Dynamic imaging of the immune system: progress, pitfalls and promise

A form of fluorescence microscopy in which out-offocus signals are rejected by an aperture that restricts all light from reaching the detector except that originating from the focal plane of the excitation spot.

A fluorescence-imaging technique that takes advantage of the fact that fluorescent molecules can absorb two photons nearly simultaneously during excitation before they emit light. This technique allows all emitted photons to contribute to a useful image.

An imaging method that depends on the threedimensional detection of (positrons) radiation from a probe that is typically localized to a cell by direct ex vivo labelling or in situ metabolic conversion of a precursor compound.

A method that uses detection of changes in the alignment of protons in a strong magnetic field when they are perturbed by radio wave pulses to generate structural information about an object in that magnetic field.

• Shorter wavelength and higher resolution

• Less expensive and easier to maintain lasers

• Better performance in some tissues (such as the skin and liver)

Lymph nodes,

Thymus,

Liver,

Central nervous system,

Bone marrow,

Skin,

Spleen,

Gut,

Eye,

Kidney

Conditions: Explant and intravital

Comments:

• Intravital imaging of inguinal and popliteal lymph nodes

• Any lymph nodes as an explant, including pancreatic

• Oxygen level and perfusion important if explant is submerged

A technique that uses photons emitted by the process of luminescence, rather than fluorescence, to obtain an image of cells in a living animal. This method is extremely sensitive and non-invasive but generates data of much lower resolution than microscopebased fluorescent imaging.

The introduction of a transgene into a precise location in the genome, rather than a random integration site.  knocking-in uses the same technique of homologous recombination as a knockout strategy but the targeting vector is designed to allow expression of the introduced transgene under control of the regulatory elements of the targeted  gene.

A method for creating genetically altered mice in which very large segments of mouse genomic DNA are propagated in bacteria and used to achieve physiological patterns of gene expression. This technique avoids the need to create knock-in mice by homologous recombination in embryonic stem cells.

Retroviral or lentiviral vectors that contain mutations that inactivate the enhancer element in the 3′ LTR (long terminal repeat). Because the sequence of the 3′ LTR is used to reconstitute the 5′ LTR during reverse transcription, these vectors ‘self-inactivate’ the 5′ LTR enhancer before integration into the host-cell DNA. This allows exogenous gene regulatory sequences downstream of the 5′ LTR to control gene expression after integration.

A quantitative representation of the wavelengths (energies) of the photons emitted from a fluorescent compound  after it is excited by shorter wavelength (more energetic) photons from an illumination source.

The wavelength of incident light that is best absorbed by and causes maximal emission from a fluorescent compound.

• Higher throughput

• Relatively free of movement artefacts

• Defined environment

• Access to different surfaces of tissue

• Pharmacological studies

• Acute cell addition

• Imaging human biopsies possible

• True in vivo observations

• Physiological oxygen levels and metabolism

• Vascular and lymphatics intact

• Neural innervation

• Vascular and lymphatics present but no flow

• Lack neural innervation

• Processes stop during death and are restarted by oxygenation and/ or perfusion

• Lower throughput

• Motion artefacts from breathing and blood flow

• Anesthaesia effects

• Surgical trauma

• Access of inflammatory cells might cause progressive damage

A nanocrystalline semiconductor of extremely small size (10–50 nm) that results in its absorption of incident photons, followed by the emission of photons at a slightly longer wavelength. Because of a phenomenon called the quantum confinement effect, the colour (wavelength) of the emitted light is determined by the size of the nanocrystal.

An objective lens for a microscope that is optimized for use with its front surface in contact with an aqueous solution, because there is an improved match in refractive index between the glass and buffer solution that limits spherical aberration in the image.

The non-radiative production of frequency-doubled polarized light emission from a highly ordered (anisotropic) material on illumination by a laser beam. In practical terms, the production of polarized light emission from extracellular matrix materials such as collagen when subjected to two-photon illumination in the absence of fluorochrome labelling.

The concept that measurement of the properties of an object, in particular momentum and position, cannot be accomplished with complete accuracy. Sometimes used (with some license) to encompass the ‘observer effect’, which indicates that the mere attempt to measure such properties changes them from their intrinsic state.