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...homology arms (60-70 bp) is added to the complex. This complete transfection mix, i.e. csRNP plus ssODN... Cas9 RNP complexed with 2-part cr:tracrRNA tags genes more effectively than Cas9 complexed with sgRNA...immunoprecipitated - along with its associated protein complexes - from the cell-lysate using antibodies against...against the target protein. The captured protein complexes are then analysed by mass spectrometry to identify...recombinant Cas9 protein to form the ribonucleoprotein complex (csRNP, Figure 1). We use in-house purified Cas9... cost-effective and highly active. After csRNP complex formation, a single-stranded donor DNA (ssODN) ...experiment depends largely on how efficiently these components are delivered into cells; for each cell type,...

...sometimes you want a tool that is a little more compact — more of an antibody fragment rather than the ...Consider the classic IgG antibody (Figure 1): it is composed of four peptide chains (two identical heavy chains...The Fc region is the “tail” of the antibody and comprises portions of the constant regions of the two heavy...on the other hand, are the “arms” of the Y and comprise the entire light chain and a variable and constant...portion. For example, Fab fragments are molecules composed of just the Fab portion of the antibody. Fab fragments...fragments. Size: ~50 kDa. Fv - These fragments are composed of just the variable domains of the heavy and ...Variable (scFv). Size: ~25 kDa.   Figure 2: Comparison between a full IgG antibody and common antigen...

... These are sandwich, competitive, direct, and indirect. Sandwich and competitive ELISA use antibodies ...et al., 2019.   Competitive ELISA Similar to a sandwich ELISA, the competitive ELISA is also used ... will bind to the competing antigen in the wells while the protein-antigen complexes will be washed away... be adapted to a competitive format. Figure 2: Overview of using Competitive ELISA to quantitatively...epitopes on the target protein, so they do not compete with each other. This antibody is also conjugated...protein of interest in the original sample. The competitive ELISA is best used for detecting antigens that...sandwich ELISA. While the most common type of competitive ELISA is a modification of the sandwich ELISA...

...structurally resemble any other known proteins; they are completely unique to the CRISPR system (Nishimasu et al....conformation.   Fun Fact! Even though the Cas9:RNA complex is well described in the closed conformation, recent...electron microscopy studies have also found Cas9:RNA complexes in the open conformation (Cofsky et al., 2022)...guide RNA, prearranged for base pairing with a complementary DNA strand. These 10 nucleotides are the “seed...pyogenes Cas9 in the apo state (PDB ID 4CMP, top) compared to the guide RNA-bound state (PDB ID 4ZT0, bottom...the PAM sequence and doesn’t mind if the PAM complementary strand is mismatched.   Figure 5: S. ... found a PAM site, Cas9 needs to check for complementarity with its guide RNA. But how to test DNA bases...

... fragment of the NanoBiT® complex, which, upon binding to its complementary partner LgBiT, reconstitutes...expressed at their endogenous levels. HiBiT is compatible with both strong and weak promoters, and even...phototoxicity, perturb biological processes, and compromise cell viability (Laissue et al., 2017). HiBiT ...add-mix-measure method. NanoBiT® luciferase activity is compatible with both intra- and extracellular environments...also be combined with the HiBiT tag to provide complementary validation of protein expression and localization...often intrinsically disordered and structurally complex. The tumor suppressor TP53 (p53) is one example...or co-cultured across very narrow filters, complementation in recipient cells proved that the enzyme was...

...a single enzyme. Compared with previous research, which required protein compartments to co-localize multiple... antibodies for aminoacyl tRNA synthetases Nanocompartments for yeast bioproduction pathways The CRISPR...Research Center developed a pooled plasmid library encompassing all possible amino acid mutations of the receptor...many aaRSs exist within a multi-tRNA synthetase complex, the team investigated whether the generated antibodies...antibodies were able to immunoprecipitate the entire complex in three different cell lines. The Gräslund lab...Clouse Claudia Vickers’ lab has created a novel nanocompartment for transporting proteins in yeast by using... anchors to VP1, which self-assembles into a compartment encapsulating the protein of interest (Figure...

