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Overview

Low Magnification "Dry" Lenses

In cases where extra space between the objective and the specimen is required to work with the sample, a dry lens can be used, potentially requiring additional adjustments of the correction collar to account for differences in imaging chambers. Special objective lenses are designed with correction collars that correct for spherical aberrations while accounting for the cover slip thickness. In high numerical aperture (NA) dry objective lenses, the correction collar adjustment ring will change the position of a movable lens group to account for differences in the way the outside of the lens focuses light relative to the center. Although lens aberrations are inherent in all lens designs, they become more problematic in dry lenses where resolution retention is key.^[7]

Oil Immersion High NA Lenses

Oil immersion is a technique that can increase image resolution by immersing the lens and the specimen in oil with a high refractive index. Since light bends when it passes between mediums with different refractive indexes, by placing oil with the same refractive index as glass between the lens and the slide, two transitions between refractive indices can be avoided.^[8] However, for most applications it is recommended that oil immersion be used with fixed (dead) specimens because live cells require an aqueous environment and the mixing of oil and water can cause severe spherical aberrations. For some applications silicone oil can be used to produce more accurate image reconstructions. Silicone oil is an attractive media because it has a refractive index that is close to that of living cells, allowing it to produce high resolution images while minimizing spherical aberrations.^[7]

Water Immersion Lenses

Live-cell imaging requires a sample in an aqueous environment that is often 50 to 200 micrometers away from the cover glass. Therefore, water immersion lenses can help achieve a higher resolving power due to the fact that both the environment and the cells themselves will be close to the refractive index of water. Water immersion lenses are designed to be compatible with the refractive index of water and usually have a corrective collar which allows for adjustment of the objective. Additionally, because of the higher refractive index of water, water immersion lenses have a high numerical aperture and can produce images superior to oil immersion lens when resolving planes deeper than 10µm.^[7]

Dipping Lenses

Another solution for live-cell imaging is the dipping lens. These lenses are a subset of water immersion lenses that do not require a cover slip and can be dipped directly into the aqueous environment of the sample. One of the main advantages of the dipping lens is that it has a long effective working distance.^[9] Since a cover slip is not required, this type of lens can approach the surface of the specimen and as a result, the resolution is limited by the restraints imposed by spherical aberration rather than the physical limitations of the cover slip. Although dipping lenses can be very useful, they are not ideal for all experiments since the act of "dipping" the lens can disturb the cells in the sample. Additionally, since the incubation chamber must be open to the lens, changes in the sample environment due to evaporation must be closely monitored.^[7]

Phototoxicity and Photobleaching

The rise of confocal microscopy is closely correlated with accessibility of high power lasers, which are able to achieve high intensities of light excitation. However, the high power output can damage sensitive fluorophores and are usually run significantly below their maximum power output.^[10] Over exposure to light can result in photodamage due to photobleaching or phototoxicity. The effects of photobleaching can significantly reduce the quality of fluorescent images and in recent years there has been a significant demand for longer-lasting commercial fluorophores. One solution, the Alexa Fluor series, show little to no fading even at high laser intensities.^[11]

Under physiological conditions, many cells and tissue types are exposed to only low levels of light.^[12] As a result, it is import to minimize the exposure of live cells to high doses of ultraviolet (UV), infrared (IR), or fluorescence exciting wavelengths of light, which can damage DNA, raise cellular temperatures, and cause photo bleaching respectively.^[13] High energy photons absorbed by the fluorophores and the sample are emitted at longer wavelengths proportional to the Stokes shift.^[14] However, cellular organelles can be damaged when the photon's energy produces chemical and molecular changes rather than being re-emitted.^[15] It is believe that the primary culprit in the light induced toxicity experienced by live cells is a result of free radicals produced by the excitation of fluorescent molecules.^[12] These free radicals are highly reactive and will result in the destruction of cellular components, which can result in non-physiological behavior.

One method of minimizing photo-damage is to lower the oxygen concentration in the sample to avoid the formation of reactive oxygen species.^[16] However, this method is not allows possible in live-cell imaging and may require additional intervention. Another method for reducing the effects of free radicals in the sample is the use of antifade reagents. Unfortunately, most commercial antifade reagents cannot be used in live-cell imaging because of their toxicity.^[17] Instead, natural free-radical scavengers such as vitamin C or vitamin E can be used without substantially altering physiological behavior on shorter time scales.^[18]

References