...excitation microscopy is a fluorescence imaging technique that allows imaging of living tissue up to about one... technique and the in vivo live-cell imaging (intravital imaging) studies it’s enabled are revealing the...immune cell mobility than the static microscopy images of chemically coloured lymphoid organs did in the...Two-photon microscopy was the next revolution in cell imaging, giving researchers the ability to monitor cellular...than any other cell type in the body! Intravital imaging powered by two-photon microscopy has uncovered ...infected tissues. Before the use of two-photon imaging, our understanding of pathogen-immune cell interaction...Neutrophils travel to the sites of infection or damage (Peters et al. 2008, Chtanova et al. 2008, Kamenyeva...

...fluorescent protein fusions for imaging Fluorescent proteins are fantastic imaging tools but their use is limited...for imaging. These authors set out to design a better fluorescent protein that could be co-imaged with...of researchers wanting to perform whole-animal imaging, however NLuc’s emission peak is at 460 nm thus...thus making it non-optimal for in vivo imaging. The solution is to couple Nluc with a red-shifted fluor....LumiFluors Schaub, F.X., et al. (3) designed two imaging probes. One consists of NLuc fused to enhanced ... The isolated cells were injected into mice and imaged after i.p. or i.v. administration of the furimazine...of why a BRET reporter can improve deep tissue imaging. The constructs for GpNLuc and OgNLuc are available...

...photo-bleaching or photo-damage than in conventional laser scanning microscopy . Since image acquisition is performed...milliseconds to acquire one image, thereby significantly reducing the time it takes to image through a large z-...the acquired image (5).  A way to get around this problem is to illuminate and acquire images of a sample...the 3DISCO (3-dimensional imaging of solvent cleared organs) method of imaging rodent brains and spinal... axial resolution and reduce phototoxicity for imaging live specimens, they combined light sheet microscopy...sequential multiple-view reconstruction (5). In order to image large tissue such as cleared rodent brain, the Ultramicroscope...point-by-point scanning along the entire specimen makes imaging too slow and unwieldy for large specimens that ...

...lens which magnifies the image, then the ocular lens, where the image is further magnified before it finally...10x magnification. To determine the final magnification of your image, multiply the magnification of your...now) is the magnification power, such as 4x, 10x, or 20x. A 4x objective magnifies the image four times...properties of a microscope are magnification (the ability to make an image larger) and resolution (the ability...your eyes. These lenses determine the magnification of the image and the resolution your microscope can...10x objective magnifies it 10 times, and so on. The ocular lens also provides magnification and the power...also 10x, you would actually be seeing a 100x magnified image of that object! Equipment Light Microscope ...

...with information about images that a sighted reader would glean from the image itself.  As we incorporated...website through the use of alt text to describe images in online spaces. Alt text is used by screen readers...Remember, our goal is to use words to describe the image in a way that helps visually impaired readers receive...information as sighted readers.    Figure 1: An image from AAV Purification by Iodixanol Gradient Ultracentrifugation... page? What are they hoping to get from it? The image in Figure 1 appears on our protocol page for AAV...Second, you’ll identify your purpose in using that image. Try using the questions: What is the goal, who ...fundamentally change about their knowledge?    Here, this image is being used to convey the difference between a...

...non-dividing cells for a period of years without damaging the host cell. In contrast, viral particles produced...

...Two-photon image of miRFP670nano3-expressing neurons in the neocortex of a living mouse. Image courtesy ... for efficient binding of endogenous BV.  When imaging NIR FPs, consider using light sources that produce...can also be used in crosstalk-free three-color imaging with green and red GFP-like FPs (Shcherbakova et... et al., 2016), as well as in four-color imaging (cyan, yellow, red, and near-infrared) (Qian et al., ...red-shifted light. The same NIR FPs and biosensors can be imaged at spatial scales from sub-cellular to whole-body...can be combined in two-color and multicolor NIR imaging.  Small miRFPnano A separate class of NIR FPs, ...proteins as tags and biosensors for multiscale imaging. Nat Commun 7, 12405 (2016). Qian, Y. et al. A ...