...endogenously expressed SCF complex members, an E3 ubiquitin ligase complex. Finally, when auxin is added...can offer over other protein control methods and compare the commonly available types of degron tags. What...removed. The degradation and reversal are rapid compared to other systems because the system modulates ...other systems reviewed here. The only required components are 1) a SMASh-tagged protein and 2) asunaprevir...fusion protein with the endogenous CRBN E3 ligase complex which will polyubiquitinate the fusion protein....target protein-of-interest and the CRBN E3 ligase complex leading to ubiquitination and proteasomal degradation...reviewed here. The AID system relies on three components. First, the protein of interest must be AID-tagged...

...experiment requires delivery of, at minimum, two components: the Cas9 protein and a guide RNA (gRNA) targeting...valuable to scientists, there are some potential complications that must be considered when using this method...alternative approach, which avoids many of these complications, is to directly deliver a ribonucleoprotein ...ribonucleoprotein (RNP), consisting of the Cas9 protein in complex with a targeting gRNA, to your cells of interest. Cas9...cleaving genomic targets with similar efficiency as compared to plasmid-based expression of Cas9/gRNA and can...CRISPR experiment is the generation of the RNP complex.  While one option is to purchase the Cas9 protein... by commercial vendors such as IDT. These two components are then incubated together to form the RNP. ...

...the viral components are small enough to enter the nucleus through the nuclear pore complex. This feature... methods. Across these methods, the following components are needed: γ-Retroviral transfer plasmid encoding...similar to lentiviral packaging methods. The three components described above (envelope, packaging, and transfer...slightly different isoforms of these packaging components. Therefore, lentiviruses may not be efficiently...interchangeably. Are γ-retroviruses replication incompetent? Yes, as with lentiviruses, the genes required...plasmid backbone is meant to be descriptive of the components found within the backbone. For example: pLXSN...particle containing Matrix, Capsid, and Nucleocapsid components. Pol Precursor protein containing Reverse...

.... They are made up of compensation beads and a conjugated antibody. Compensation beads are a mixture between... single color control prior to compensation (B) and after compensation (C) with APC-Cy7.   FMOs FMO...your experiment, and they are non-specific. The compensation beads can bind to any primary antibody you add...up your voltages and can also be helpful for compensation. You should have a single color control for ...They may also be used as a starting point for compensation (Fig. 1B-C). However, it’s important to note...and not experimental samples, the voltages and compensation you set from your single color controls may ... the reference plots provided by the antibody companies or check the literature. Pro tip! Early gates,...

...Delivering CRISPR components to plant cells Once you have selected the correct CRISPR components for your application...deliver these components to your target cells. Remember, efficient delivery of CRISPR components is essential...organisms, some plant-specific changes to CRISPR components are necessary to enable genome editing in plant... academic researchers through Addgene. CRISPR components for plant genome engineering CRISPR can be used...genome of plant cells. The structure of the gRNA (composed of a ~20 nucleotide targeting sequence and ~75...line will result in a failed experiment. CRISPR components can be expressed stably or transiently depending...delivery method and cell type in question. CRISPR components can be delivered and expressed transiently using...

...time. But biology is complicated — so many different parts and species, with components quickly losing activity...Chemistry reactions, one component bearing an azide reacts covalently with another component bearing an alkyne... a good tool for sticking together biological components. Nearly all of us played with LEGO as a child...alkyne; the two components lock together rapidly and selectively in all sorts of contexts. But the reactive...reactive groups of Click Chemistry are almost completely absent from living systems. What are the possibilities...generating product without a lot of left-over starting components. High rate constant— time is money and things...utilization and development of genetically engineered components, like protein tags (Figure 2). Through the work...

... Note: hypoxanthine (shown) is the nucleobase component of the nucleoside inosine. Created with BioRender.com...and one-ring pyrimidine base. This is a far more complex chemical task, not a job for a single enzyme. ...bind DNA, Cas9 requires (1) a guide RNA, (2) a complementary DNA sequence, and (3) a protospacer-adjacent...removed the uracil glycosylase inhibitor (UGI) components, which inhibit BER during cytosine-to-uracil ...developed two fusions of a CBE with other BER components: the DNA repair protein rXRCC1, or the DNA binding...separate and preventing repair based on the complementary strand. This could explain why the edited U ...original G is not primarily converted back to a complementary C. Optimized base editing with eOPTI-CGBE and...

