Category Archives: Whole tissue imaging

Re-Evaluating the Spherical-Nucleic-Acid (SmartFlare) Technology

I co-authored a Correspondence pre-print article that puts into question the Smartflare technology. SmartFlares (the commercial name of NanoFlares) are gold nanoparticles covered in  oligos specific to a certain mRNA of interest (aka spherical nucleic acids). Supposedly, cells internalize these particles and, once the mRNA hybridize to the oligo, a complementary fluorecently labeled oligo is being unquenched and “flares”, indicating the presence of said mRNA. In this post I want to briefly mention the main topics of our pre-print, and expand on some points. I encourage readers to comment here or on Pubpeer on our article.

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Imaging with CRISPR/Cas9

The hottest buzz-word in biology today is CRISPR: an adaptive immune system in bacteria and archea. At its basis is a nuclease, named Cas9, which is targeted to DNA by a short single-guide RNA (sgRNA). This turned out to be a very useful system for genome engineering in any organism due to its specificity (provided by the sgRNA) and its simplicity (all you need is to express the Cas9 and sgRNA in the cell). However, this system can also be used for other purposes. One such use is modulation of gene expression, for example by targeting a nuclease dead Cas9 (dCas9) fused to a transcription activator or repressor to promoter regions. Another such use is for imaging.

Here, I’ll described how Cas9 can be used to visualize specific DNA loci or specific RNA transcripts in fixed and live cells.

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ASCB15 part 1

The ASCB meeting brings scientists from all levels to talk about cell biology, which is actually almost anything “biology”. But there’s also a full program dedicated to other matters, like science careers, science publishing, science communications and science policy. This is also a great venue for companies to show their products, and for organizations/institutions to recruit new members. If I remember the numbers correctly, there were over 550 oral presentations and over 2,700 posters. I overheard someone saying there were ~6000 people attending the meeting. I typically go to RNA meetings that are mostly in the lower 100’s of participants. So, to me, that’s a large meeting.

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sequencing localized RNA in single cells by FISH

To celebrate the 2-year anniversary of this blog, lets talk about the new Science paper in which the authors claim to performs in situ single cell, single molecule  RNA sequencing. So what’s the big deal? Well, RNA sequencing (RNA-Seq) has become a very common method to study gene expression. In many cases, RNA-Seq uses cell extraction from an entire cell population -thus averaging the RNA content of each individual cells. In recent years, single-cell RNA-Seq is becoming more feasible (for example). In this case, cells are sorted via flow cytometry so that one can sort individual cells into designated wells in a multi-well plate. Thus, RNA from a single cell can be sequenced. Though this process is becoming both efficient and accurate, you loose information about the cellular localization of the RNA. FISH is a method that enables to determine the accurate localization of the target mRNA. However, FISH is limited to only a small number of mRNAs. Using color barcoding of the FISH probes can increase the complexity that is achieved (i.e. – one can simultaneously detect multiple types of RNAs) but these are still only a few compared to the entire transcriptome. This new paper in Science combines FISH with RNA sequencing to give Fluorescent in situ sequencing (FISSEQ). How did they do that? First, they generated cDNA by performing reverse-transcription in fixed cells with random primers. The cDNA was then circularized and then amplified. One of the nucleotides has a reactive group so that the cDNA is cross-linked to the surrounding macromolecules. Thus, the cDNA is localized to the same location as the RNA.  The primer has an adapter sequence that can be used as template for sequencing or for FISH. They show some 3D FISH images of cells and tissues using a probe for this adapter. Pretty pictures, but not much info there. From here on, it gets trickier. For the sequencing, they used the SOLiD method. One problem was to reduce the spot density so that it will be sufficiently low to distinguish single molecules. If I understand them correctly, they modified the SOLiD sequencing probes so that the sequencing primers have mismatches that reduce the efficiency. The second problem was to identify auto fluorescence and other fluorescent artifacts in their images. Rather than fine-tuning a specific detection threshold, they relied on the fact that they are monitoring sequences. That means that the fluorescence at each spot should account for a known sequence (i.e. the colors pattern should change based on the sequence), whereas auto fluorescence should remain stable. So, they actually used no threshold at all.

The sequencing reaction cycles and images of the first 15 cycles in primary fibroblasts. Source: Lee JH et. al. (2014) Science 343:1360.

The sequencing reaction cycles and images of the first 15 cycles in primary fibroblasts. Source: Lee JH et. al. (2014) Science 343:1360.

