...introduced at ~2 copies per cell per color to achieve the highest color variations. Adjust the plasmid concentrations...Takeshi Imai from Kyushu University. Stochastic multicolor labeling is a powerful solution for discriminating...neuronal reconstruction. To achieve stochastic multicolor labeling, Brainbow used the Cre-loxP system to...with different copy numbers, producing dozens of color hues (Livet et al., 2007; Cai et al., 2013). However...introducing a mixture of XFP genes. Stochastic multicolor labeling is possible when each of the XFP genes...genes is introduced at ~2 copies per cell per color, following a Poisson distribution. To overcome the ...Richi, Marcus N. Leiwe, and Takeshi Imai. "Bright multicolor labeling of neuronal circuits with fluorescent...

...way they generate their color: they strongly absorb visible light to give colors in ambient light. In contrast...fluorescent jellyfish, bioluminescent phytoplankton, and colorful corals, to name a few. What’s behind these sights... In 1987, scientists described an unusual blue-colored protein from the jellyfish Cassiopea xamachana ...used for mimicry and camouflage. An anemone’s red colored tentacles resemble worms which attract fish for...Figure 1). Figure 1: The sea anemone's red color can be used for mimicry. Image: Bernard Spragg. ...chromoproteins absorb visible light and give off color in ambient light, it gives scientists the ability... For each chromoprotein, the lab determined the color intensities, maturation times, and fitness cost ...

...least a couple of decades. Green is the classic color, but fluorescent proteins are available in a variety...iRFP720, as photoacoustic contrast agents for two-color imaging in animals. The approach relies on ultrasound....1918.) Near-infrared fluorescent proteins for multicolor in vivo imaging. Shcherbakova and Verkhusha (...

...shown in the darker colors and the mCherry spectrum is shown in the lighter colors; excitation spectra...in choosing fluorescent proteins for your multi-color imaging experiment is to be aware of what fluorescent...fluorescent proteins Commonly used filter sets for multicolor imaging include ones designed for CFP, YFP, and...compartments. I hope this sheds some light on multicolor imaging with fluorescent proteins. With the right...right choice of fluorescent proteins, imaging four colors simultaneously should be pretty straightforward... multi-color imaging...

...to a rainbow of colors. With three copies of the construct, one would expect ten colors (see table to the...distinct colors due to higher copy number. The name Brainbow is truly fitting for this colorful technology...and high stability of colors. Sanes and Lichtman estimate that Brainbow’s colorful labeling has decreased...will highlight the evolution of the  Brainbow multicolor labeling system - a perfect example of the continued... Brainbow-2.1 construct can express one of four colors (n-GFP, RFP, YFP or M-CFP.) The construct contains...Cre-mediated excision removes one segment, eliminating two color possibilities. The remaining cassette can invert...the mouse brains, they found many more than 3-4 colors due to the tandem integration of multiple copies...

...variety of colors when a combination of XFP are expressed. Each cell remains the same color for its entire...Stochastic multicolor labeling is a popular technique in neuroscience and developmental biology. This...containing fluorescent proteins (XFP) of different colors to label an organ or entire organism. Because each...entire lifetime and daughter cells retain the same color, allowing for the fate mapping of cell populations...persistently fluorescent during development. Stochastic multicolor labeling systems, many based on Brainbow, now...protein expressed in a single cell maintains its color over the entire length of the cell, even axons of...improving the level of detail possible for stochastic multicolor labeling of the brain. Labeling Drosophila neurons...

...CRISPR to label multiple loci in distinct colors. To create a colorful Cas9 toolbox, Ma et al. turned to SpCas9...nucleome. CRISPRainbow: Scaling up to 6 colors + white Color Hairpin Blue MS2-MS2 Green PP7-PP7 ...number of limitations. Since color is specified by PAM sequence, each color requires a different Cas9 ortholog...Colorful CRISPR technologies are helping researchers visualize the genome and its organization within...track multiple genomic loci over time using all the colors of the CRISPRainbow. Figure 1: CRISPRainbow...chromosome-specific loci in the U20S cancer cell line./ Each color represents a gRNA targeting a specific chromosome...different fluorescent protein to create three distinct colors. The specificity of these orthologs is key: since...