...approach to monitor the composition of spatiotemporally defined protein complexes. Nature communications...can be part of multiple dynamic and overlapping complexes that have distinct functions. Many existing methods...proteins that interact with a particular protein  complex as opposed to an individual protein. The Bethune... when the bait is part of a particular protein complex. Not performed in a native cell context. For AP-MS...controllable version of the BioID protocol. The critical component for both BioID and Split-BioID is BirA*, a promiscuous...experiment is called a protein fragmentation complementation assay (PCA). In the Split-BioID method, each...another example of a protein fragmentation complementation assay that can be used to study binary protein...

... in proximity by the looping of chromatin, the complex of DNA and proteins that forms chromosomes. These...of a stable cell line that co-expresses three components: dCas9 with an added biotin acceptor site BirA...genomic location of interest  When these three components are co-expressed, the gRNA targets the dCas9 ... of the CAPTURE method. After protein and DNA complexes are cross-linked, DNA is digested into smaller.... This is cumbersome and also makes it hard to compare results across multiple targets because different...with primary cells or rare cell populations. Key components of the CAPTURE 2.0 system To improve upon the... Using this tag reduces the number of CAPTURE components that need to be delivered to cells from three...

...point is there are many ways to quantify the composition of a viral vector solution, and generally these...only 20% of recombinant AAV2 particles were full, compared to 50% of wild-type AAV2 stocks (Grimm et al.,...understand what these values mean and how they can be compared.   Figure 2: Label that comes on Addgene...affected by many experimental factors. To list some complicating factors for quantifying AAV: qPCR efficiency...the AAV genome, caused by repeats and self-complementarity of the AAV genome and the ITRs. qPCR efficiency...individual sample, this will reduce convenience and comparability because each sample with be quantified with ...linear region of the assay. This may be due to competition for binding sites between the primers and the...

...immunoglobulins, to isolate a specific protein out of a complex mix. Using this technique, users can look for the...the antibody or antibody-target protein immune complexes, wash away unbound protein, elute and measure ...Protein extracts from cells or tissues contain a complex mix of proteins seen as green squares, purple circles...protein mix and antibody-target protein immune complexes are allowed to form. Following incubation, beads...beads are used to immobilize the antibody-protein complexes, and as in the first method, unbound proteins ... need to choose the protein type that is most compatible with the host species and isotype of their primary...developed, which can be used for most antibodies compatible with either Protein A or G.  While commonly used...

... plasmids with the same ori are incompatible because they will compete for the same machinery, creating...their copy number, ori, and incompatibility groups. Note the A -C compatibility grouping is an arbitrary ...origin of replication/replicon. The replicon is comprised of the origin of replication (ori) and all of ...origin of replication wisely and avoid plasmid incompatibility In other words, the best choice of ori depends... or not you need to consider your plasmid's compatibility with one or more other plasmids. Generally speaking...plasmids for an experiment, make sure they have "compatible" ori's. See the table below for more details....details.   Common Vectors Copy Number+ ori Incompatibility Group Control pUC ~500-700 pMB1 (derivative...

...gene-of-interest, all the way to the complex multi-TU constructs that compose genetic circuits and metabolic... regulators, and lots of viral components. This kit is also compatible with the Joint Modular Cloning ...noticed that studies frequently made comparisons between CRISPR components expressed from different plasmid...extensive with a supplemental add-on kit and is compatible with E. coli and/or cell-free systems. Protein... backgrounds, MoClo can be invaluable for more complex editing and screening experiments in organisms ...nature of the Fragmid system allows for direct comparisons of CRISPR parts. The Fragmid system was used ...plasmid backbone elements impact viral titer and to compare different Cas enzymes in all-in-one AAV delivery...

...effects, ABEs also compare favorably to other methods. In a head-to-head comparison with Cas9, Cas9 modified...double-strand breaks (DSBs) preferred by researchers. Compared to non-homologous end joining (NHEJ), HDR occurs... Note: hypoxanthine (shown) is the nucleobase component of the nucleoside inosine. Created with BioRender.com...represent the fourth generation of base editors. Compared to BE3, BE4 offers a 2.3 fold decrease in C→G ...frequency of 1.2%. Although the lab did not conduct comprehensive genome-wide studies of ABE specificity, their... 40 new ABE8 variants (Gaudelli et al., 2020). Compared to ABE7.10, ABE8s resulted in 1.5-fold more editing...higher editing efficiency at non-NGG PAM variants compared to ABE7.10. ABE8s have an improved base editing...

