Immunofluorescence Microscopy

Specificity of Antibodies

Immunofluorescent labelling has established as a mainstay method for specifically labelling biological macromolecules, by virtue of the exquisite specificity offered by antibody binding sites for their corresponding antigens. Fixed cells and tissues are very common specimens for immunological labelling. In such specimens, fluorescently labelled antibody conjugates are an important tool for determining both the presence and the sub-cellular localization of an antigen, and in some cases can also be quantified to yield relative concentrations of sub-cellular biomolecules.

The most common protocols for Immunofluorescence labeling are Direct and Indirect. Direct labelling involves incubating the sample with a fluorophores-conjugated antibody that is specific to the antigen of interest. With Indirect labelling, the specimen is incubated first of all with an unconjugated antibody, specific for the antigen under study. Then a second fluorophore-conjugated antibody is introduced that is specific for the first antibody.

Immuno-labeling has proven highly effectual in helping determine the localization and function of sub-cellular proteins. One method is to first bring about expression in the cell of an epitope-tagged protein, without affecting cell physiology, making use of an appropriate expression vector. A raised antibody, specific to this epitope, can then be used to specifically label the expressed protein of interest, either by direct or indirect means.

Detecting Immunofluorescence

Immuno-labeled specimens are examined under a fluorescent microscope, often by widefield epifluorescence microscopy. Widefield epifluorescence microscopy has developed into a universally accessible technique for study of fluorescently labeled cells and tissues. Over the last few decades, this drive has been accelerated by improvements in fluorescent probes, labeling chemistry, optical instrumentation (such as filters and objectives) and detector technology.

The widefield technique involves flood-illumination of the field of view by a wavelength or small wavelength range (often though use of an excitation filter and arc lamp). The stoke-shifted fluorescent emission transmits though the dichroic, that was initially used to reflect the shorter wavelength excitation light onto the sample, gets optically filtered once again by an emission filter (often called barrier filter) and is then focused onto the camera detector.

Often, specimens are labeled with multiple fluorescence labels, some by immuno-specific means, some not (e.g. the DNA-intercalating dye DAPI). The idea is then to record a series of images, each corresponding to a fluorescent label.

One must therefore make use of a series of epi-fluorescent filter blocks (excitation, dichroic and emission filters) that are specific to the dyes of interest. It is also possible to use mutli-band dichroic filters that do not have to be swapped out for each image.

Optimizing Immuno Detection

The image quality of widefield epifluorescence microscopy on immunofluorescent samples can be compromised in a number of ways:

1. For each dye probed, the ideal situation is to achieve an image that is untarnished by the emission of the other dyes. This is rarely achieved in practice, due to the spectrally broad nature of both excitation and emission bands, resulting in spectral overlap between dyes, i.e. filtered excitation light for one dye will excite a little bit of a spectrally neighboring dye, and a filter to collect emission light of a specific dye will also collect a small percentage of light from other dyes. The solution to achieving highest contrast, is to use specific sets of narrow-band excitation or emission filters, positioned to have minimal spectral overlap with the other labels. This can compromise signal to noise however, since the narrow-bandpass nature of such filters means that only a fraction of the available excitation or emission light is being transmitted to the sample or CCD respectively.

2. A common issue with immunofluorescent labelling, is non-specific binding (also known as background staining) of antibodies to regions of the cell that they are not specific to via their antigen-binding site, instead attaching via other types of molecular interaction. This can result in reduced contrast and can be particularly problematic when trying to find weakly expressed proteins for example. Fortunately the problem is addressable and there are a number of well-documented precautions that can be taken during sample preparation to ensure that non-specific binding is minimized. One very effective step towards minimizing this form of background is to employ direct labelling protocols. Whilst indirect labelling can boost overall signal intensity, the use of secondary antibodies results in extensive non-specific binding, compromising signal to background ratio. Therefore it is more beneficial to make use of a sensitive detector combined with direct protocols.

3. A common concern is fading of the signal. Often to achieve an adequate signal to noise, a high intensity excitation light must be employed, that also causes accelerated photobleaching of fluorophore labels. "Anti-fade" mounting media agents can be used that retard the rate of photo-bleaching, but the most effective ones invariably cause emission intensity to deplete also, and can sometimes result in higher photon background and diffused fluorescence.

4. Another negative effect of widefield is "out of focus fluorescence". This is due to light from outside of the focal plane making it through to the detector. One way to avoid this completely is not to use a widefield technique, but instead make use of a highly optimized epi-fluorescent confocal set-up. With widefield though, it is possible to apply deconvolution algorithms to collected images to minimize the effects of out of focus haze and improve contrast. For such algorithms to be successful, signal to noise and resolution need to be optimal

Affordable EMCCD for Enhanced Immunofluorescence

As discussed above, there are a number of factors involved with collecting high-contrast widefield epifluorescent images that require signal to noise to be maximized:

  • Narrow band excitation and emission filters, that give best spectral separation of multiple labelled specimens, also severely restrict the number of emission photons that eventually make it to the detection camera.
  • A more sensitive detection technology means that direct labelling protocols can be used, markedly reducing level of non-specific background, and improving photon signal-to-background ratio.
  • Use of deconvolution algorithms to remove "out-of-focus fluorescence" requires maximum signal to noise and resolution from the images.
  • Some epitope-tagged proteins of interest can be weakly expressed within the cell, resulting in correspondingly weak emission signal.
  • It is highly desirable to make use of shorter exposure times and reduced illumination power, resulting in reduced bleaching rates and higher sample throughput, especially when multiple fluorophores are to be imaged in turn. A more sensitive detector would also correct for the weaker intensity emission resulting from use of anti-fade agents. Photodamage of cells and tissues is also of concern when imaging multi-labelled specimens, especially when having to excite with higher-energy photons (e.g. Cy-2 dye).
  • Situations in which inefficient uptake limits the amount of labelled antibody within the cell.

    • All of the above gives rise to the desirability of a flexible camera technology, capable of operating effectively under both "standard" and "ultrasenstitive" modes should the need arise, highlighting the potential of EMCCD technology for advanced immunofluorescence microscopy.

    As shown in the figure on the right, EMCCD is ideal for significantly enhancing the signal-to-noise of weak epifluorescent signal from immuno-labeled samples. This particular test was performed under extremely weak illumination power combined with very short exposure times, but could equally represent the effect of a minimally present antigen, such as weakly expressed protein.

    EMCCD is a novel detector technology for incorporation into your Immunofluorescence microscopy set-up. EMCCD cameras use an on-chip amplification technology that can be accessed to amplify the signal above the read noise floor. In light-starved instances, one simply needs to apply some EM gain to amplify the previously undetectable signal into a respectable marker.

    The real beauty of the EMCCD is in it's flexibility to operate as a standard high Quantum Efficiency (QE) CCD, or as a single photon sensitive power-horse when the signal is weak and/or exposure times need to be reduced. One can also preserve immuno-labeled specimens from the effects of photo-bleaching by filtering the power of the illumination light, and compensating with higher EM-gain.

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