...later. Single Colors Single color controls contain, as the name suggests, a single color from your antibody... single color, fluorescence minus one (FMO), and isotype. You should always have single color controls...single color control for every fluorophore you intend to use in your panel. Using single color controls...from your single color controls may not be accurate for your samples. The single color controls should ... flow plots for a single color control. A) A flow plot showing a single color control for the APC channel...can bind to any primary antibody you add. Single color controls are used to help you set up your voltages...achieved by adjusting your voltage using your single color controls (Figure 1A), instead of using your actual...

...diversity of colors AND sparse labeling of neurons , Chan et al separated color labeling and color expression...these AAV’s be used for?  1. Multi-color labeling of neurons Multi-color labeling of neurons is for more ... high levels of color diversity so that cells are labeled with their own unique color; and 2) low levels...labeling, without loss of color diversity.  With this two part system, color diversity is maintained at... neurons. A simple but flawed approach to multi-color labeling is to inject a mix of AAV vectors that ...multiple viral particles and labeled a variety of colors (figure 4b). But notice in figure 4b how difficult...AAV and red, green, or blue are the predominate colors expressed (figure 4c).   AAV Vector  Percent...

... decided to jump into one of those unexplored areas with our color-changing flower, but to do that we’...plants can be challenging, there are a lot of unexplored areas in plant biotechnology that are ripe with...specifically for consumer sales. Flowers that change color throughout the day cannot be developed through conventional...years of basic research standing behind it. Our color changing flower involves two basic components: the...these components to develop flowers that change color. Without this basic research – this comprehensive...have also been patented. In order to construct our color changing flower we are carefully making our way ...Revolution Bio had to address as we developed the color-changing flower, and we’d love to hear about the...

... to Image>Color>Replace Red with Magenta. Or you can use Color>Split Channels and then Color>Merge Channels... are accessible to all viewers. Use a colorblind-friendly color palette for display (like green-magenta-blue...secondary antibodies, and image all the different colors in one session – fast and easy! Just make sure ...isotypes and that each secondary has a different color fluorophore that you can image separately on your... the images of each labeled target as different color channels and analyze the results. Depending on the...show each channel in individual panels, then the multicolor overlay; this helps all viewers, especially if...automatically from the microscope software, you can always recolor them! In Fiji, for example, you’d open the offending...

...and color One of the best ways to grab your audience’s attention is with the background and color of your...Depending on the color you select for the background, you’ll want to make sure that the color of your font...background, but two common approaches are either a solid color, or a “themed” background that is relevant to the...but beware. A distracting background or clashing colors can take away from what should be the focus of ...the data and your story. If you choose a single-color background but want to add a bit more “flash,” you... in all of our illustrations above) so that the color fades to a lighter or darker version of itself from... is compatible. Use high-contrast between these colors so that your text is easily read. Similarly, be...

...background on the color spectrum and the principles of excitation and emission. The visible color spectrum is...principle of color emission. As you can see in Figure 1, emission spectra of neighboring colors can and do... Figure 1: Emission spectra and respective colors of a few commonly used fluorophores. Each fluorophore...flow-cytometry-fluochrome-brightness-spillover Excitation Laser Color Very Bright Bright Moderate Dim Ultraviolet...fluorophore-conjugated antibodies together in the same color panel. In this case, make sure to compare the excitation... issue. Make sure to check out our blog post on color compensation to learn more about them. Alright, ... dead cells present and it is fine to use a dim color, like APC-Cy7. Next comes our transduction reporter...

...Massachusetts Medical School developed a dCas9-based multicolor labeling system to visualize chromosomal loci...two different hairpins will produce a secondary color (yellow, cyan, or magenta) depending on the hairpin... containing all three hairpins produces a white color. While CRISPRainbow allows for simultaneous visualization...inserted into the sgRNA generates blue, green, or red colors for labeling. A combination of MS2, PP7, or boxB...sequences in the same sgRNA will generate additional colors. Image from Ma et al., 2016 with permission. ...fluorescent ligands to the loci of interest. Dual color detection In order to label two loci simultaneously...CRISPRainbow) to 3-fold with CRISPR-Sirius. Triple color detection With a third sgRNA containing alternating...