...many opportunities to learn from other biotech companies at workshops and can use what we learn to improve...office space shared by numerous emerging biotech companies. The space was modern with clean bold colors of...hallway for people to take a break and relax. Companies at LabCentral get access to a variety of experimental...how LabCentral aims to enable emerging biotech companies, I was excited to go there because both LabCentral...must first fragment a DNA sample into many small components. Without proper processing, these small DNA strands... workshop participants from different biotech companies to interact and learn more about one another. ...expand my skills and network with other technicians/company representatives. I got to talk with a technician...

...recently developed a photosensitizer composed of the protein complex complex and the MG-2I-dL5** fluorogen that...thus forming the full FAP-TAP complex (MG-2I-dL5**).   A FAP-TAP complex will light up if excited with...cells. The photosensitizer used in this work is composed of two parts: 1) an activatable protein (the FAP...-TAP MG-2I-dL5** MG-2I-dL5** is a new FAP-TAP complex where dL5** is the FAP. It’s job is to bind MG-...fluorogenic dye.  MG-2I is the TAP portion of the complex and is derived from the malachite green (MG) dye...modified with the addition of two iodine residues. Compared to MG, MG-2I has increased ROS generation when...with these fluorescent proteins. He et al also compared MG-2I-dL5** head-to-head with KillerRed, a genetically...

...discovery in the 1990s and quickly rose to popularity. Compared to CRISPR, RNAi methods are often quicker and ...create pri-miRNA, which is then processed by a complex called Drosha to pre-miRNA. miRNA plasmids are ...RNA duplexes bind to the RNA-induced silencing complex (RISC). After binding to RISC, the RNA duplex is... can simply block translation machinery. The composition of the RISC varies, but at the very minimum requires...recruits other silencing proteins. Figure 1: Comparison of shRNA and miRNA processing and silencing pathways...typically range from 19–22 nucleotides and are 100% complementary to their target sequence. However, you can’t... day. You need to include both sides of the complementary RNA duplex and the hairpin loop sequence (Figure...

...isopropyl β-D-1-thiogalactopyranoside). (To be completely accurate, IPTG binds and inactivates the lac ...to a lacZΔM15-mutant bacterial cell in trans complements the mutation allowing for a functional enzyme...enzyme. This process is called α-complementation. The system described above was put to practical use in the... inserted it into a plasmid, creating an α-complementation cloning vector. When a cloning reaction goes...interrupted as shown in cell A, and thus will not complement the cell's β-galactosidase mutation. An unsuccessful...functional β-galactosidase enzyme through α-complementation (cell B). As shown on the representative plate... use a proper plasmid (i.e. contains the α-complementation cloning MCS): pGEM-T, pUC18 and pUC19, and ...

...cerevisiae yes - This can complement ura4- S. pombe, but the complementation is weak. yes   LYS2 L-lysine...vector propagation. This post, along with a future companion post on mammalian vectors, will catch you up on...vectors? Yeast are eukaryotes and thus contain complex internal cell structures similar to those of plants...in the lab typically contain all the necessary components to allow shuttling between E. coli and yeast ...an organism to synthesize a particular organic compound required for its growth. Many auxotrophic strains... including a copy of a functional gene which complements the host’s auxotrophy. When grown on media NOT...LEU2 L-leucine no S. cerevisiae yes - This can complement leu1- S. pombe, but multiple copies are required...

...of the protein MLH1, a component of the MutSα–MutLα mismatch repair (MMR) complex, to temporarily inhibit...45-fold) higher across different genomic sites compared to PE1 (Anzalone et al., 2019). PE3: Resolving...recognized as an MMR intermediate, and the MutSα–MutLα complex may either selectively excise the edited DNA flap...the unedited strand. Temporarily inhibiting this complex may allow time for 5′ flap exonucleases and DNA...nuclease activity (Chen et al., 2021). In a system as complex as prime editing, every efficiency counts! The ...deliver with AAVs but still efficient at long and complex edits. The team also developed PE6e-g editors with... the truncated pegRNA can still guide Cas9 and compete with full pegRNAs for access to the target site...

