PMC

a | Epifluorescence microscopy. b | Confocal microscopy. c | Total internal reflection fluorescence (TIRF) microscopy. d | Two-photon laser scanning microscopy (TPLSM). Illumination is depicted in yellow and the detection volume is labelled. The images show fluorescent clusters of linker for activation of T cells (LAT) in Jurkat T cells activated on antibody-coated coverslips.

Several model systems have been developed to enable lymphocyte imaging at improved resolution. These models use a surrogate substrate for activation instead of an antigen-presenting cell (APC). a | Beads, typically coated with stimulatory antibodies, can be easily prepared and customized but do not allow rapid imaging of cell activation. b | Supported lipid bilayers that incorporate peptide–MHC molecules and other molecules such as integrins provide a better surface for imaging. Although the molecules are mobile, the bilayer lacks diffusion barriers that may be present in real cells. c | Coverslips that are coated with stimulatory molecules, including peptide–MHC molecules and antibodies, also allow controlled concentrations of a wide range of ligands and optimal surface imaging. However, the bound molecules are immobile.

a | Fluorescent resonance energy transfer (FRET) occurs when a suitable donor is in close proximity to an acceptor fluorophore; the process is illustrated here by arrows showing donor excitation, donor emission, energy transfer and acceptor emission. For bimolecular FRET, synthetic fluorescent probes (green and red stars) or fluorescent proteins can be used, whereas FRET sensors primarily incorporate fluorescent proteins. See for further details on FRET measurements. b | Fluorescence recovery after photobleaching (FRAP), showing fluorescent proteins in a region of membrane undergoing selective photobleaching, followed by the recovery of fluorescence in this area as a result of repopulation with unbleached mobile molecules. c | Single-particle tracking (SPT), showing a single fluorescently tagged molecule as it is tracked over time.

These techniques are needed to image cellular structures beyond the diffraction limit of light. a | Super-resolution light microscopy techniques — photoactivated localization microscopy (PALM), fluorescence PALM (FPALM) and stochastic optical reconstruction microscopy (STORM) — are based on stochastic detection of single particles (red stars) and their subsequent bleaching and localization (white circles). Localized molecules are summed over time to reveal sub-diffraction-sized features. b | Structured illumination microscopy (SIM) uses a sequence of illumination patterns to decode cellular features (blue line) with different components of spatial orientation. Summing all of the resolved spatial components results in a super-resolved image of the cellular feature (solid grey line). c | Stimulated emission depletion (STED) microscopy uses two concentric focused illumination beams (yellow and orange cones) to reduce the effective volume of detection. The illumination beams are scanned across the cell to resolve small cellular features. d | Transmission electron microscopy (TEM) uses a beam of high-voltage electrons to image features within thin layers or membrane sheets of cells.

The study of lymphocyte activation requires observation of samples that vary in size over six orders of magnitude. This figure shows the T cell receptor (TCR)-mediated signalling pathway and microscopy techniques used at three levels of sample size. a | In whole organisms or intact tissues, encounters between lymphocytes and antigen-presenting cells (APCs) can be studied in situ using two-photon laser scanning microscopy (TPLSM). The specimen size is tens of millimetres. b | Cell–cell interactions and many subcellular details can be monitored using a large number of conventional microscopy techniques. These samples are typically several micrometres in size, but some details are at the diffraction limit of light scale. c | To observe molecular detail, high- and super-resolution imaging techniques are required. The samples here are individual molecules, only a few nanometres in size. DIC, differential interference contrast; LAT, linker for activation of T cells; PALM, photoactivated localization microscopy; SEM, scanning electron microscopy; SIM, structured illumination microscopy; STED, stimulated emission depletion; TEM, transmission electron microscopy; TIRF, total internal reflection fluorescence; ZAP70, ζ-chain-associated protein kinase of 70kDa.