All microscopy, including: immuno-fluorescence, confocal, IHC

• Martin Friemel

  Division of CFP and YFP flourescence

  Hi guys, i've a question and hope someone has experience with this. We're studying 2 proteins in HeLa-cells, which we tagged with YFP and CFP respective. The cells are fixed in Molwiol. We want toHi guys, i've a question and hope someone has experience with this. We're studying 2 proteins in HeLa-cells, which we tagged with YFP and CFP respective. The cells are fixed in Molwiol. We want to show, that they partly co-loaclize but also have different localizations. We're using a ZEISS CLSM710. We tried already linear unmixing by some reference-spectra and also multi-track imaging. But i've still the impression still get some YFP-Flourescence, where we just have to see CFP. Could it be that we excite the YFP with 458nm Argon-laser in the multi-track approach? Or could that be FRET CFP-->YFP, which we can see as YFP-flourescence? Is there any approach to avoide this? Perhaps give some quenchers to the fixation medium? Bests Martin! Bests

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    Manel Bosch replied

    Thanks George, I think it is worth trying it "in vivo".

    3 days ago
• Khairiya Ahmed

  Hi A good protocol to isolate T cell from solid tissue?

  please dose anybody know how to isolate T cell from solid tissue? thanks

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    Khairiya Ahmed replied

    Thanks

    5 days ago
• Zena Khaznadar

  A good protocol to stain primary NK cells activating receptors for IF?

  I've been trying to see the positioning of NK's activating receptors in a synapse for long time. But my staining fails on primary NK cells, it worked on NKL cell line that shows more receptors. AnyI've been trying to see the positioning of NK's activating receptors in a synapse for long time. But my staining fails on primary NK cells, it worked on NKL cell line that shows more receptors. Any ideas? Should I look for other antibodies? (perhaps the staining I see on NKL isn't specific, my isotypic control is negative though). Should I use another method to amplify the signal. A colleague suggested using two antibodies (dual link), does it sound like a good idea? Anyone has an experience with it?
• William Guilford

  Designing a 2X magnification changer

  This question is a little embarrassing for me since I'm sometimes a go-to guy for microscope optics questions, but here it goes... I need to double the magnification of the image from the side-portThis question is a little embarrassing for me since I'm sometimes a go-to guy for microscope optics questions, but here it goes... I need to double the magnification of the image from the side-port of an Olympus IX70. This means putting a magnification changer between the 160 mm tube lens of this infinity-corrected system and the 1" faceplate of my camera. I tried a "2X extender" from Leeds Micro, but unfortunately the aperture is too small. I'm not only getting vignetting, but I'm losing too much light. The question is, can someone direct me to a vendor for a large aperture c-to-c mount magnification changer (zoom)?, or... Can someone tell me for sure if I can put a simple Galilean expander (set to infinity) between the tube lens and the camera?, or... Can someone give me advice on how best to design this bugger? If the light weren't converging I'd know just what to do, but the light is converging so I'm not certain.

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    Amany Shakeir replied

    I'm sorry I do not know much about the power of magnification

    16 days ago
• Jesica Canizo

  Problems with sample Mounting

  Has anyone a good tip to do nice mounted sample for immunocytochemistry? I have tried to have good images, but, sometimes, my embryos are close to the boundary of the mounting reagent and I haveHas anyone a good tip to do nice mounted sample for immunocytochemistry? I have tried to have good images, but, sometimes, my embryos are close to the boundary of the mounting reagent and I have problems with fluorescence there; other times I have bubbles, even when I try not to form them when I put the embryo over the slipe or put the coverslip. Sometimes the mounting reagent deform the shape of my embryos and they dont look well!, I need help. One other question: is there a way to replace the mounting reagent (vectashield or the other of invitrogen that are both very expensive) for some lab preparation that conserves fluorescence for several months?

