Key Points
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Two main techniques have been used to analyse the immune system. In vivo experiments examine cell populations in their natural environment, but they cannot provide information about individual cells. In vitro experiments determine subcellular and molecular details, but they cannot replicate adequately the physiological environment. Therefore, new techniques, involving fluorescent labels, are required to analyse single cells in intact tissues in real time.
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Three main problems limit optical resolution when attempting to visualize fluorescently labelled cells in living tissues. First, the high-numerical-aperture lenses that are required have a narrow depth of field, so that fluorescent label from above and below the focus obscures the image. Second, biological tissues scatter light, which reduces image contrast. Third, the intense excitation light causes photodamage and photobleaching.
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A partial solution is provided by confocal microscopy, which provides a sharp 'optical section' at a given depth by rejecting out-of-focus fluorescence from above or below the focal plane. But, light scattering limits the depth of imaging and the specimen is susceptible to photodamage.
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Two-photon microscopy has the advantages of greater imaging depth (owing to reduced light scattering of the long wavelengths that are used for excitation) and minimal photobleaching (as excitation is confined to the focal plane).
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Two-photon excitation involves the simultaneous absorption by a fluorophore of energy from two photons, each of which contributes one half of the total energy required to induce fluorescence. The probability of two-photon excitation falls off rapidly from the focal point.
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Pulsed lasers (such as the titanium-sapphire laser) are required to provide the necessary high photon density at the focal spot without vaporizing the specimen. The peak power during a pulse is extremely high, but the average laser power is relatively low.
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The choice of objective lens is also important to maximize the numerical aperture while maintaining imaging depth. 'Dipping' water-immersion objectives provide the best compromise.
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Two-photon microscopy allows imaging in as many as six dimensions (three spatial dimensions, time, intensity and wavelength of more than one probe). This creates problems in terms of storage, analysis and representation of the data that will require new solutions, such as the use of virtual-reality technology.
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As an example, two-photon techniques have been used to examine the movement of T cells in lymph nodes and their response to antigen. These studies have indicated that both the single-encounter model and the serial-encounter model of antigen presentation might be relevant physiologically.
Abstract
Many lymphocyte functions, such as antigen recognition, take place deep in densely populated lymphoid organs. Because direct in vivo observation was not possible, the dynamics of immune-cell interactions have been inferred or extrapolated from in vitro studies. Two-photon fluorescence excitation uses extremely brief (<1 picosecond) and intense pulses of light to 'see' directly into living tissues, to a greater depth and with less phototoxicity than conventional imaging methods. Two-photon microscopy, in combination with newly developed indicator molecules, promises to extend single-cell approaches to the in vivo setting and to reveal in detail the cellular collaborations that underlie the immune response.
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Handbook of Fluorescent Probes and Research Products
Glossary
- FLUORESCENCE RESONANCE ENERGY TRANSFER
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(FRET). This is used to measure protein–protein interactions microscopically or by a FACS-based method. Proteins fused to cyan, yellow or red fluorescent proteins are expressed and their interactions assessed by measuring the energy transfer between fluors, which can occur only if the proteins interact physically.
- BRIGHT-FIELD MICROSCOPY
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The original, and most commonly used, form of microscopy, in which the specimen is viewed by transmitted light from a condenser lens.
- NOMARSKI OPTICS
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One of several implementations of differential interference contrast (DIC) techniques for use with bright-field microscopy. DIC converts optical gradients in a specimen (differences in thickness, slope or refractive index) to differences in intensity, which gives a pseudo three-dimensional appearance to objects that would otherwise appear featureless.
- FLUORESCENCE MICROSCOPY
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The visualization of fluorescence in a specimen — either natural autofluorescence or after staining with fluorescent probes. Usually, this is achieved using an epi-fluorescence microscope, in which the excitation light is directed by a dichroic mirror through the objective lens, and fluorescence emission is viewed through the same lens after blocking excitation light with a barrier filter.
- FURA-2 RATIOMETRIC IMAGING
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Calcium imaging using the calcium-sensitive dye fura-2. When excited at short ultra-violet wavelengths (∼350 nm), the fluorescence of fura-2 increases with increasing calcium concentration, whereas fluorescence decreases with increasing calcium concentration at longer wavelengths (∼380 nm). By forming a ratio of successive images obtained at each wavelength, it is possible to obtain a measure of absolute free calcium concentration.
- DECONVOLUTION MICROSCOPY
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The use of software to correct partially for diffraction-limited blurring by a microscope and thereby enhance the resolution of three-dimensional images obtained by wide-field or confocal microscopy.
- CONFOCAL MICROSCOPY
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A form of fluorescence (or reflected light) microscopy in which out-of-focus signals are rejected by measuring through an aperture that restricts all light except that originating from the focused excitation spot.
- LIPID-BILAYER IMAGING
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This involves phospholipid with protein constituents deposited in a bilayer on a glass surface; it is used to study cell–cell interactions, such as the immunological synapse.
- COLLAGEN GEL MATRIX
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A culture system consisting of a three-dimensional collagen environment that mimics a physiological substrate for the study of immune-cell motility and function.
- CFSE LABELLING
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CFSE (5,6-carboxyfluorescein diacetate succinimidyl ester) is a green-fluorescent dye that binds covalently to intracellular proteins, providing a stable cell label. When a cell divides, the dye is diluted equally in the daughter cells and fluorescence is halved with each generation, which allows up to 7–10 cell divisions to be detected by flow cytometry.
- NUMERICAL APERTURE
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(NA). The light-gathering ability of a microscope objective. The NA is equal to the refractive index of the medium multiplied by the sine of half the angular aperture.
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Cahalan, M., Parker, I., Wei, S. et al. Two-photon tissue imaging: seeing the immune system in a fresh light. Nat Rev Immunol 2, 872–880 (2002). https://doi.org/10.1038/nri935
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DOI: https://doi.org/10.1038/nri935
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