...photostability. It enables sustained deep-tissue imaging in vivo. Image reproduced from Liu et al. 2022, under ...fluorescence lifetime imaging. B) LiLac responses are highly resistant to pH changes. Image adapted from Koveal...intracellular loop of various G-protein-coupled receptors. Image adapted from Ino et al. 2022, under CC-BY license... sensitivity compared with earlier SF-iGluSnFR. Image from Aggarwal 2022.   Find iGluSnFR3 plasmids here...improved activation kinetics and localization for imaging synaptic transmission. bioRxiv 2022.02.13.480251...-2P (Liu et al. 2022) optimized for two-photon imaging. They use a unique and powerful high-throughput...RTK activation by shift in cellular localization. Image adapted from Farahani et al. 2022, under CC-BY license...

...Likewise, the switch from ensemble imaging to single-molecule imaging places a greater burden on the photon...protein (GFP) sparked a renaissance in biological imaging. Suddenly, cell biologists were no longer beholden...to the tag. Like most good ideas in biological imaging, the initial breakthrough for in-cell labeling ...antibody and oligonucleotide labels for fixed-cell imaging, not for live-cell applications. Thus, these fluorophores...properties inside living cells, especially for advanced imaging experiments. We continue to develop derivatives...can provide a fresh infusion of photons for your imaging experiment. Innovative labeling strategies and ...us to push further the frontiers of biological imaging.  *Worried about your budget? Email janeliafluor...

...water.)  Step 5: Image   Figure 1: The ExM process allows you to physically magnify a sample so you...ExM) promises an easier, more accessible way to image biological features previously only visible via ...planning your own ExM experiment or evaluating ExM images in a new publication.         Why Expansion Microscopy...Super-resolution microscopy (SRM), a family of imaging techniques developed to break through the resolution...straightforward answer to the space problem. Instead of imaging smaller, what if we just made the sample bigger... microscope.  Along with using more accessible imaging equipment, ExM protocols utilize relatively inexpensive... you don’t have to optically magnify quite so much. Created with biorender.com Steps 2-4 are not found...

...switches to any color or tag you want? It sounds like magic, but it’s real! In this post, we’re going to talk...substrate. Then, all it takes is a bit of chemistry magic to produce a wide range of useful molecules linked...and black is the fusion tag or labeling system. Image adapted from Lavis, 2016, under CC-BY. Common...structures or low levels of expression with the same imaging workflows you already use. Or you can really capitalize...   Figure 2: Two-color super-resolution imaging of ER and mitochondria reveals dynamic organelle... cell ER labeled with Halo-Sec61β with SiR-CA (magenta) and outer mitochondrial membrane labeled with ...with SNAP-OMP25 with Atto590-BG (green), imaged with stimulated emission depletion (STED) nanoscopy. Scale...

...substance (fluorophore) that has absorbed light or electromagnetic radiation at another wavelength (Heinrichs,...molecules are critical for accurate and reliable imaging and assay results (Hayashi-Takanaka et al., 2014... important for intracellular fluorescence live imaging ​​(Hayashi-Takanaka et al., 2014). Dyes can also... cell membranes can prove useful for live-cell imaging. Experimental context and ligand binding: The context...matching dye/ligand choices For effective live-cell imaging, the use of dyes that exhibit low toxicity and ... 123 (R123) (Liu et al., 2017). In multi-color imaging, it is essential to select dyes with different ...differentiation between multiple dyes during analysis. Image resolution is impacted by several factors. Firstly...

...shown to be effective for imaging single molecules and for live-cell imaging in C. elegans. Other groups...riboswitches can be used as stable metabolite sensors for imaging in live cells. By placing Spinach downstream of...search for something more stable for quantitative imaging, Ming Hammond’s group created Spinach2 using site-directed... its length reduces artefacts during live cell imaging. Bunch of baby spinach, or BoBS, contains tandem... intracellularly due to a lower dependency on magnesium and higher thermal stability, making it a potentially...about 1.8-fold, and may be a better choice for imaging than the monomer. Broccoli has been developed into...photostable and useful for both live and fixed cell imaging. New ingredients in the aptamer soup In addition...

... using widefield imaging. Our choice for a photostable protein for live cell imaging would be mTurquoise2... will photobleach at some point. For live-cell imaging, it is desirable to have fluorescent proteins that.... The best fluorescent proteins for live cell imaging can be excited many times, thereby producing a ...power is important since different fluorescence imaging strategies use widely varying amounts of excitation...light (for a brief time), whereas in widefield imaging fluorophores are excited with relatively low light...is a key requirement for quantitative live-cell imaging, it is important to be able to quantify the photostability...experiments’, it is advisable to perform time-lapse imaging of cells producing the fluorescent protein of interest...