... using cesium-chloride gradients, water-saturated phenol, sodium acetate, and chloroform are added to ...material. Then, the homogenate was loaded onto cesium-chloride gradients and ultracentrifuged for up to 20 hours... protocol with guanidinium thiocyanate-phenol-chloroform. In this method, samples are still homogenized...ultracentrifugation!), the layers of phenol and chloroform separate, and RNA is retained in the top, aqueous...centrifugation and the supernatant is extracted with chloroform as in the above no-kit method.  Researchers have... trick is to precipitate the RNA using lithium chloride. LiCl solutions selectively precipitate RNA, but...Isolation by Acid Guanidinium Thiocyanate–Phenol–Chloroform Extraction. Analytical Biochemistry 162:156–159...

...added to the wells. This reaction produces a colorimetric reaction that reflects the concentration of .... The reaction is stopped by the addition of hydrochloric acid (for more details on this, see below). ... concentration or antibody specificity via a colorimetric reaction. Image from Boguszewska et al., 2019... concentration or antibody specificity via a colorimetric reaction. Image from Boguszewska et al., 2019...added and the reaction can be visualized via a colorimetric reaction. The direct ELISA is often used when...

...no one has ever seen before (flowers that change color throughout the day) and doing this with very little.... Our scientific training was put to use as we explored ideas, planned out potential strategies for pursuing...Anything was fair game for speculation - in fact, our color changing flower project was inspired by a Facebook...upon the idea of building a flower that changed color throughout the day. It would be a beautiful GMO,...we didn’t figure out how we would accomplish the color change until a few months later. RevBio accelerates...clue how to run a business. SynBio Axlr8r thought color-changing flowers was a great business idea as well...the funds needed to build a flower that changes color, but the experience is invaluable. This opportunity...

...fluorescence states, they can change from one fluorescent color to another, or their fluorescence can be switched...PC-FPs). PC-FPs can convert from one fluorescent color to another (e.g. green to red, or cyan to green)...Protein Oligomeric State Activating Light Pre/ Post Color Contrast (Fold Change in Fluorescence) Brightness... a red fluorescent state make them useful for 2-color imaging with green PA-FPs. Table 2: Properties of...experiment even begins! If you are performing multicolor experiments, you will want to be sure that the...Photoactivatable mCherry for high-resolution two-color fluorescence microscopy." Nature methods 6.2 (2009...

...limitation of this system is the single-color labeling; multicolor labeling would enable the visualization...every week! One of the newest applications is a colorful system that uses fluorescently labeled Cas9 to...may be altered in disease states. dCas9 gets a colorful makeover Even without its nuclease activity, catalytically...the cell cycle or other stimuli.   To create a colorful Cas9 toolbox, Ma et al. turned to SpCas9 and its...a different fluorescent protein to create three colors. The specificity of these orthologs is key: due...excited to see what’s coming next! References Multicolor CRISPR labeling of chromosomal loci in human ...

...Glutamate indicators with increased sensitivity and tailored deactivation rates Addgene's own empty lentiviral...Glutamate indicators with increased sensitivity and tailored deactivation rates By Mike LacyGlutamate is one...Reporter (iGluSnFR) designed and improved by the Loren Looger and Kaspar Podgorski Labs now has a new version...Glutamate indicators with increased sensitivity and tailored deactivation rates. bioRxiv, 2025.03.20.643984.../10.1101/2025.03.20.643984.  Build your way: Explore Addgene's new empty backbones By Meghan Rego You...cell line creation (Figure 2). Looking for more? Explore our empty and EGFP retroviral plasmid set, our ...(TMT) mass spectrometry (MS) in HCT116 cells (colorectal cancer). The bottom panel depicts the identification...

...Nagai laboratory has assembled a collection of multicolored nano-lanterns for use in various applications...In 2015, Takai et al. expanded the Nano-lantern color palette to include cyan and orange Nano-lanterns...previous Nano-lanterns by 2-4 fold and permit five-color fluorescent imaging! These eNL constructs use the...appropriate for use with Nano-lantern technology. Multicolor imaging using Nano-lanterns requires a linear...linear unmixing algorithm to separate the various colors. In fluorescent imaging, you use the excitation wavelength...linear unmixing algorithm to decode the individual colors. The development of this algorithm is explained...Expanded palette of Nano-lanterns for real-time multicolor luminescence imaging.” Proc Natl Acad Sci U S...

