...practical due to the low intensity of luminescent proteins. To make luminescent imaging a reality, Addgene depositor...with Venus. This luminescent technology is based on the principle of bioluminescence resonance energy ... a few drawbacks associated with luminescent imaging: A luminescent substrate is required for imaging...Researchers have long been interested in using luminescence to get around these issues, but this solution...fusion proteins. Solving the problems of low luminescence By itself, Renilla luciferase has a low quantum... hasn’t stopped some scientists from using chemiluminescent proteins for imaging, but it has precluded...orange Nano-lanterns and showed that these three luminescent proteins can be used together in cells. As Nano-lanterns...

...scientists. Seeing inside these specimens has offered illumination on biochemical processes that are crucial in...85% of its original value after 60 minutes of illumination, highlighting its reliability for long-term ...to a specific stimuli, which leaves only a small volume of fluorophores in an excited state, effectively...

...eScience Labs as part of their curriculum by either taking existing curriculum and customizing it for schools...the entire curriculum development team at eScience Labs. She still gets to write curriculum, but has taken...Caitlin’s take on her job as an SME: “I write curriculum for students taking science courses online, which...student the best possible experience.” Managing curriculum development About a year after Caitlin was hired...

...compared to fresh columns, regenerated columns had similar DNA yields. Additionally, columns regenerated up...times had similar DNA yields as fresh columns, suggesting that columns can be reused at least five times....Before you reach for that silica spin column, stop to consider some ways to purify DNA without a kit....bunch of unused buffers because you’ve run out of columns. Kit-less DNA purification methods avoid many ...without a kit as well as one way to reuse silica columns from DNA purification kits. Kit-free alkaline lysis...protocol from Addgene follows a similar workflow as a column-based plasmid extraction kit. First, you lyse the...available kits but instead of binding DNA to silica columns, it’s bound to glass syringe filters. Normally ...

... the lab has dubbed these fusions LumiFluor Reporters. The LumiFluor reporter constructs were designed...fluorescent donor for a bioluminescent donor, making BRET probes. Here the bioluminescent donor is unaffected...deep sea shrimp to a compact powerhouse of bioluminescence, NLuc is 100-fold brighter than our more common...wavelengths should be used. Researchers often use bioluminescence to overcome these issues as traditional luciferases...excites the fluor through a process known as bioluminescent resonance energy transfer (BRET). The fluor...a tissue-penetrating wavelength above 600 nm. LumiFluors Schaub, F.X., et al. (3) designed two imaging...is an excellent BRET donor due to its bright luminescence. Intracellular Calcium Sensor Yang, J. et al...

...disadvantages of luminescence versus fluorescence.  Table 1: Properties of Luminescence versus Fluorescence...Secreted Luminous shrimp(Oplophorus gracilirostris) 455 19 Coelenterazine None Yes Luminous Japanese... the emission of photons. The most familiar bioluminescent organism for most people is the firefly (Photinus...surprisingly it is also the most commonly used bioluminescent reporter. This beetle emits a yellow-green ...as substrates. Production of photons by this bioluminescent reporter occurs slower than fluorescent-based...the different mechanisms to produce photons, chemiluminescent reporters are generally less bright than fluorescent...Fluorescence   Luminescence Fluorescence Source of Emitted Photons Chemical reaction High-energy photons...

.... coli strains. Increase the volume Starting with a larger culture volume can improve plasmid yield. Our...yields. Why would volume make a difference? In the simplest of terms, more volume = more cells = more plasmid...plasmid. When increasing the culture volume, be aware that a higher volume of lysate passed over the binding... prep. Check the bacterial strain Increase the volume Improve the culture media Let it incubate Check... Our lab often doubles the volume of culture used for inoculation if we get inadequate plasmid DNA yields...purify! Additionally, if you've reached your lysate volume limits, the copy number of some origins of replication...indicate the number of cells present in the given volume.  Let oxygen in! Make sure your culture is getting...

... common chemiluminescent methods for detection (though the enzymes used for chemiluminescence can be conjugated...classes of reporters: chemiluminescence enzymes and fluorescent proteins. Chemiluminescence enzymes are cheap...agents. Chemiluminescence kits When activating your enzyme, make sure to choose a chemilumescence kit appropriately...multiplexing, and the solutions used to provide the chemilumescence may be light-sensitive. They also have a poor... after the reaction has started. Types of chemiluminescence enzymes The two most common enzymes are horseradish...have a ten-fold greater dynamic range than chemiluminescence enzymes, making it easier to accurately visualize...loss of signal. While not as sensitive as chemiluminescence enzymes, they are more likely to be compatible...

...Location: tail vein or retro-orbitally Gauge: 27–30 Volume: < 0.2 ml Skill required: beginner (retro-orbital...scruffing Location: peritoneal cavity Gauge: 25–27 Volume: < 2–3 ml Skill required: intermediate Intramuscular...mechanical restraint Location: thigh Gauge: 25–27 Volume: < 0.02–0.05 ml Skill required: beginner Subcutaneous...restraint Location: back or hind legs Gauge: 25–27 Volume: < 2–3 ml (~1 ml per site) Skill required: beginner...anesthesia Location: between skin layers Gauge: 26 Volume: < 0.05 ml Skill required: advanced Intrathecal...Restraint: anesthesia Location: spinal cord Gauge: 30 Volume: up to 0.01 ml (10 µl) Skill required: expert ...Location: trachea Gauge (tracheal tubing): 23–20 Volume: < 0.07 ml Skill required: advanced Intranasal ...

