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As seen on the "Cell Imaging" Facebook Page Jason A.

Kilgore Molecular Probes Tech Support, Life Technologies

One-Hundred Imaging Tips

100 Imaging Tip of the Day: If your tissue is already labeled with a fluorescent dye (such as neuronal tracers or injected cells tracked with dyes), be aware that paraffin processing may extract the dye. Consider cryosections instead. 99 Imaging Tip of the Day: Use of BrdU is a well-established method for determining cells that have proliferated, but it requires denaturing DNA with HCl, which can affect cell morphology, and the use of antibodies for detection, which requires time and can add background. Consider Click-iT EdU, which incorporates the same way but doesn't need HCl or antibodies, just a 20-minute "click" reaction to detect: 98 Imaging Tip of the Day: Tissue is particularly bad for autofluorescence, but paraffin sections are worse than cryosections. Consider using autofluorescence reduction methods (like sodium borohydride treatment), amplification methods (such as avidin-biotin), and avoiding direct conjugates (which are dimmer). 97 Imaging Tip of the Day: Is your dye stock in DMSO? Be aware that DMSO will freeze in the refrigerator and condense a bit, giving the impression that you received a solid or too low a volume. DMSO takes a while to thaw completely, so give it time at room temperature or with gentle warming before opening and using. 96 Imaging Tip of the Day: Autofluorescence is a particular problem for plant samples, due to phycobiliproteins, chlorophyll, xanthophylls, carotene, and various flavinoids fluoresce in a variety of wavelengths. Make sure to have a nodye control to test for this. Images of plant autofluorescence: 95 Imaging Tip of the Day: When imaging cells with microplates, choose a type with the least autofluorescence in your wavelength by testing them first with a water blank. Also, opaque walls will prevent bleedthrough between wells. 94 Imaging Tip of the Day: DNase I conjugates have been used for labeling Gactin in fixed and permeabilized cells, and has relatively low affinity to F-actin

confluencies. 84 Imaging Tip of the Day: Some traditional flow cytometry dyes are not as useful for microscopy. volumes. It is retained for just a few hours and isn't bound covalently (and thus isn't as likely to perturb cells).abcam. called "dye-dye quenching. too. but it can be pumped out of cells over time. DNase I conjugates may label nuclei." If this is a problem reduce your dye-to-protein molar ratio. 93 Imaging Tip of the Day: DiOC6(3) is a traditional dye for labeling endoplasmic reticulum. 85 Imaging Tip of the Day: Be aware that when you conjugate a protein with a dye. like FluoCells cell and tissue slides or FocalCheck bead slides. 86 Imaging Tip of the Day: AM ester dyes and indicators can sometimes have difficulty in getting a uniformly-dispersed final solution or uniform loading. If this 87 Imaging Tip of the Day: When a protein is over-labeled. usually due to photostability problems or filter . the proximity of the dyes to each other can lead to quenching of the overall fluorescence. 90 Imaging Tip of the Day: Calcein AM is often used as a live cell indicator or for short-term cell tracking and cell mobility testing.pdf 88 Imaging Tip of the Day: Useful numbers for cell culture (seeding densities. which is an optimized formulation of Pluronic compounds. 92 Imaging Tip of the Day: Good link for info on using antibiotics and antimycotics in cell culture: http://bit. too. you always risk losing some or all of the protein's functionality by steric hindrance of active sites (especially with high degree-of-labeling) or changes in protein conformation (if reduction is required.pdf ). ER-Tracker dyes are far more selective.(unlike phalloidin conjugates). labeling mitochondria and other structures. leading to decreased signal. Some assays purposely self-quench proteins to show a dequenching fluorescence upon completion of the assay (such as this one: http://probes. use of Pluronic F-127 (a non-ionic surfactant polyol) can for instance).ly/iKyKnv 91 Imaging Tip of the Day: Need a quick sample to throw on the fluorescent scope to demonstrate the hardware? Commercial slides are available. However. You can also make quick samples using autofluorescent pollen or flower petals.invitrogen. but it's not very selective. etc): http://bit. 89 Imaging Tip of the Day: A link for general troubleshooting of whole mount immunolabeling problems: too. PowerLoad.

