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FAQ

Fluorescence observation

  • Q Does the fluorescence intensity reflects the concentration of the target?
    A

    The answer is no.

    Most of our products are activatable probes that irreversibly fluoresces upon reaction with the target, except for some reversible products such as AcidiFluor series or QuicGSH. Therefore, fluorescence intensity reflects the sum (integral) of the target concentration, from the time point of loading the reagent to the time point of the detection. The slope (differential) of the fluorescence intensity change reflects the target concentration.

    The reasons for the reversibility are (1) short lifetime of the targets such as reactive oxygen species or nitric oxide, and (2) low concentration of the targets such as Fe2+. It is not easy to design reversible fluorescent probes for these targets with practical sensitivity.

     

  • Q Instead to observe relative concentration, can I determine the absolute concentration?
    A

    For cell impermeable activatable probes, it is possible to determine absolute concentration of extracellular target. In this case, prepare a calibration curve using the standard substance of known concentrations. On the other hand, it is difficult to determine intracellular target concentration using cell permeable fluorescent probes, except for some reagents which are specially designed for intracellular concentration measurements. The reason for the difficulty is that users cannot determine the intracellular probe concentration.

  • Q Tell me about the organic solvent to dissolve fluorescent probes.
    A

    Many fluorophores and activatable probes have low solubility to water. Therefore, to prepare a solution in millimolar-cocentrations, use organic solvent described in the protocol document in each products. For most products, dimethyl sulfoxide (DMSO) is a first choise. For some produicts, N,N-dimethylformamide (DMF) is recommended.

    For NHS products, anhydrous DMSO is recommended, because NHS is unstable in aqueous solutions. Use commercially available anhydrous DMSO, or prepare anhydrous DMSO by adding vacuum dried molecular sieves 3A to DMSO.

    Quality of organic solvents usually reduces upon moisture absorption, oxidation, or UV irradiation. Degradated organic solvents increase the fluorescence background or reduce the reactivity when use with some activatable probes. To avoid the degradation, it is recommended to dispense DMSO to small vials and store in a deep freezer and use up each of the vial after once the vial have opened. Avoid to store organic solvents in moist conditions, in high temperature, and under strong lights. 

  • Q Can a probe be applied to fixed cells or paraffin sections?
    A
    In most cases, it is difficult to use activatable probes for paraffin sections, except for SaraFluor labeled antibodies. (SaraFluor series products are not activatable probes).

    In addition, applications of activatable probes to fixed cells are not always successful. Most activatable probes reacts with intracellular ions or reactive oxigen species, which localization and concentration are kept by living cellular activities. Concentrations of these substances may be decreased in dead (fixed) cells. Therefore preliminary experiments to evaluate the detectability of the target is essential.

    In some cases, it is possible to detect fluorescence of activatable probes, when it is reacted in living cells and fixed after the reaction.  Even in those cases, membrane permeabilization using alcohols or detergents is not recommended. In all cases, preliminary experiments to evaluate the detectability of the probes is strongly recommended.

     

  • Q What kind of dish is suitable for observation?
    A

    Usually, glass bottom dish is made for fluorescence microscopy observation. Use glass bottom dish that fit the stage of your microscope. In some cases, you may use multi-well plates instead of glass-bottom dish.

    Note that most of the biological microscope lenses are designed to use with coverglass of 0.17 mm in thickness. It is recommended to use with No. 1 or No. 1S coverglass or equivalent glass. 

     

  • Q Is it necessary to replace the solution, before observing the cells treated with fluorescent probes by a fluorescence microscopy?
    A

    It is not always necessary to replace the solution before observation, in case of high-efficiency fluorescence probes which do not emit fluorescence light before reacting with target substances. The signal could be detected even when the unreacted probes remain in the culture medium. However, it might be necessary to replace the solution to remove fluorescence substances, especially when the medium containing phenol red interferes optically with the fluorescent observation. (Please refer the below illustration.) In this case, by using the medium which does not contain phenol red, it is able to observe without the solution replacement. It is recommended to determine the solution condition, based on preliminary experiments with the target cells and the medium.

     

    The fluorescence spectra of DMEM medium when exciting at different wavelengths (slit width 20 nm). Fluorescence emission from green to red were observed when exciting with the blue light.

  • Q When adding a fluorescence probe into cells, is it possible to dilute with cell culture medium instead of buffers such as HBSS?
    A

    It is possible in most cases.
    Especially in case of ROS detection probes, ROS increases in a starvation condition for certain types of cells. Therefore it would be better to add the fluorescence probe into the culture medium, not the buffer solution such as HBSS. However, components (such as phenol red which is used as an indicator) in the medium might influence the detection. Therefore, please follow the  instruction manual or literatures.

Others

  • Q I'd like to know molecular weight, formula weight, structure of the reagent
    A

    Formula weight (FW) is printed on the package of the reagent. This weight is formula weight including salts associated to the molecule, which differs from molecular weight.

    In general, we do not disclose the structural information, except for some products which structure is shown in the Protocol document.

     

  • Q Can the reagent which is indicated to be stored at 4℃ be stored in a freezer?
    A

    Yes, except for antibody products. For antibody products, it may damage the reactivity. Please be careful.

  • Q How can I order a research reagent?
    A

    Please contact our distributors listed in this page.

  • Q I could not find my question in this list, yet.
    A

     Contact us if your question has not been solved.

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