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    Jesica Canizo replied

    yeah! I usually do that too! but this time they havent answered me yet! :S

    23 days ago
• Tejabhiram Yadavalli

  Difference between a 200 KV and 300 KV TEM

  What are the differences in the functionality, resolution and type of samples that are used in a 200 kV and 300 KV TEM? I would like to know the technical differences, what are the exact change inWhat are the differences in the functionality, resolution and type of samples that are used in a 200 kV and 300 KV TEM? I would like to know the technical differences, what are the exact change in image resolutions and lattice parameters, SAED patterns or EBSD patterns etc.

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    Rene Frank replied

    Have a look at an excellent review on the properties of electron microscopes by Richard Henderson in Q. Rev. Biophys (1995) 28 (2): 171-93. This deals directly with the effects of different energiesHave a look at an excellent review on the properties of electron microscopes by Richard Henderson in Q. Rev. Biophys (1995) 28 (2): 171-93. This deals directly with the effects of different energies on resolution and other effects (eg. charging of the specimen). http://www.ncbi.nlm.nih.gov/pubmed/7568675

    Apr 26, 2012
• Rishabh Jain

  Is there a way to do high resolution imaging of human skin in low vacuum in Scanning Electron Microscope?

  ESEM is not available. Au or C coating can be done.

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    Vladimir Dusevich replied

    Low vacuum mode does not preserve wet specimens. You will work at pressure about 1 Torr in contrast to atmospheric pressure of 760 Torr. So pressure in the specimen chamber will be just 1/760 of theLow vacuum mode does not preserve wet specimens. You will work at pressure about 1 Torr in contrast to atmospheric pressure of 760 Torr. So pressure in the specimen chamber will be just 1/760 of the atmospheric pressure, about the same as for high vacuum mode. The same is true for water – residual partial water vapor pressure will be just 1/760 of normal one, so water will evaporate as fast as in high vacuum mode and specimen will dry out really fast. If you do not have ESEM capabilities you’d better work in high vac mode. Best preservation of surface – no specimen treatment at all. Just air dry it and coat with either Au or C (Au could be better). Sure, bacteria morphology will be not as good as after proper preparation (fixation, dehydration, etc.), but even air dried bacteria and fungus are still very much recognizable. If your main problem (as I understand) is preservation of organisms on a surface, then do not treat it with solvents at all!

    Apr 26, 2012
• Ele Mira

  Vegetal Tissue preparation for TEM

  I have some problems in the embedding process in foliar tissue of tropical species, and particularly with species containing glands and trichoms. After fixing with glutaraldehyde, deshydratation andI have some problems in the embedding process in foliar tissue of tropical species, and particularly with species containing glands and trichoms. After fixing with glutaraldehyde, deshydratation and bath in epoxypropylene, I use the low viscosity resin kit from agar and despite that, the infiltration in the samples is pretty bad. I guess some molecular exchanges occurs during the impregnation and I would like to know if it's possible to avoid them. Has anyone some experience in vegetal tissue preparation ?

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    Wolfgang H. Muss replied