...include the ImageJ plugins JACoP and Coloc 2, BioImageXD, and customized CellProfiler pipelines. Image analysis...examine your images using subjective judgement by simply “looking at them”. Modern cameras and image analysis...organelles. Take a sneak peak inside a cell with the images from the Allen Cell Explorer (1). When studying...subjective color intensity perception. Open source image analysis software tools can be used for colocalization...quantitative colocalization analysis methods and useful image analysis software tools. Pitfalls, limitations,...for general problems associated with fluorescent imaging such as bleed-through (the incorrect detection ...should use and how to select FPs for muli-color imaging. Furthermore, one must bear in mind that the resolution...

...collected by centrifugation (agarose beads) or a magnet (magnetic beads), providing a solid support while unbound...surface of magnetic beads, and are therefore completely accessible. The yields from magnetic beads are ... The beads are collected by centrifugation or a magnet. 4) Unbound proteins are washed away. 5) The target...protein is immobilized, or tethered, on agarose or magnetic beads and then incubated with a protein mix. During...often called the capture antibody, to agarose or magnetic beads in a way that also allows the antibody to...recombinant Protein A/G are conjugated to agarose or magnetic beads and allowed to bind to the antibody being...antibodies can be directly conjugated to agarose or magnetic beads with commercially available chemical agents...

... the smFP structure resembles images of the deity (see comparison image above). The church of the Flying...cutting-edge imaging technologies such as array tomography, super-resolution STORM imaging, and electron...antibodies. The most famous ones are influenza hemagglutinin (HA), myelocytomatosis viral oncogene (myc),...signal, they cannot be detected directly in live imaging experiments. It is therefore difficult to use epitope... fluorescent and can therefore be used in live imaging experiments crucial to studying neuronal circuits...light in only a single channel. In multi-color imaging experiments, the second color is usually provided... difficult to use in a multicolor/multichannel imaging set-up. In contrast, smFP tags can be recognized...

...at Addgene, can be used for ratiometric in vivo imaging. Griesbeck explains, while these Twitch sensors...commercially available beam splitters. In addition to imaging neurons, Griesbeck adds that this color change ...neuroscience and immunology. Don’t be shy: Ratiometric imaging for beginners Griesbeck says his calcium indicators...may have a tendency to shy away from ratiometric imaging, he recommends they give it a shot. “Beginners ...sensor proteins, particularly for the intracellular imaging of transition metal ions, such as zinc, copper,...including his zinc sensor and his genetically encoded magnesium sensor, the first of its kind. “I hope by depositing...ratiometric calcium sensors for functional in vivo imaging of neurons and T lymphocytes. Nat Methods. 2014...

...for in-culture work and 2P in vivo imaging, PDGFR for bulk 1P imaging or in new preps. Using iGluSnFR3 ...iGluSnFR3: Improved Glutamate Indicators for Synaptic Imaging However, the first two generations of iGluSnFR ...experiments requiring high signal to noise ratios, imaging of high frequency glutamate signaling events, and...sensing glutamate using both 1-photon and 2-photon imaging.   Figure 1: 1P and 2P excitation and emission...SF-Venus-iGluSnFR.A184V, iGluSnFR3 v82, and iGluSnFR3 v857. Image adapted from Aggarwal et al., Figure 1   Increased...titration curves for the different iGluSnFR variants. Image adapted from Aggarwal et al…, Supplementary Figure... and ∆F/F0 for the different iGluSnFR variants. Image adapted from Aggarwal et al…, Supplementary Figure...

...Metal spatula Razor blade Plastic tray Gel imaging system ImageJ or similar software Reagents Purified recombinant...the sink. Take a brightfield image of the gel with an appropriate imaging system. Recombinant antibody... QC and would not be used. Section 3: Image Analysis Use ImageJ or a similar photo software to determine...bands to the overall lane. Import the gel image into ImageJ. Select File . Select Open . Choose the location...location of the file to open. Change the image type to 8-bit . Select Image . Select Type . Choose 8-bit . Determine... using ready-to-use biosafe Coomassie stain and ImageJ software. Protocols...250 stain (Addgene uses SimplyBlue SafeStain) and ImageJ software. The sample is separated by denaturing...