images. By publishing all of our data alongside the study [1], we enable other scientists to check and challenge our conclusions and propose alternative hypotheses. PLoS One is a particularly good forum for our article because of its commenting platform where this discussion can continue in the open after the publication of the article.
[1]. DOIs of the data:

10.6084/m9.figshare.1088379, 10.6084/m9.figshare.875584, 10.6084/m9.figshare.875630, 10.6084/m9.figshare.875545, 10.6084/m9.figshare.875477, 10.6084/m9.figshare.874219, 10.6084/m9.figshare.874153, 10.6084/m9.figshare.874033, 10.6084/m9.figshare.873852, 10.6084/m9.figshare.1088399, 10.6084/m9.figshare.1246458, 10.6084/m9.figshare.1246609,
10.6084/m9.figshare.1246622, 10.6084/m9.figshare.1246660, 10.6084/m9.figshare.1246696, 10.6084/m9.figshare.1246707

TAT and HA2 Facilitate Cellular Uptake of Gold Nanoparticles but do not Lead to Cytosolic Localisation

That is the title of a paper now published at PloS One. I am particularly pleased at the publication of this paper and would like to congratulate and thank its authors. They have all moved on from Liverpool. The joint first authors are Umbreen Shaheen and Yann Cesbron (currently in Rennes, France). Third author is Paul Free, now at IMRE in Singapore. The data were previously published at Figshare (links below).

PS: As we are today the second of April and not the first, you can be confident that this is not an April Fool post.

Yann Cesbron

Yann Cesbron

Umbreen Shaheen

Umbreen Shaheen

Paul Free

Paul Free

DOIs of the data:

10.6084/m9.figshare.1088379, 10.6084/m9.figshare.875584, 10.6084/m9.figshare.875630, 10.6084/m9.figshare.875545, 10.6084/m9.figshare.875477, 10.6084/m9.figshare.874219, 10.6084/m9.figshare.874153, 10.6084/m9.figshare.874033, 10.6084/m9.figshare.873852, 10.6084/m9.figshare.1088399, 10.6084/m9.figshare.1246458, 10.6084/m9.figshare.1246609,
10.6084/m9.figshare.1246622, 10.6084/m9.figshare.1246660, 10.6084/m9.figshare.1246696, 10.6084/m9.figshare.1246707