invitrogen. but after a certain time period looks dimmer. it may be that the dye is being actively extruded out of the cells. Use of probenecid may help stop the active extrusion and retain the dye in the cytoplasm. Pacific Blue. But for archiving of 74 Imaging Tip of the Day: Good article from BioProbes 62 on autophagy assays for live cells: http://bit. All other mountants will lead to loss of initial intensity over days to OC-1041 78 Imaging Tip of the Day: How to choose the right filter set: 80 Imaging Tip of the Day: A protocol for embedding tissues for cryosectioning: OC-1042 79 Imaging Tip of the Day: Causes of aggregation for microspheres: http://molecularprobestechnologynetwork. 81 Imaging Tip of the Day: Qdots are the most photostable fluorophores. 82 Imaging Tip of the Day: If you are using a avidin-biotin amplification strategy. you need to mount in Qmount mounting medium. such as 77 Imaging Tip of the Day: An example protocol for immunocytochemical labeling of cultured cells: be aware that cells have endogenous biotin which will need to be blocked (particularly in mitochondria).invitrogen. 83 Imaging Tip of the Day: If you are labeling with an indicator and allophycocyanin (APC). and your initial signal looks good.invitrogen. Commercial endogenous biotin blocking kits are OC-1031 76 Imaging Tip of the Day: Causes and fixes of photobleaching summary for microscopy: http://molecularprobestechnologynetwork. Examples include R-phycoerythrin (PE).com/docs/D OC-1043 75 Imaging Tip of the Day: Good technical note about the structure and classification of antibodies: http://bit.

make sure to use a brand of plate that has low autofluorescence in your wavelength. Don't fix with solvents. splotchy. which will lead to more bleedthrough problems between imaging channels. to prevent cross-talk between wells. 68 Imaging Tip of the Day: Examine your fluorescent filters 70 Imaging Tip of the Day: Good table of scavengers of reactive oxygen species: http://bit. though. If the coating is irregular. to improve signal-to-background ratio. 71 Imaging Tip of the Day: Great article summarizing fluorescent dyes for use in plant samples: 69 Imaging Tip of the Day: Problem with your antibody conjugate coming off its target (off-rate) after labeling and mounting your sample? Consider postlabel fixation with formaldehyde to cross-link it in place. then it is degrading. 67 Imaging Tip of the Day: For AM ester and diacetate dyes. leading to loss of the dye. . remember that different cell types have different levels of esterase activity. However. scan a water blank for each brand that you have available and use the one with the lowest value. including methanol in methanol-stabilized formaldehyde (formalin) or permeabilization with Triton X-100. subsequent exposure to detergents or solvents will delipidize the sample. and storing cold. 66 Imaging Tip of the Day: There is no reliably *fixable* organic dye for labeling lysosomes. so you'll need to optimize label time and dye concentration for your system. 72 Imaging Tip of the Day: Phalloidin conjugates are an excellent way to label F-actin in fixed and permeabilized cells.73 Imaging Tip of the Day: If you are imaging with a plate reader. If in doubt. or has a "bull's-eye" pattern. mounting in a solidifying mounting medium. Opaque walls are a plus. 65 Imaging Tip of the Day: Good site for introduction to basic statistics: http://www. CellLight Lysosome fluorescent protein products will transiently transfect mammalian cells for lysosome labeling using a BacMam construct and can be subsequently formaldehyde-fixed (and is nonperturbing to viability).usablestats. no antibody 64 Imaging Tip of the Day: If you are using lipophilic dyes. This will lead to a wider bandwidth.