...projects in introductory labs. Genome projects use computational methods to identify genes by their similarities...abnormalities due to the deletion (i.e. the plasmid complements the deletion). If the phenotype is corrected,..., well-studied and inexpensive. In addition, a complete set of well-annotated deletion strains is available... the absence of methionine (i.e. the plasmid complements the deletion). Find the Plasmids Here Plasmid...blotting in other experiments (Figure 2).   Complementation reveals conservation of function Students have...Addgene, to successfully analyze Met protein complementation. In the procedure diagrammed in Figure 3, students...promoter controls MET/Met gene expression, complementation should only occur when galactose replaces glucose...

... for a new generation of ABEs), consists of 3 components:  a deaminase (TadA, originally from E.coli,... or Cas nickase)  a guide RNA that targets the complex of TadA and dCas to the genomic DNA of interest...effect of removing the wild-type TadA from the complex the researchers developed a second set: The ABE8...resulted altogether in 40 new ABE8 variants. When comparing on-target DNA editing efficiency in a human cell...found that the smaller version, ABE8.x-m, showed comparable editing behavior to ABE8.x-d. Overall, ABE8s ...showed a median increase of 1.94x editing activity compared to ABE7.10. Depending on the position in the protospacer...line while reducing indel frequency more than 90% compared to ABE7.10. Choosing Cas9 proteins to increase...

... human body is made up of organs, cells comprise compartments and structures, called organelles. Take ... the subcellular localization and composition of cellular complexes are important steps in understanding...that the fusion protein localizes properly by comparing the signal from the fusion itself to the wild-...and potentially which other proteins it forms complexes with. These “colocalization” studies give insights...within the same subcellular structure or protein complex. By coexpressing both the marker protein and the...possible to determine the location of proteins within complex structures. In order to interpret colocalization...quantifiable results and statistics that can be used to compare between different experimental setups. A colocalization...

...contains a composite of GenBank, EMBL, DDBJ, and PDB sequences and may be the most comprehensive for searching... Tool (BLAST) provided by NCBI. BLAST helps us compare the sequencing results of the plasmids in our repository...deletions, or insertions. We use blastp or blastx to compare our sequencing results to protein sequences to ...page, the first decision to make is whether to compare a Sanger sequencing result to a single known reference...BLAST search to perform and will save you time compared to other types of BLAST searches. If you do not...megablast, but can return longer alignments to compare with your sequencing trace file. Unlike megablast...consistently run our blastx searches with the “Low complexity regions” filter unchecked so that these regions...

... most successful computer languages are free to use. Of the 15 most popular computer programming languages...engineered DNA systems capable of storing data and computing inside living cells. He's also a co-founder of...digital genetic switches that enable cellular computing. These logic gates built from transcriptors are...are available at Addgene as a BIOFAB kit comprised of well-characterized bacterial transcription and translation.... What the BioBricks Foundation is seeking to accomplish is to make the engineering of biology much easier...carefully engineered to support reliable functional composition. In other words, these parts can be used over...logic shows up in mechanical systems, plumbing, computers, and now through BIL gates we've implemented them...

... as the building block of our understanding of complex biological processes. Many tools exist to perturb...silencing,” of any gene that contains sequence complementary to the dsRNA (Wilson and Doudna, 2013). RNAi... translation, or chromatin modifications of complementary gene sequences (Figure 1). As a result, gene...unmodified dsRNA elicits immunogenic reactions that complicate the effectiveness and interpretation of RNAi.... of interfering RNAs.   RNAi vs. CRISPR: Complementary techniques It is fair to say that CRISPR has...Stainier, 2020). Additionally, a study that directly compared CRISPR to RNAi in the context of a high throughput...data generated from both methods did a more comprehensive gene list emerge that accurately reflected the...

...scientific art competitions where creations can go on display for all to enjoy. The largest competition of this... University’s Annual Art in Science competition. This competition has gained so much public interest for...moonlighting as artists can compete in the Nikon Small World Photomicrography Competition - a 40 year old international...voyage. Previous destructive techniques were incompatible with the need to conserve these unique samples...been featured in Time magazine . Other smaller competitions are now taking place and gathering momentum,...international competition that last year received over 2,000 entries. Do you mix your passion for art and...