They them showed a few applications of this method. One interesting feature was to show that the nucleus is enriched for non-coding and antisense RNAs compared to the cytoplasm that is enriched with mRNAs. Unfortunately, the nuclear/cytoplasm dichotomy was the only “localization” aspect in their paper. It would have been much more interesting to show mitochondrially-localized vs ER localized vs plasma membrane localized etc… Or, look at highly polarized cells like neurons and use FISSEQ to look at somatic vs dendritic or axonic RNAs. Another experiment they did was to look at the gene expression changes in response to a wound healing model. They found the expected increase in genes related to cell migration, and with some genes differentially expressed only in the migrating cells (at the “wound”) compared to contact inhibited cells that are close to them.  Again, It would have been even better if they could show the subcellular of these mRNAs – are they at focal adhesion sites or other unique sites in the migrating cells? Their results on the whole seem convincing, but I didn’t check all the little technical details.  One thing which is missing is a high resolution image showing the sequencing of just one RNA, compared to an auto fluorescence spot. All their images show one or many cells with multiple spots in different colors – but that is it. The authors say upfront that this is just a demonstration of their method, and that they expect it to improve in coming years, just like what happened with next-generation sequencing. We’ll see… ResearchBlogging.orgLee JH, Daugharthy ER, Scheiman J, Kalhor R, Yang JL, Ferrante TC, Terry R, Jeanty SS, Li C, Amamoto R, Peters DT, Turczyk BM, Marblestone AH, Inverso SA, Bernard A, Mali P, Rios X, Aach J, & Church GM (2014). Highly multiplexed subcellular RNA sequencing in situ. Science (New York, N.Y.), 343 (6177), 1360-3 PMID: 24578530   [post-pub note: this post was also published at the RNA-Seq blog, here].

Feb 2015 – the detailed protocol was published at Nature protocols.

This month’s Nature methods (part 1): Spinach, blue transcription & photoacoustic imaging

This month’s Nature Methods issue has several interesting imaging items & articles, including two super-resolution reviews, two optogenetics articles, and more.

This post will be dedicated to three items in the “tools in brief” section.

Blue transcription

Optogenetics usually refers to control of ion flux via light sensitive channels. However, there are other light-responsive molecules. The item titles “Optimized optogenetic gene expression” describe a work from Kevin Gardner’s lab. They fused the transcription activating domain of the protein VP16 to the protein EL222. EL222 is a light-oxygen-voltage protein from the bacterium Erythrobacter litoralis. This protein binds to DNA when illuminated by blue light, and detaches from the DNA when the light is removed. Using this system, they could induce and repress transcription of a specific gene of interest (harboring a specific promoter recognized by EL222) in mammalian cells in tissue culture and in zebra-fish embryo. This can be a great tool.

Zebra fish egg and embryo harboring  an mCherry gene under the control of the VP-EL222 (or not) under dark or blue-light conditions. Source: Motta-Mena LB et al. (2014) Nat Chem. Biol. 10:196.

Zebra fish egg and embryo harboring an mCherry gene under the control of the VP-EL222 (or not) under dark or blue-light conditions. Source: Motta-Mena LB et al. (2014) Nat Chem. Biol. 10:196.

Photoacoustic imaging

Fluorescent molecules absorb light, and then emit light at a different wavelength. Photoacoustic molecules absorb light and emit sound waves. This is called the photoacoustic effect. This effect can be utilized to image inside whole animals, and the hope of the field is to get deep tissue penetration and a high resolution. The item titled “Activatable photoacoustic probes” presents a paper by the J. Roa’s lab at Stanford university. They developed a new polymer which absorbs at near-infrared (thus allowing good tissue penetration) and these produce a higher signal than commonly used materials for such imaging. They were also able for the first time to create a photoacoustic sensor of reactive oxygen species. This new field is very interesting and very exciting.

Spinach2

Spinach may deserve its own post, but briefly, Spinach and Spinach2 are RNA aptamers that can be used for the genetic encoding of fluorescent RNA. This aptamers form a unique structure which binds a specific molecule which then fluoresce. However, the optical properties of this dye were not suitable for common microscope filters. So now the group that developed Spinach developed several new dyes to enhance the fluorescent range of Spinach2.

The main problem I have with Spinach is that most of their work is based on an artificial RNA composed of 60 repeats of CGG trinucleotide and the ribosomal 5S rRNA. I haven’t followed the literature of Spinach much, but haven’t seen any single molecule imaging using Spinach. but, I guess I owe Spinach a post of its own.

ResearchBlogging.orgTools in brief (2014). Chemical biology: Optimized optogenetic gene expression Nature Methods, 11 (3), 230-230 DOI: 10.1038/nmeth.2867
Tools in brief (2014). Sensors and probes: Expanding Spinach2’s spectral properties Nature Methods, 11 (3), 230-230 DOI: 10.1038/nmeth.2865
Tools in brief (2014). Imaging: Activatable photoacoustic probes Nature Methods, 11 (3), 230-230 DOI: 10.1038/nmeth.2868
Pu K, Shuhendler AJ, Jokerst JV, Mei J, Gambhir SS, Bao Z, & Rao J (2014). Semiconducting polymer nanoparticles as photoacoustic molecular imaging probes in living mice. Nature nanotechnology PMID: 24463363
Motta-Mena LB, Reade A, Mallory MJ, Glantz S, Weiner OD, Lynch KW, & Gardner KH (2014). An optogenetic gene expression system with rapid activation and deactivation kinetics. Nature chemical biology, 10 (3), 196-202 PMID: 24413462
Song W, Strack RL, Svensen N, & Jaffrey SR (2014). Plug-and-Play Fluorophores Extend the Spectral Properties of Spinach. Journal of the American Chemical Society, 136 (4), 1198-201 PMID: 24393009
Strack RL, Disney MD, & Jaffrey SR (2013). A superfolding Spinach2 reveals the dynamic nature of trinucleotide repeat-containing RNA. Nature methods, 10 (12), 1219-24 PMID: 24162923

Looking at single mRNAs in neurons hints at memory formation

It is postulated that learning and memory are modulated by synaptic plasticity – molecular changes  that result in changes in the synapse morphology and signaling capacity. Local protein translation is considered important for synaptic plasticity. Two works from our lab were published last month (back to back!) in Science. Both papers deal with how beta-actin mRNA localization and dynamics in neurons may account for local protein translation upon stimulation, and hence, may supply insight into memory formation.

