Deep cell imaging | Deep cell imaging

Key Words: Live cell imaging, confocal, fluorescence, deconvolution, infra-red imaging, water immersion

Definition:Deep cell imaging refers to technologies that enable deeper observations into tissues, for example, confocal and multiphoton (2 /3-photon) laser scanning microscopy

TECHNOLOGY:

Imaging live cells deep in tissues is difficult to accomplish using light microscopy because light scatters as it travels through tissue. Deeper imaging can be realized with:

1. Confocal microscopy, where the use of a pinhole reduces light from outside the plane of focus. Thin optical sections acquired by scanning through a thick specimen can be digitally reconstructed in 3-D to enable visualization of deep structures / dynamic events.
2. Multiphoton imaging exploits the greater transparency of tissues to infrared (IR) light. IR light, however, does not have the energy required to excite fluorophores unless two or more photons of light interact with a molecule simultaneously to achieve twice (or more) the energy and a proportional reduction in wavelength. This can be achieved by focusing ultra-short pulses of light (10-13 seconds or less) through the microscope objective. Excitation only occurs where two (or more) photons interact reducing potential photodamage above and below the plane of focus. Efficient IR transmittance through the lens is a key factor, and depends on the types of glass used in manufacture.
3. High performance objectives (for, example, high IR transmittance, high N.A., long working distances, water immersion, water dipping) enable deeper, higher resolution imaging into tissues.
4. Digital deconvolution can be used to used to improve background noise and, hence, greater depth resolution in fluorescence widefield, confocal and multiphoton imaging.

APPLICATIONS:

Deep cell imaging is particularly important in the study of brain structure, nerve cells, the study of angiogenesis in cancer, and molecular trafficking, where continuity of observation in the Z-direction is important. Deep cell imaging with light microscopy has potential for non-invasive studies in living specimens, optical biopsy and disease diagnosis.

MICROSCOPE CONFIGURATION:

Nikon offer a number of solutions for improved deep cell imaging including the FN-1 physiology work station, confocal imaging solutions (C1 Plus, C1, C1si and LiveScan Sweptfield system) and a variety of high resolution objectives with high IR transmittance ideal for multiphoton applications. Plan Apochromat VC lenses, for example, not only work well in the violet part of the spectrum, but also perform well in the IR enabling greater depth penetration in the specimen and enabling multiphoton applications. A Plan Apochromat VC water immersion lens is also available (VC60 x W.I. N.A. 1.20). Ideal for deep tissue imaging, this objective reduces optical aberrations caused by phase changes through different media.

RECOMMENDED SYSTEM:

Consult you local Nikon representative for advice on imaging solutions matched to you needs.

LINKS:

Laser scanning confocal microscopy

Multiphoton imaging

Water immersion objectives