such as low-impact 61 Imaging Tip of the Day: An interesting guide to using calcium indicators: http://bacterio. 62 Imaging Tip of the Day: Important criteria for choosing a calcium indicator include desired Kd value (calcium concentration range). For those be aware that the reagent will bind to any amine groups.microscopyu.imagescience. 57 Imaging Tip of the Day: The most nuclear-selective dyes for cells are DAPI and Hoechst dyes. and whether you want ratiometric or nonratiometric. 56 Imaging Tip of the Day: Problems with your cell culture? Here is a troubleshooting guide: http://tools.63 Imaging Tip of the Day: If a mounting medium is advertised as "antifade".uam.pdf. but likely won't be as effective as having an antioxidant present as though their selectivity for one or the other will vary between dye options and conditions. 59 Imaging Tip of the Day: A good review article for neuronal tracing: http://www.neuroanatomy. or proteins in e%20Troublshooting%20Guide. and adjust your microscope eyepieces and chair height appropriately. and moving things on your lab bench to be within easy reach. Here is a good table for many dyes: http://bit. Here is a good link for other suggestions: http://dohs. All others will label both DNA and stel. If it's just because it hardens.nih.pdf 58 Imaging Tip of the Day: Excellent site for all things related to microscopes: www.od. that will including on the cell surface or a poly-L-lysine coating on the substrate. which will cause "false .pdf 55 Imaging Tip of the Day: Don't underestimate the importance of good ergonomics in the lab to prevent injuries. check to see why.htm 54 Imaging Tip of the Day: Trypsin is typically used to detach adherent cells.cbm. comfortable lab stools. an RNase wash first (for fixed and permeabilized cells) will typically be needed to insure clean nuclear labeling.ors. But this temporarily disrupts the plasma membrane. desired excitation/emission Take frequent stretch breaks.pdf 60 Imaging Tip of the Day: When labeling cells with a succinimidyl ester (such as CFDA SE). Another for how to do the analysis: http://www.

50 Imaging Tip of the Day: For taking digital microscopic images.invitrogen. to unmask antigens in samples for immunolabeling. 457nm laser works great. 47 Imaging Tip of the Day: Be sure to rest and get enough sleep." and shouldn't be used as long-term cell tracers. 46 Imaging Tip of the Day: Doing two-photon microscopy? Here is a good link for two-photon absorption cross-sections for dyes: http://www. or even 488nm. 48 Imaging Tip of the Day: Nucleic acid Thus. but it is only semi-permeable to live cells. UV or 405nm is optimal. chemicals and solvents. others not.cornell. regardless of emission wavelength. 51 Imaging Tip of the Day: There are a lot of apoptosis assays. If you don't have UV or Lack of sleep reduces your powers of observation.html 45 Imaging Tip of the Day: Antigen retrieval techniques.dead" readings with dead cell indicators. but is far more cell-permeant. and we scientists often work with dangerous biohazards. or most hydrophobic barrier pens (the ImmEdge pen sold by Vector Labs is one that doesn't quench). HCS. See page 16 in BioProbes 52 for a list of some options for various stages of apoptosis: http://www. they are not necessarily well-retained after fixation. are not "fixable. but the best .html . an optimized exposure time is typically one which allows the brightest pixels of the area of interest without over-saturating the pixel values. Use your lowest available laser line or excitation filter. or flow cytometry. typically involve heating the sample. some cell types work fine. Wash out trypsin well and allow a resting period before labeling. like DAPI or propidium iodide. 52 Imaging Tip of the Day: DAPI is a great nucleic acid stain. or false-positive annexin V readings. for all Qdots. for a given filter and objective. 53 Imaging Tip of the Day: Qdot quantum dots can be quenched by heavy metals. It can also lead to clumsiness.drbio. (Sleeping with your head against the microscope in that dark imaging room doesn't count!). casein-containing blocking solutions. Consider using Hoechst 33342. 49 Imaging Tip of the Day: The lower you excite Qdot nanocrystals the better. mainly for microscopy of live cells. which labels DNA the same way and has the same spectrum. do not covalently bind to DNA. and they have an off-rate over time. leakage of esterases for AM ester dyes.