...concentrate in a smaller volume. Consequently, you will end up with a considerably lower volume than with the unconcentrated...polyethyleneimine, calcium phosphate or liposomal methods), the volume of the reaction, and the timing and number of harvests...ultracentrifugation, centrifugation on a sucrose gradient, spin columns, and polyethylene glycol (PEG) precipitation. All...this in mind, if the experiment requires a certain volume of virus, consider scaling up by either increasing...require some initial optimization; cell number, media volume, and plasmid concentrations will need to be optimized...number, plasmid concentration, and experimental volumes accordingly. While you may need to make some final... you don’t anticipate that you will need large volumes of lentivirus routinely, you may opt for simply...

...molecules within a volume. In many biological research situations, the relevant volume is that of the cell...molecules are homogenously distributed throughout the volume and can freely tumble in 720°. However, if the ...structures including organized smooth endoplasmic reticulum (OSER) whorls. 2)A dimerizing protein of interest...For a membrane POI localized to the endoplasmic reticulum, the normally spiderweb-like pattern of tubules...membranes termed Organized Smooth Endoplasmic Reticulum or OSER if a dimerizing FP is fused to the POI...describing the FPs above including molecular sizing columns and native gels fail to detect EGFP dimerization...control of secretory proteins in the Endoplasmic Reticulum and live cell imaging approaches. Erik is also...

...restricting fluorescence to specific excitation volumes to enhance spatial contrast (i.e. optical sectioning... light exits the microscope objective evenly illuminating the entire (wide) field of view. Fluorescent...and focused onto a camera for visualization. Illuminating a sample in the opposite direction to fluorescence...fluorescence collection is referred to as “Epi” illumination. Therefore these are sometimes call Epi-fluorescence...location in the sample. As more than one hole is illuminated at a time, the image can be acquired more quickly...project excitation light through the entire axial volume of a sample. Therefore, when a large number of ...constantly exposed to light, not just those in the focal volume. This leads to more rapid photobleaching and a ...

...organized format.  Columns. You can choose from either a two, three, or four column style, and in all ...; the reader will navigate the poster from column to column left-to-right, which is the natural way that...success of this style is clear borders around your columns, and good spacing between them so that they are... some Powerpoint based templates with different column numbers for a good starting point. Adobe also offers...central box is then flanked by two equally-sized columns, where the introduction, methods, conclusions, ...horizontally. Instead of displaying your information as columns, you may choose to display it as rows. It can be...

...CRISPR/Cas9 for gene disruption in M. abscessus  Bioluminescent reporter of mechanical forces   Epitope tag...e0033523. https://doi.org/10.1128/jb.00335-23.  Bioluminescent reporting of mechanical forces with PILATeS...expressed separately) assemble and catalyze the luminescence reaction (Figure 5). The researchers used the.... Upon binding, LgBiT and BiT Tag catalyze a luminescent reaction with the substrate furimazine. Right...coverslip creates mechanical tension, and the luminescence colocalizes with the focal adhesion marker paxillin...compatible with common microscopy methods. Plus, the luminescence wavelength (460 nm) is shorter than many popular... (510 nm), allowing co-imaging. And because bioluminescent markers do not need to be excited by an external...

...Spelman focused a laser beam into a thin sheet to illuminate a fluorescent sample and captured the reflected... lens oriented perpendicular to the plane of illumination (i.e. light sheet).  This is in contrast to ...described the single-plane or selective-plane illumination microscope (SPIM). In order to improve axial...the laser light sheet, typically 2-6  microns, illuminates only one thin plane of the sample surrounding....  One limitation of LSFMs using single side illumination, especially with large tissue, is that any obstacles...concentration of fluorophores) cast shadows along the illumination path. The shadows appear as stripes in the acquired... (5).  A way to get around this problem is to illuminate and acquire images of a sample from two opposing...

...incorporate more science and technology into the school curriculum. Our Parent Teacher Association (PTA) has been...been instrumental in supplementing that curriculum. It’s through the PTA that I recently started coordinating...Lab Middle school: supplementing the science curriculum Sharing my expertise with our district middle...tried contacting our school district’s science curriculum coordinator with minimal luck, but eventually...speaking on topics that would compliment the STEM curriculum. As it happened, I’d met some of the 8th grade...said “it was such a valuable extension to our curriculum and offered engaging examples of how understanding... who work hard maintaining a current, dynamic curriculum, would be hard-pressed to maintain the pace of...

...Luminescent molecules are very useful tools because we can easily detect and measure the light they emit...emit. Proteins that give off light include chemiluminescent proteins, like luciferases, as well as fluorescent...Protein (GFP). These molecules occur naturally in bioluminescent organisms, but their real power lies in the...interesting. Although humans have known about bioluminescent organisms and their characteristic radiance..., in the 1960s, Osamu Shimomura studied the bioluminescent properties observed in the crystal jellyfish...Lukyanov realized that, although they are not bioluminescent, some types of corals and anemones are vibrantly...

...to as many lab alumni as you can or schedule video/calls if they are not local. Alumni meetings can be... too long or do they leave prematurely? Do lab alumni stay in the same field taking projects with them...remaining close colleagues of the PI? Or do lab alumni leave the field or get pushed out by the PI? What...One scientist who followed my advice to interview alumni of the lab was told that the PI removed a woman... happen to you. As you speak to lab members and alumni, don’t ever disregard warning signs of unethical...supporting career development and growth. Have lab alumni gone into both academic and non-academic careers...

...YFP and use an affinity column for purification. After binding YFP to the column, you wash several times... use OptoNBs. You fill a column with OptoNB coated beads and wrap the column with blue LED lights. When...their target protein is altered by blue light illumination. This ability to reversibly bind and release...pathway. What’s next for OptoNBs? While OptoNBs illuminate a path to light-controlled protein binding, they...