...just make one fusion protein that switches to any color or tag you want? It sounds like magic, but it’s ...protein gives you the flexibility to choose different colors or ligands for different experiments without having...haloalkane dehalogenase that binds a synthetic chloralkane (CA) ligand. The current standard version known...  (TMP-tag) Few Easy HaloTag 295 Chloralkane (CA) Many Easy CLIP-tag 182 Benzylcytosine...single-molecule techniques.   Figure 2: Two-color super-resolution imaging of ER and mitochondria ...fluorescent protein) enables applications like multi-color imaging (Figure 2). If you’re working with purified...separately before reconstituting, choosing your colors depending on the needs of your experiment (Figure...

...single-color APC control, with APC and APC-CY7 plotted against each other. Since it is a single-color control...Compensation example with single color control. Flow plots showing an APC single-color control sample before (A...are only positive for APC (Figure 1B). For single-color samples, you can only change the compensation values...generally recommended to start compensating with single-color controls before moving onto your experimental samples...experimental samples without needing to use your single-color controls as a starting point. Applying compensation...

...the emission color (emission wavelength range) FPs are usually classified by emission color as outlined...familiarize yourself with the available FP emission colors and 10 points to keep in mind when choosing an ...Photoconvertible: These proteins can switch their color when activated by a specific excitation wavelength...Fluorescent Timers (FT): These proteins change their color over time. Therefore, these can be used as “timers...narrow enough to distinguish between closely related colors. Furthermore, most FPs  have a broad range of emission...proteins as biomarkers and Biosensors: Throwing color lights on molecular and cellular processes” Curr...

...only a single channel. In multi-color imaging experiments, the second color is usually provided by red fluorescent...of existing FP and epitope tags, researchers in Loren Looger’s lab have developed new molecular tags combining...antibodies, making them difficult to use in a multicolor/multichannel imaging set-up. In contrast, smFP...specific reactivities making them more useful for multicolor imaging experiments than standard FPs. The Looger...Looger Lab was thus able to easily perform a four-color labeling experiment using smFPs and standard neuronal...and detection: fluorescent protein structure and color variants." Cold Spring Harbor Protocols 2009.12 ...

...their lab coats handling glassware with brightly colored hazardous liquids. Working in a lab is nothing ...Research assistant Typically has a Master's or Bachelor's degree Generally a short-term position (1-2 years...biofuel production. There’s a lot out there worth exploring! Being excited about the science allows you to...and drive, but also makes for an experience more tailored to personal interests. Talk to people, ask questions... importance of building a network of mentors. Explore available resources! Many online resources and...Scientist While Poor” Keep an open mind as you explore opportunities The world of science is vast and...bench in academia or industry, so feel free to explore! Addgene has an entire page dedicated to different...

...Biology at the University of Colorado Anschutz Medical Campus in Aurora, Colorado, USA. The primary focus ...Biology at the University of Colorado Anschutz Medical Campus in Aurora, Colorado, USA.     Richard Gronostajski... of a common laboratory chemical, guanidine hydrochloride, as a potential treatment for a large number...be rescued by supplementation with guanidine hydrochloride (GdnHCl). Rescue occurs through guanidinium ...

...fluoresce one color but slowly mature until they predominantly fluoresce a second color. This change is...fluoresce red. The ratio of the mature color (red) to the immature color (green or blue) within a population... al. ""Fluorescent timer": protein that changes color with time." Science 290.5496 (2000): 1585-1588. ...et al. "Monomeric fluorescent timers that change color from blue to red report on cellular trafficking....

...General protein expression BL21 (DE3) pLysS* Chloramphenicol (pLysS) pLysS expresses T7 lysozyme to reduce...Expression of toxic proteins BL21 (DE3) pLysE* Chloramphenicol (pLysE) pLysE has higher T7 lysozyme expression...Expression of insoluble proteins  Rosetta2 (DE3)* Chloramphenicol (pRARE) Good for “universal” translation; contains...Expression of eukaryotic proteins Lemo21 (DE3)* Chloramphenicol (pLemo) Rhamnose-tunable T7 RNAP expression...T7 expression. The pLys plasmid contains a chloramphenicol resistance cassette for positive selection ... Julian Taylor-Parker...

...spectra. These probes can be combined in two-color and multicolor NIR imaging.  Small miRFPnano A separate.... They can also be used in crosstalk-free three-color imaging with green and red GFP-like FPs (Shcherbakova...Shcherbakova et al., 2016), as well as in four-color imaging (cyan, yellow, red, and near-infrared) (Qian et...monomeric near-infrared fluorescent proteins for multicolor imaging across scales. Nat Commun 11, 239 (2020..., V.V. Near-infrared fluorescent proteins for multicolor in vivo imaging. Nat Methods 10, 751-754 (2013...

