The most important component for live-cell imaging is the microscope objective and particular attention should be given to the wide range of available choices. Selection begins by identifying the specimen requirements for three parameters: the numerical aperture (NA), the working distance, and the magnification. Of these, the numerical aperture is the most critical feature because this value determines the lateral and axial resolution limits and the amount of light collected (in effect, the available resolution and contrast). Objective numerical aperture ranges from 0.1 to 1.45 with higher values corresponding to greater resolving power. Those objectives considered high numerical aperture (greater than 1.0) have a short working distance (maximum focal distance from the coverslip) and require a special immersion medium, such as water, oil, or glycerin, between the front lens element and the coverslip. The working distance is often overlooked in objective selection, but becomes very important for imaging thicker plant and animal tissue slices. Magnification determines how large the specimen will appear, but can also be considered as a gauge for how much of the specimen area is viewable in the microscope. In general, magnification increases with numerical aperture and decreases with greater working distance.
Microscopes designed since the mid-1990s employ the modern infinity-corrected optical system, which replaces the fixed tube length (usually 160 millimeters) that broadly governed microscope design for most of the twentieth century. Infinity-corrected objectives generally feature higher light transmission, longer working distance, and are not compatible with older microscopes because of the requirement for a tube lens located elsewhere in the microscope frame. Objectives are corrected for chromatic aberration over a wide range from a single focal plane for red and green (achromats) to complete convergence of violet, blue, green, and red (apochromats). Intermediate correction factors are also available. In the lowest correction level, achromats, and even for some highly-corrected apochromat objectives, the axial focus position for blue and ultraviolet light is different from green and red light, even in objectives where differently colored objects co-align in the lateral (x-y) field. This artifact leads to serious problems when determining the relative positions or intensities of two fluorescent probes or when conducting ratio imaging experiments. In addition to chromatic correction, spherical aberrations in the objective can be corrected to achieve a flat visual field (termed plan correction). Almost all modern objectives are also corrected to remove off-axis aberrations, such as coma and astigmatism.
The microscope manufacturers offer a wide variety of objectives having nearly the same numerical aperture, magnification, and working distance. In general, these objective designs incorporate a tradeoff between light transmission efficiency and better optical correction, or the preservation of light polarization. The additional glass lens elements required for correcting chromatic and spherical aberrations tend to lower transmission efficiency (in effect, reduce brightness). In contrast, objectives designed for fluorescence microscopy (appropriately termed Fluor or Fluar) transmit more light over a broader spectral range, but are often poorly corrected for spherical and chromatic aberration. The absolute light transmission efficiency of an objective tends to be a closely guarded manufacturer's secret. Objectives designed for transmitted light (DIC, HMC, and phase contrast) and polarized applications may be highly corrected, but less efficient for light transmission. Polarized light objectives employ strain-free glass components that preserve the azimuth of linear polarization. Phase contrast objectives contain a dense phase ring near the rear focal plane that has only a slight effect on transmission efficiency (5 to 15 percent reduction). In general, fluorescence objectives are the best choice for live-cell imaging, unless high-resolution DIC or polarization is required for the experiment.
Illustrated in Figure 2 are a series of high-performance objectives designed for, among other applications, producing images having optimal signal-to-noise in live-cell imaging. The 63x plan neofluar mixed media immersion objective in Figure 2(a) is equipped with a correction collar to offset variations refractive index as a function of the immersion medium and to minimize spherical aberration at higher operating temperatures (37° C). The numerical aperture of this objective (1.30), although slightly lower than comparable oil immersion objectives, is sufficient to ensure high resolution imaging at wavelengths spanning the violet into the near-infrared spectral regions. The highly corrected plan apochromat 63x (Figure 2(b)) oil immersion objective has a numerical aperture of 1.4 and produces very bright images in fluorescence imaging mode, but does not exhibit parfocality between visible and near-infrared images. The 100x objective (illustrated in Figure 2(c)) produces high-resolution images with superior chromatic correction (similar to the 63x water and oil immersion versions). For electrophysiology, the 63x water dipping objective (Figure 2(d)) is excellent for high magnification imaging using upright microscope frames in patch-clamping experiments.
The optical design of many objectives requires that they be carefully matched to coverslips having a specific thickness (usually 170 micrometers), a value that is usually inscribed on the barrel of the objective (see Figure 2). Virtually all microscopes constructed for the life sciences are equipped by default with objectives that are corrected to use number 1.5 (170 micrometer thick) coverslips. For critical experiments, coverslips having very strict specifications are available. Inadvertently using a number 1 or 2 coverslip with these objectives can markedly degrade image quality, depending upon the magnification factor. In contrast, objectives designed for electrophysiology studies (Figure 2(d)), termed water dipping, do not require a coverslip. These objectives have a ceramic or composite polymer nosepiece and can be dipped directly into the culture medium (or buffer) while working with a living specimen.
Oil immersion objectives are designed for imaging thin specimens that are attached or firmly pressed onto the coverslip. If a liquid medium resides between the specimen and coverslip, or if imaging through more than 20 micrometers of cell volume, the light-focusing properties of the oil immersion objective begin to degrade, and in the worst case, add noise (improperly focused light) to the image. This concern is very often realized when imaging thick tissue slices and plant specimens. Water and glycerin immersion objectives were developed for such conditions because the hydrophilic immersion medium refractive index more closely matches that of the culture medium and cytosol. Regardless of the fact that water and glycerin immersion objectives have lower numerical apertures (1.2 to 1.3) than oil immersion objectives (up to 1.45; see Figure 2), the signal-to noise ratio and resolution for the image may be significantly improved under live-cell imaging conditions. Flat-field (plan) correction is often poor with these objectives, but a typical laser scanning confocal microscope or imaging camera captures less than the central two-thirds of the viewfield, a region where most objectives feature a flat field, irrespective of the correction factor. In cases where the imaging medium contains sufficient high molecular weight components to significantly alter the refractive index, water should be replaced with the imaging medium between the coverslip and the front lens element (for water immersion objectives) and the correction collar used to correct minor spherical aberration artifacts.
In conclusion, several critical guidelines should be considered when choosing objective priorities for live-cell imaging. In all cases, the investigator should use the highest numerical aperture objective that permits adequate working distance to focus through the features of interest in the specimen. In addition, one should choose the magnification factor that will project only the region of interest from the specimen onto the detector. If the specimen is in direct contract with the coverslip (as is the case for adherent cell cultures) an oil immersion objective will usually provide the optimum light collection efficiency. In cases where a liquid medium exists between the specimen and coverslip, or where the microscope must be focused through a large cell or tissue volume, both the conventional and dipping water immersion objectives usually produce superior contrast at nearly comparable resolution. For objectives that feature a correction collar for eliminating spherical aberration when using varying coverslip thicknesses and immersion media, the optimum setting should be carefully determined. Also, when imaging single fluorophores in live-cell scenarios, it is often wise to choose an objective that features high transmission rather than extensive color correction (which is not necessary for single-color imaging). Finally, applications requiring short (less than 380 nanometers) or long (more than 600 nanometers) illumination wavelengths, as well as polarized light, require specialized objectives having optical materials that remain transparent throughout these regions of the spectrum.
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