40 Imaging Tip of the Day: Need to make your own conjugate using a reactive dye (as opposed to buying a kit)? Here is a good selection tool: http://igene. Make sure to have an unlabeled sample for an autofluorescence control. Not all antigens require Nitrile is a good option. too. not only can there be refractive index problems. and isn't important for most fluorescent dyes. 37 Imaging Tip of the Day: A good site discussing cleaning of microscopy lenses: http://www. . What matters is the fluorescent properties and functionality.kcdigestivehealth. like DiI C18. here is a good link on the topic: http://www. 42 Imaging Tip of the Day: Make sure to clean your objective very well if you change from one immersion oil to another.invitrogen. buffer. imaged the same If two unlike oils mix. Though 38 Imaging Tip of the Day: Autofluorescence of samples can interfere with fluorescent 43 Imaging Tip of the Day: Lipophilic cyanine 39 Imaging Tip of the Day: Lectin conjugates will label organelles in fixed and permeabilized cells.utah. Here's a good link for ways to reduce autofluorescence: http://www.html . such as ER and Golgi but be aware that their selectivity varies between cell types. though. Be aware. 44 Imaging Tip of the Day: Interesting interactive page illustrating cell structure and organelles: http://learn. Here is one guide to different techniques: http://www.pdf. but some will react with each other to form a sticky substance that can damage your lens.genetics. and timing differ from antigen to antigen. have often been used for tracking cells. the color can vary in production from lot-to-lot. Often there are better dye options. when viewed by eye in room light? Don't worry.html 36 Imaging Tip of the Day: Regarding latex lab glove allergy. be aware that some people can have an allergy to nitrile 41 Imaging Tip of the Day: Is your fluorescent dye solution a slightly different color than older lots. that these dyes float in membranes and can be transferred from one membrane to another if cells fuse in any fashion.uhnresearch. and can be lost from the cell if the membranes are compromised or permeabilized.

and it is a good idea to compare backgrounds of different oils initially on your system. for molarity calculations. too.35 Imaging Tip of the Day: Beware of very new or very old mercury bulbs: their intensities can waver. 30 Imaging Tip of the Day: For finding dyes to match your flow cytometry laser lines. be sure to have the lot number and order date handy. for instance for phagocytosis assays. If you do fluorescent microscopy. first aid kit. called DailyCalcs.invitrogen. the first thing you should do is explain the safety rules and familiarize them with locations for safety equipment (emergency phone number list. but a great one is BODIPY TR methyl ester. 33 Imaging Tip of the Day: When calling for Tech Support for a specific product.Par. emergency shower. If it is for troubleshooting a product you already possess. 31 Imaging Tip of the Day: Some immersion oils can have autofluorescent background. spill leading to quantitation errors. and have a variety of choices. and unit conversions: . cell culture specs. dilutions. fire extinguisher. be sure to have a catalog number handy. 32 Imaging Tip of the Day: Want to quench dyes in the extracellular media.dat/O-072942-Flow-Instrumentation-Guide.ehs. which won't overlap spectrally and can label live or formaldehyde-fixed samples. biohazard and chemical disposal stations).ufl.File. by company: http://www. evacuation route. most lipophilic cyanine dyes will work. here is a really good guide. which lists most of the common flow cytometers and matches dye options: http://www.pdf 29 Imaging Tip of the Day: Always use gloves in the lab. Let new bulbs burn in before using. 27 Imaging Tip of the Day: A great science calculator for your phone or iPod Touch. make sure your immersion oil is validated or designed for fluorescence. Here is a link to glove compatibility charts. and replace old bulbs according to manufacturer guidelines for max number of hours (old bulbs will grow dim as well). even with buffers.83037 . gloves. But which gloves? Make sure the type of glove you use is compatible with your chemical or solvent. formula weight.htm 28 Imaging Tip of the Day: Safety first! Anytime a new person starts work in your lab. 34 Imaging Tip of the Day: If you want to visualize an entire GFP-transfected embryo or whole-mount with a general counterstain. but not in the cells? Trypan blue is a common option.