...them for use in your lab. Structural Comparison Functional Comparison Advantages of Nanobodies How to Use...for a comparison of nanobody, HCab, and traditional IgG antibody structures. Functional comparison Strong...nanobodies and how their structure and function compare to IgG antibodies, as well as how to produce them...Nanobody Toolbox Find Nanobody Plasmids Structural comparison As the name suggests, nanobodies are smaller ...incubated together without forming large oligomeric complexes. Antibodies are also too large to be expressed... antibody incubation. See figure 4A below for comparison of this one-step staining with nanobodies vs ...localization patterns as cells stained for one target (compare nanobodies staining in figure 4A vs 4B). How to...

... function as a kind of “GPS for the genome” to compute coordinates for genetic elements like protein, ...action at a distance.” Through the marvel of DNA compaction, an enhancer can increase the expression of a...millions of base pairs away. (For details on DNA compaction and the structural proteins that make it possible...appendix DNA is a 3D fractal). Enhancer biology is a complex and dynamic field. We’re going to focus on a tried...details. DNA is a 3D Fractal Figure 2: DNA compaction. Graphics courtesy of beyondthedish (left) and...People are wrong. In reality, cellular DNA is a complex three-dimensional globule of chromatin. Chromatin...tertiary structures (globules) to achieve exquisite compaction—on the order of 700 terabytes per gram—and form...

...But what exactly is an antibody, and how is a component of the immune system useful as a research reagent...heavy chains, and the rest of the Fab regions are completed with the Ig light chains. The variable region ...production strategy has advantages and disadvantages compared to each other. Polyclonal antibodies These antibodies...Single-chain variable fragment (ScFv) Figure 2: Comparison between the IgG antibody and scFv. Because... their lower affinity for their target protein compared to whole antibodies (Crivianu-Gaita 2016). Get...Variables (scFvs). Nanobodies Figure 3: Comparison of the Hcab and nanobody. Nanobodies are the...fluorescence. Western blots are frequently used to compare relative levels of protein expression between cell...

...derived from the more complex type III-A and III-B systems. Though it was composed of many protein subunits...might be phylogenetically classified as the more complex Class I systems that contain multiple subunits,...evolution, were also working on the project. Using computational observations, Koonin and Makarova found that...the domains into a single protein, shedding the complexity of its ancestors. They classified the system ...system contains two pieces: a single protein, computationally described as a fusion of four Cas7 proteins...significantly reduced amount of off-target toxicity compared to other RNA editing methods — streamlining experimental...mammalian cell lines. However, in those same comparisons, the AbuGoot lab reported no detectable effect...

... advise pharmaceutical companies, biotech start-ups, health insurance companies, and governments on the...the part of the company involved: Research & Development (R&D) teams at pharma companies carry out preclinical... acquisitions of or collaborations with other companies. Medical Affairs groups work on building awareness... patients, shaping discussions with insurance companies about the pricing and coverage of drugs, and developing... teams, reflecting the need for all parts of a company to collaborate whenever it faces an important challenge...investments over the next 3, 5, or 10 years. Complicating these challenges is the fact that they occur...their jobs most, get pleasure from taking up such complex challenges, quickly breaking them up into smaller...

...Table 1: Taking it a Step Further - Big Company v. Small Company Big (e.g. Pharma) Small (e.g. Biotech...advancement in one company, good performers in for the long haul Limited movement in one company, likely will...academic silo to tenured professor.  Non-academia encompasses millions of choices including pharma, tech transfer...options in nonprofit science. No one list can ever encompass them all. We can’t designate non-academic jobs...and teach students Collaborations may be less “complicated” - for example, easy to initiate a collaboration...More sources of training and resources within the company Must find own sources of knowledge often externally...every 3-6 years—a short and exciting ride Complicated matrix structures are common Small integrated...

...protein. Proteins located in membrane-bound subcompartments, like the mitochondria or nucleus, will likely...loading buffers, as most Bradford assays are not compatible with detergents like SDS, while BCA assays are...their differing chemistries. You therefore cannot compare Bis and Tris gels or blots to each other. Once ...protein-based blocker is either degraded or not completely mixed together.   Commercially available chemical-based...lower cost, and smaller size. However, HRP is not compatible with sodium azide, a common microbial agent used...phosphates have a linear reaction rate and are compatible with antimicrobial agents. Chemiluminescence ...chemiluminescence enzymes, they are more likely to be compatible with stripping and probing.   Imaging At the ...