Researchers use microscopes as essential tools for advancing their science, and objective lenses are crucial components of these systems. Many applications benefit from high-quality images with a large field of view. The objective lens is the fundamental building block of any imaging system. The ultimate purpose of an objective lens and an optical microscope is to provide useful magnification that allows minute specimens to be observed in great detail, thus exposing a hidden world of invisible objects that would otherwise remain unseen. Microscope objectives are perhaps the most important components of an optical microscope because they are responsible for primary image formation and play a central role in determining the quality of images that the microscope is capable of producing. Objectives are also instrumental in determining the magnification of a particular specimen and the resolution under which fine specimen detail can be observed in the microscope. It is the most difficult component of an optical microscope to design and assemble, and is the first component that light encounters as it proceeds from the specimen to the image plane. Objectives derive their name from the fact that they are, by proximity, the closest component to the object (specimen) being imaged.

Common issues caused by objective lenses

Lens errors in modern optical microscopy are an unfortunate problem caused by artifacts arising from the interaction of light with glass lenses.There are two primary causes of non-ideal lens action: Geometrical or Spherical aberrations are related to the spherical nature of the lens and approximations used to obtain the Gaussian lens equation; and Chromatic aberrations, which arise from variations in the refractive indices of the wide range of frequencies found in visible light. Blue light is refracted to the greatest extent followed by green and red light, a phenomenon commonly referred to as dispersion.

Another artifact that results from using lenses with curved surfaces is the field curvature. Two points in the specimen, one in the center and one on the edge, will not be focused on the same plane resulting either a sharp focus in the center or on the edges.

Design characteristics of the objective that set ultimate resolution limit of the microscope

Three critical design characteristics of the objective set the ultimate resolution limit of the microscope. The illumination wavelength λ, the angular aperture θ, and the imaging medium refractive index n. The human eye responds to the wavelength region between 400 and 700 nanometers, which represents the visible light spectrum that is utilized for most microscope observations. This is the first crucial component determining the resolution. Another component is the refractive index of the imaging medium. Objectives are designed to image specimens either with air or a medium of higher refractive index between the front lens and the specimen. The field of view is often quite limited, and the front lens element of the objective is placed close to the specimen with which it must lie in optical contact. A gain in resolution by a factor of approximately 1.5 is attained when immersion oil is substituted for air as the imaging medium.

The last, but perhaps most important factor in determining the resolution of an objective is the angular aperture, which has a practical upper limit of about 72 degrees (with a sine value of 0.95). When combined with refractive index, the product is the numerical aperture.

A new objective manufacturing technology

A novel new proprietary lens polishing technology has enabled lens manufacturers to develop convex lenses with ultra-thin edges as well as ultra-thin concave lenses. The lens' shape captures light at a wider angle than conventional lenses. Because they are so thin, more lenses can be packaged in each objective housing, which increases the NA, image flatness, and chromatic correction range.

Conventional objective lens manufacturing technology once forced a tradeoff between numerical aperture, image flatness, and chromatic correction, making it difficult to improve all three in one objective. In the past, engineers concentrated on making objectives that were exceptional in one of the three areas so that users could choose the objective that best suited their application. These objectives were engineered to acquire high-resolution images in a relatively narrow area (smaller FOV), which could introduce issues when using image analysis software since the processing algorithms normally assume that the images have no optical aberration or peripheral darkening. It is difficult to obtain reliable, accurate processing data unless the objectives deliver high-resolution, high-quality images over a large FOV. The new advanced manufacturing technology solves these challenges.

To see how the improvements in flatness, chromatic correction and numerical aperture affect the image quality, we can look at a few examples.

Better image flatness improves whole slide imaging

Comparison of brightfield images between conventional 20X objective (above) and X Line 20X objective (below).

The new objective manufacturing technology has improved image flatness compared to conventional objectives, which is beneficial for whole slide imaging applications. For instance, an Olympus X Line objective, the UPLXAPO20X, has a numerical aperture of 0.80 which significantly improved image flatness in the entire FOV and improved resolution when compared to a conventional objective. These improvements yield better image quality in each image as well as more efficient acquisition of tiled images.

Numerical aperture improvements

article imageConventional UPLSAPO20X (NA 0.75)

 

article imageX Line UPLXAPO20x (NA 0.80)

Numerical aperture determines the resolution of the image and the brightness. Improvements in lens manufacturing have led to increases in NA, which correlates to less photons needed to illuminate the sample reducing phototoxicity and photobleaching during fluorescence observation. The images are consistently bright over the entire FOV, which leads to more accurate analysis.

Reliable multicolor fluorescence images with high NA

The new objective manufacturing technology used in the Olympus X Line objectives has improved chromatic correction in the range of 400–1000 nm provide accurate data of multicolor fluorescence observation. With wide chromatic correction, all the signals from different colors are colocalized.

Summary

Manufacturers are at the forefront of designing high-quality objectives using breakthrough techniques and digital technology to improve numerical aperture, image flatness, and chromatic correction. The next generation objectives help create reliable quantitative and qualitative imaging results.


Alket Mertiri, PhD, is associate product manager, Scientific Solutions Group at Olympus Corporation of the Americas.