but this can be problematic due to extremely fast endocytosis and poor retention after washing. make sure to have a no-dye control with and without the . or by using a reactive dye such as an Alexa Fluor succinimidyl ester derivative. consider both the spectral overlap of the donor and acceptor dye. for instance: http://bit. though initial intensity will be reduced. as well as the R(0) value [the distance in Angstroms at which fluorescence resonance energy transfer from the donor dye to the acceptor dye is 50% efficient (Förster radius)]. but the tradeoff is a loss of resolution. like GFP. wheat germ agglutinin conjugates. Many labs use clear nail polish. Some have been used for plasma membrane labeling of cultured Technical note on FRET and other R(0) values: http://bit. 20 Imaging Tip of the Day: FM dyes (like FM 1-43) are lipophilic stains good for endocytosis or synaptic vesicle studies. (applied with a cotton-tipped applicator stick) which hardens instantly and is 23 Imaging Tip of the Day: Photobleaching of samples using confocal? If use of an antifade mountant or a more stable dye isn't an option. but this has solvents which can kill live cells and quench some Invitrogen Primary Ab Selection Tool: http://tinyurl. 22 Imaging Tip of the Day: If you image samples in buffer or a non-curing mounting medium. Here is a table of R(0) values for some Alexa Fluor dye pairs. 19 Imaging Tip of the Day: If you are testing a drug treatment with a fluorescent assay. or CellLight BacMam products. You can also lower the laser 25 Imaging Tip of the Day: Good sites for finding primary antibodies: Linscott's Directory: http://tinyurl. We recommend using melted paraffin. which will label mainly intracellular proteins in live 26 Imaging Tip of the Day: An amazing resource for spectral information on just about any fluorescent dye: PubSpectra: http://home. which will label proteins on the surface of the bacteria (alive or dead).com/4mbj9ya 24 Imaging Tip of the Day: When choosing a FRET pair. A faster scan rate and less average dwell time can help. 21 Imaging Tip of the Day: Tracking bacteria with dyes can be accomplished by using CFDA SE. Better options: CellMask Plasma Membrane Stains.earthlink. try adjusting you have to seal the coverslip edges. Biocompare: http://tinyurl.

and spinning the mountant stock prior to usage. by eye). as some drugs can have fluorescence of their own that can bias your reading. Potential causes: freezing. pulling a slight vacuum on your tissue in buffer prior to mounting. 2011. i. If there's a pellet. a-c). the fused cells will appear in both colors with flow cytometry (or yellow. 16 Imaging Tip of the Day: For testing cell fusion assays. but it's usually reversible. imagined in the FITC channel at 60x. they're probably aggregated. 18 Imaging Tip of the Day: Bubbles in your mounting medium upon coverslipping? Could be from the angle you mounted it. Combat by mounting the coverslip at an angle. or small concentration of detergent (such as 0. or you can image in HBSS or DPBS. as it can quench many dyes. they'll aggregate irreversibly. or bubbles in the mountant stock solution. high DMSO concentration (>2%). Spin them down before using. Wash well and proceed with restaining and remounting. microbial contamination. To avoid confusion.e. This can happen during shipment during snowy weather too. labeling membranes. maybe to re-stain the sample or get rid of bubbles? Simply soak the slide in warm PBS for 30-120 minutes with gentle rotation. 15 Imaging Tip of the Day: A typical imaging lab can produce thousands of images a year. 13 Imaging Tip of the Day: Microspheres can sometimes aggregate in the vial. and field "a" of the sample (if you image multiple fields per sample. 17 Imaging Tip of the Day: Don’t do your live cell fluorescent imaging in media that contains phenol red. . on February 16. here is an example template we've used for labeling images: "12_AbA5ug_FITC_60x_021611a.tif" would refer to an image of slide 12. it is important to mark the two cell populations with tracking dyes. Combat with appropriate storage. high salt concentration. A few mounting media brands can also outgas upon curing. air trapped in your sample (particularly tissue). or simply small diameter. 12 Imaging Tip of the Day: Don't freeze your Qdot nanoparticle products. 14 Imaging Tip of the Day: Are you using a curing. testing "antibody A" at 5 ug/mL.drug. If successfully fused. like DiO C18 (green) for one population and DiI C18 (red-orange) for the other population. bath sonication. Perhaps the best option is to use lipophilic cyanine dyes. extremes in pH.1% Tween-20). aqueous mountant but later realize you need to remove the coverslip. Most common media are available without it. The mountant will dissolve and the coverslip will slide off.