    Dear Anirban, (as well as dear Ele Mira), first of all: thank you very much for your kind reply, also entitling me as "Mister" and "Dr. Wolfgang" MUSS (latter is my family name). I am a veryDear Anirban, (as well as dear Ele Mira), first of all: thank you very much for your kind reply, also entitling me as "Mister" and "Dr. Wolfgang" MUSS (latter is my family name). I am a very informal person and therefore you do it quite right if you just address me as a colleague "Wolfgang", be it in success as well as in (scientific) doubt- as you all I understand too this Research Gate Forum to be an interesting possibility and opportunity for rapid informations on specific or global themes which might arise during your studies, research projects etc, and I think also this to be a very informal matter. So hopefully is not angry about being addressed/entitled only with his/her first name...that includes me too. As to Anirban's question me suggesting an alternative for Toluene (AS INTERMEDIUM for resin embedding): IMHO (in my honest opinion) there are several possibilities: depending on the resin you use (yours is Araldite CY212 ): a) without an intermedium from 100% EtOH (or acetone) into resin mixture (e.g. 1:1) : EtOH (needless to say that that approach needs good knowledge about the physics of dehydration-embedding in resins and chemicals/resins used, and at least the lab climate should be relatively arid) b) from 100% EtOH (or Acetone) into propylene oxide (PO) OR acetonitrile (AN= methyl cyanide). To stay in Acetone for afterwards mixing it with Araldite (but perhaps as well as for other resins like Durcupan, Epon, Epon substitutes, etc.) is not really recommended as the literature read so far reports of brittleness of blocks produced (problems in polymerization). PO is the chemical usually used as an intermedium in TEM preparations but meanwhile - at least in Western Countries - has been marked (not forbidden) too as being carcinogenic like Toluene, so you should take care of with safety actions when working with it. Acetonitrile is a not commonly used <intermedium> candidate since it is miscible with EtOH, Acetone and other organic solutions, as well as the resins. It is not classified a carcinogen (despite being a cell tox, = methyl cyanide), has a remarkably lower vapor pressure than PO, so you need not stringently to apply harsh safety issues when working with it. On the other hand (especially important for tropic regions or areas with high relative humidity) the 100% miscibility with water is a weak spot since you won’t like to adsorb water again in your embedding procedure/resin and you should take action to get rid of the solvent out of the resin properly (i.e. by means of a light bulb over the open specimen vials in a specimen rotator). But for Labs with moderate room climate this should be no issue. If you would like to know more about this method I certainly can give you proper informations how this is done routinely in my/our Lab since 1985 instead of using PO any more. c) as another method of processing AND embedding I could offer to you a “rapid processing/dehydration and embedding method” which is done by dehydration in/with 2,2-DMP (dimethoxypropane) acidified (method on request) which is as old as my “student and professional career”(if I had any): originally reported from the 1970ies, and used not much around the EM-Labs in the world, I guess, but always good to know such a method exists: the molecular stoichiometric dehydration 1:1 by acidified 2,2 DMP means that this organic solution brought into specimen vessels containing tissue specs and water/buffer, or also say 50% EtOH, will react 1:1 / molecule H2O in the tissue, generating Acetone and Methanol as reaction product. Acidified 2,2-DMP can also be used to be mixed with the pure resin (and therefore you don’t need toluene or PO for intermedium). Alas two points: if you use acidified 2,2-DMP as a dehydration agent you should be aware of the fact that your specs should be free of PO4-ions (which is of no means when using sodium-cacodylate buffer) since such would be precipitated by the generated Methanol (so after osmication you should do 1-3x a wash with A.(bi)dest.). I understand that not only the non-rehydrating/immiscibility properties of Toluene might be an issue for using it in your area and on the other side also perhaps the cheapness of getting the substance. And I know by myself that it might be necessary to use that stuff anyway if there is no possibility of using a substitute for that chemical. Additional questions (e.g. concerning the techniques or how to do it I am willing to answer to the best of my knowledge. Best regards and wishes, Wolfgang

    Apr 23, 2012
• Zalan Szabo

  Imaging bacteria

  Are there good ways to embed live bacteria for microscopic imaging? I tried agar(ose) which works okay, but seems a bit fussy.

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    Wolfgang H. Muss replied

    Dear Zalan, it would be of interest to know which kind of >microscopic imaging< you are going to do. As an electron microscopist I have done several preparations from cultures done in theDear Zalan, it would be of interest to know which kind of >microscopic imaging< you are going to do. As an electron microscopist I have done several preparations from cultures done in the Bacteriology Department, and used fixed (e.g. meningococci) culture with the underlying culture substrate to process that for a) resin semithin sections (imaged by LM) as well as ultrathin sections (TEM). Due to the fact that chemical fixation always induces so called fixation artifacts it may be necessary to know also the strain you want to image (usually - with conventional fixatives and processing - the bacterial membrane is well preserved but not the bacterial body itself, nor - in most cases - the membrane-adhering glycoconjugates/glycocalyx. So always you have to consider a very special fixative - if you are not going to use e. g. HPF and Cryo-TEM for such. In case you only would like to image bacteria by LM-methods, pelleting by centrifugation in agarose (as you tell us above) might be working but perhaps needs some modifications (temperature during centrifuging, time, g-forces, etc., as well as a suited fixative and suited staining procedure). Best wishes and regards, Wolfgang MUSS, PhD, Salzburg-Austria

