Water Immersion Objectives | Water Immersion Objectives

Key Words: Live cell imaging, GFP, N.A., oil immersion, resolution, spherical aberration, Kaede, deep cell imaging, water dipping objectives

Definition:Microscope objectives designed for use with water as the immersion medium over cover-slipped specimens

TECHNOLOGY:

All immersion techniques are designed to realize the maximum N.A. of the lens and increase resolution. In live cell imaging cells are usually immersed in culture medium within a culture dish. Regions of interest may be 50-200 microns away from the cover glass. High N.A. oil immersion objectives are not ideal for imaging focal planes that are not immediately next to the cover glass because aberrations (particularly spherical aberrations caused my differences in refractive indices in the optical path) increase with imaging depth. When cells need to be observed deep within tissues, water immersion lenses show superior performance in terms of resolution and aberration correction than oil immersion lenses. When water immersion lenses are used, the optical path is symmetrical: light passes from cells (watery) to glass (cover slip) to water (immersion medium) and then to glass (objective lens) again. Water immersion objectives have slightly lower N.A.s than comparable oil immersion lenses but enable high-resolution imaging through aqueous layers approximately 200-microns thick. Other advantages of water immersion lenses are that, unlike oil immersion objectives, they do not require special cleaning, there is no risk of contaminating physiological media, and water does not have inherent fluorescence that can complicate image interpretation.

APPLICATIONS:

Water immersion objectives are ideal for investigation of cellular dynamics inside living tissue where important events can occur deep within the specimen and far from the cover glass.

MICROSCOPE CONFIGURATION:

Nikon's water immersion objective, the CFI Plan Apochromat violet corrected (VC) 60x WI (N.A. 1.20; W.D. 0.27) corrects axial chromatic aberration for five lines ranging from 405-656 nm. This lens is also vignetting-free: the resolution of the objective lens is even across the entire field of view making it an ideal choice for digital photomicrography. In fluorescence imaging, certain probes require Plan Apochromat VC optics for activation and imaging. These include CFP, GFP, YFP, Cherry-FP, Tomato-FP, PA-GFP, Kaede, and PS-CFP. Plan Apochromat VC lenses 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. This objective, capable of accommodating an excitation wavelength of 405 nm and imaging from blue to red parts of the spectrum, is also useful for quantum dot deep cell imaging techniques.

Nikon also supplies a multi-immersion objectives suitable for oil, glycerin or water immersion (CFI Plan Fluor 20x, N.A 0.75, WD for oil: 0.35; glycerin: 0.34, water: 0.33mm) DIC prism: 20x, 20x-C

RECOMMENDED SYSTEM:

For maximum flexibility in live cell fluorescence applications, Nikon's Ti series inverted microscopes with C1plus, C1si or Livescan sweptfield confocal system and Nikon's CFI Plan Apochromat violet corrected (VC) 60x WI (N.A. 1.20; W.D. 0.27) objective.

LINKS:

Water immersion objectives [microscopyu]