try increased protein blocking (such as 6% BSA/10% normal serum). or reducing the secondary concentration or time. where you can mix and match color and dye options for live and fixed cell labeling and see them in a simulated cell: http://www.11 Imaging Tip of the Day: If you're looking for a tool to help choose dyes for specific organelles. For instance. antibodies validated for Western blotting or ELISA are not always successful for immunocytochemistry or immunohistochemistry techniques. or you can make direct 10 Imaging Tip of the Day: If you wish to label cell membranes in live or formaldehyde-fixed (but unpermeabilized) cells.invitrogen. be aware that instrument sensitivity is also a concern. 6 Imaging Tip of the Day: Have two primary antibodies of the same species for the same sample? To avoid cross-label.invitrogen. IgG2a. Here is help in choosing a kit for making direct conjugates: http://www. . where you can display the spectra for up to five dyes at a time and put in your filter or laser wavelengths to check for overlap: http://www. There are handy tools online. and consider that some background can be due to charge-based binding (which can be blocked with Image-iT® FX signal enhancer). be sure to check which techniques they are validated for. while wheat germ agglutinin conjugates. For instance.invitrogen. but in plant cells they will also label the cell wall. One is the SpectraViewer tool. concanavalin A 7 Imaging Tip of the Day: Problems with apparent secondary antibody background? Be sure to run a secondary-only control to insure the problem isn't from the primary. you can use isotype-specific secondaries if they are of different isotypes (IgG1. It is typically noted in the product manual or certificate of analysis. 4 Imaging Tip of the Day: When considering the range of concentration to test for your primary or secondary antibody. microscopy is typically less sensitive than flow cytometry and often requires a higher concentration. try the Stain Your Own Cell online 5 Imaging Tip of the Day: When choosing a primary antibody. 8 Imaging Tip of the Day: When choosing a dye. or CellMask™ plasma membrane stains are more selective for the plasma membrane. be sure to specify plasma membrane or all membranes. such as DiI C18. make sure it matches your filter specs or laser line. 9 Imaging Tip of the Day: Propidium iodide (non-cell permeant) and Hoechst 33342 (live cell permeable) nucleic acid stains work great for nuclear labeling of mammalian cells. Lipophilic cyanine dyes. IgG2b). will label all membranes of the cell.

make sure you do not label in the presence of serum.3 Imaging Tip of the Day: If you are labeling live cells with diacetate dyes. For live samples. 1 Imaging Tip of the Day: Photobleaching problems? For fixed samples. before switching over to your problematic dye to snap a picture. which are cleaved by cytoplasmic esterases to become active. or purchase ready-made plastic uniformity slides from distributors. more stable dye (such as Hoechst 33342) to find and orient the cell first. To correct for this. . which has esterase activity and will prevent uptake of the dye. such as many ion indicators or cell tracking dyes. you can make a uniform intensity standard slide by making a solution of a reference dye in buffer and putting it under a coverslip. try using a second. particularly for quantitation. thus limiting the time it is being exposed and fading. using an antifade mounting medium is recommended (such as ProLong Gold®). 2 Imaging Tip of the Day: Having a uniform intensity across your field of view is important.