    Apr 3, 2012
• Andrew Paek

  How to image senescent cells by B-galactosidase activity

  I need to determine whether a cell is senescent or not by B-galactosidase activity. I am using the Senescence β-Galactosidase Staining Kit from Cell Signaling. Does anyone have any suggestions onI need to determine whether a cell is senescent or not by B-galactosidase activity. I am using the Senescence β-Galactosidase Staining Kit from Cell Signaling. Does anyone have any suggestions on how I should image the cells? Is Phase contrast sufficient or do I need a specific filter?

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    Amany Shakeir replied

    For reflection, for a specific tissue cells use dye dab heamatoxin to observe the internal structures of the cell if either patients or infected tissue there are other ways to observe the cells

    Mar 30, 2012
• Dorothy Bisase

  Is there a specialist stain for cancer cell, if so which one is it, and is it the best?

  Please describe the latest most powerful microscopy of of the 21st century.

  Recent replies ⋅ Show All (1)

  • 
    Ly Tran replied

    I think Atomic Force Microscope (AFM) that enable to measure a sub-nm adhesion force as 0.1 nN AFM has been applied to study ligand-receptor single bonding like Protein - Oligosaccharides

    Mar 28, 2012
• Jackson Sage

  Alternatives to micromanipulators for use in cell capture

  Hey all, I'm wanting to look at the movement eukaryote flagella under a microscope for image analysis (the cells I will be using will be ~10µm wide). I can view the flagella by looking at cellsHey all, I'm wanting to look at the movement eukaryote flagella under a microscope for image analysis (the cells I will be using will be ~10µm wide). I can view the flagella by looking at cells trapped in the air-water interface on the slide; but their flagella do not move naturally. I know that this can be done by "holding onto" a cell through use of a micromanipulator, but I don't have one available to use - nor £10K lying around :( Does anyone have any ideas of a way around this? Or any pointers to set me in the right direction? Many Thanks for any advice :)

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    Amany Shakeir replied

    I HOPE IN SHOW types of bacillus bacteria under microscope image and send me .thanks

    Mar 28, 2012
• David Orloff

  The Cell: An Image Library has grown extensively. Check it out at

  http://www.cellimagelibrary.org

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    EP López-Damián replied

    Thank you to share. It´s really good web site!!

    Mar 28, 2012
• Jesica Canizo

  Cell viability

  Can you recommend a method to monitor cell viability in vitro, while an embryo is developing in culture. Thanks!

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  • 
    EP López-Damián replied

    Hi , May be this article can help you. If you have problems to download I have the article , you can give your email and I´ll sent you. RegardsHi , May be this article can help you. If you have problems to download I have the article , you can give your email and I´ll sent you. Regards http://onlinelibrary.wiley.com/doi/10.1111/j.1439-0264.2011.01133.x/abstract

    Mar 28, 2012

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  • 
    Thomas Manesh replied

    RNA visualization in live bacterial cells based on fluorescent protein complementation. The RNA can be tagged with an RNA aptamer that binds an RNA-binding protein with high affinity. ThisRNA visualization in live bacterial cells based on fluorescent protein complementation. The RNA can be tagged with an RNA aptamer that binds an RNA-binding protein with high affinity. This RNA-binding protein is expressed as two split fragments fused to the fragments of a split fluorescent protein. In the presence of RNA the fragments of the RNA-binding protein bind the aptamer and bring together the fragments of the fluorescent protein, which results in its re-assembly and fluorescence development.

    Mar 2, 2012