...Chlamydomonas noctigama (CnChR1), Chloromonas subdivisa (CsChR), Chloromonas oogama (CoChR), and Scherffelia... mutagenesis or gene targeting Victor de Lorenzo's lab has engineered a modular mini-Tn5 vector that... proteins in the cephalochordate Branchiostoma floridae, named bfloGFPs. They went on to characterize ...fluorescent protein dsRed that changes irreversibly its color from green to red when it is oxidized. The Timer...have enabled the group to perform independent two-color optical excitation of neurons. The paper describes... in transgenic mice, the lab established a dual-color RNAi “sensor” mouse strain in which Cre expression...University previously changed the way researchers can explore important questions surrounding serine phosphorylation...

...opsin genes, and include bacteriorhodopsins, halorhodopsins, and channelrhodopsins, the first two found... excitation, whereas yellow light activates halorhodopsins to silence neuronal activity (Figure 1) (Wiegert...neuron activity on or off (Atasoy et al., 2008; Taylor et al., 2016; Hooper and Maguire, 2016). Recombinant...al., 2018; Deubner et al., 2019). For example, Taylor and colleagues used rAAV FLEx-based vectors to ...previously subjected to steady-state general anesthesia (Taylor et al., 2016). These very same neurons also play...PMID: 24700321. PubMed Central PMCID: PMC4100247. Taylor, Norman E., et al. "Optogenetic activation of dopamine...

...Action Items:  Explore this career map to learn more where science policy roles exist. Explore these resources...diverse workforce. Out of all the career spaces I explored for my post-PhD life, the science policy community...Items:  Sign up to receive updates from ESAL and explore their Local Engagement Playbook for step-by-step...this is the most important question that you can explore. The answer will help you decide how to define ...the policy world much smoother.  Action Items:  Explore this comprehensive resource developed by Dr. Kelly...

... unexpected infiltrates the realm of focused exploration. Here, we delve into the importance of leaving...research lies in expanding the scope of your exploration. While it is essential to delve deeply into your...intersections of disciplines. Cast your net wider, explore tangential subjects, and let curiosity guide your... that embraces the unexpected and encourages exploration of tangents or seemingly unrelated ideas. It'... emerge when we allow ourselves the freedom to explore beyond the expected. By widening your search and...

...they change color as they shift from "off" to "on" in the presence of calcium. This color change can be...addition to imaging neurons, Griesbeck adds that this color change has a particular advantage for observing ...applied this “trick” to the development of additional color variants. This resulted in the construction of redCALWY... donor and acceptor domains. He says, these new colors might now enable observation of the interaction...

...retained only in some species of bacteria. The stain colored one type, Streptococcus pneumoniae, while the alcohol...stain became known as gram positive, and the decolorized as gram negative. A German scientist, Carl Weigert...types. In water, the dye dissociates into CV+ and Chloride (Cl-) ions. When the negatively charged iodine... unable to retain the stain, leading to its decolorization. In contrast, counterstain, safranin, is a ...crystal violet dye, but is easily retained in the decolorized gram negative bacteria, staining them red or ...

...wet lab wasn’t the right path for me. I began exploring alternative career trajectories within the industry...self-motivated and excelled when given the freedom to explore projects autonomously. Failing to recognize and...frustration, micromanagement, and disengagement. By tailoring my leadership approach to each person, I was able... a bit to my mentors, as they encouraged me to explore small-scale opportunities where I could make mistakes...role. My advice is: Give yourself permission to explore, reflect on your priorities, and embrace these ...

... Many thanks to our guest blogger David Mellor! David Mellor leads the policy and incentive programs ...This post was contributed by David Mellor from the Center for Open Science. In the last decade, researchers...Before coming to the Center for Open Science, Dr. Mellor worked with citizen scientists to design and implement...at https://orcid.org/0000-0002-3125-5888 and @EvoMellor.  ...

...with spectral flow—you can run a three color panel or a 40 color panel! When preparing a spectral flow ...experiment. For very large panels (upwards of 30-40 colors), spectral flow cytometry might be the better choice... Figure 3: Similarity index across 12 different colors on a three laser spectral flow cytometer. ...from a batch that is not the same as your single color control. Reference controls: Beads or cells? Compensation...