The first paper by Adina Buxbaum shows that beta-actin mRNAs in dendrites are “unmasked” upon activation of the dendrites. Using single molecule FISH, She noticed that the average number of probes bound to the mRNAs in dendrites (but not in adjacent glia cells) was lower than expects, and this number increased upon stimulation. Not only that, there were more mRNAs in the stimulated dendrites. This indicated masking by a protein “coating” that prevented FISH probe binding in the unstimulated cells. A modified FISH protocol which included a protease digestion step prior to probe hybridization showed that indeed the mRNAs were masked by proteins.

single molecule FISH for beta-actin mRNA in dendrites shows that mRNAs in unstimulated neurons are masked. A) Unstimulated neuron. B) stimulated neuron showing increased number of spots. C) Unstimulated neuron, in which the fixed cells were digested with protease prior to FISH probe hybridization. Source: Buxbaum, Wu & Singer (2014). Science Vol. 343  pp. 419-422

single molecule FISH for beta-actin mRNA in dendrites shows that mRNAs in unstimulated neurons are masked. A) Unstimulated neuron. B) stimulated neuron showing increased number of spots. C) Unstimulated neuron, in which the fixed cells were digested with protease prior to FISH probe hybridization. Source: Buxbaum, Wu & Singer (2014). Science Vol. 343 pp. 419-422

 

She further showed that this masking relates to other mRNAs, as well as to ribosomes, and that this is due to a metabolic process resulting from stimulation. Thus, this unmasking process may be a way to “activate” localized mRNAs for translation.

Apart from being a very neat paper technically and biologically, I think it was exceptionally entertaining to begin her paper by quoting an 1894 work by Cajal, the father of neuroscience.

The second paper by Hye-Yoon Park follows the dynamics of single molecule endogenous beta-actin mRNAs in neurons by live imaging, using the MS2 system. She shows movement of mRNAs along dendrites, as well as some events of merging or splitting – suggesting that some mRNAs are packed together in larger granules – which may regulate local translation. She also looked at brain slices, visualizing beta actin transcription dynamics. This is an important achievement since it is much harder to look at mRNA dynamics in tissue slices than in single cells on plate, due to background fluorescence. Though some biological insight is derived here, this is more of a “new technology” report.

Live imaging of beta-actin mRNAs in dendrites (movie. Source: Park HY et al. (2014) Science Vol. 343 pp. 422-424)

These papers are just the beginning of a long-term story of how mRNA localization and local translation are regulated in neurons.  A lot of cool experiments are being done in our lab in this regard and I’ll report more as they are published.

ResearchBlogging.orgBuxbaum AR, Wu B, & Singer RH (2014). Single β-actin mRNA detection in neurons reveals a mechanism for regulating its translatability. Science (New York, N.Y.), 343 (6169), 419-22 PMID: 24458642
Park HY, Lim H, Yoon YJ, Follenzi A, Nwokafor C, Lopez-Jones M, Meng X, & Singer RH (2014). Visualization of dynamics of single endogenous mRNA labeled in live mouse. Science (New York, N.Y.), 343 (6169), 422-4 PMID: 24458643

Open source microscope & software: openSPIM & Fiji

Just hears a great talk today by Pavel Tomancak from Max-Planck institute. He’s doing amazing work in systematic imaging of fly RNAome & proteome during development. Check out his website (click his name above).

He also talked about Fiji, which an open source software that is just like ImageJ, but with supporting community that develops new scripts & applications.

A most unusual “open source” development that he is very proud about is the openSPIM.

SPIM – Selective Plane Illumination Microscopy – is a microscopy method in which a laser beam sends a narrow light sheet to the specimen, and the objective is at 90 degree from the light sheet (there are SPIM developments with up to 4 objectives that can image from all 4 sides. See here). This type of microscopy is good for imaging thick live specimens (e.g. whole worm, fly or fish embryo etc..). Due to the thickness of the sample, wide field imaging will cause too much background.

So, there are good SPIM microscopes that you can buy from companies like Zeiss. but he developed the openSPIM, which is a build-it-yourself  SPIM microscope, that actually fits into a suitcase (he took it to South-Africa with him to show college & high-school kids). He claims that non-specialist can construct it in one hour. All the details (parts, assembly instructions (“IKEA/Lego style”) are found at the website. The cost, he estimates, is ~40,000$ (with the camera being about half the cost). He claims that the openSPIM is comparable, in quality of images, to commercial SPIM microscopes from 5 years ago.  Pretty good for standard imaging.

I like the idea.