Olympus : Objectives without Compromise
Image: Confocal image of fixed mammalian cells from Olympus
A new proprietary lens polishing technique has broken through the technological barrier limiting the optical performance of microscope objectives.
Until now, the optical performance of objectives has been limited by a tradeoff between image flatness, numerical aperture and chromatic correction.
The new lens polishing technique produces ultra-thin lenses, which allows more lenses to be packed into each objective housing unit and deliver an improvement in all three key areas of optical performance.
The proprietary lens manufacturing method was developed by Olympus, whose history in making lenses stretches back 100 years.
This new technique, which underpins the design of the X Line high-performance objectives, are highly suitable for a wide range of life science research and clinical applications.
The improved lenses benefit applications ranging from brightfield imaging (eg whole-slide scanning) to fluorescence, TIRF and super-resolution.
This article will explore what these three key areas of optical performance are, how they are improved in X Line objectives and how the objectives benefit different life science applications.
Improving Image Flatness for a Clear, Consistent View
Image flatness determines the uniformity and clarity of an image throughout the field of view.
It is important for image stitching and whole-slide imaging (WSI), which combines multiple images of the same histological ection to create a single image of the entire section.
Improving image flatness reduces artifacts at the stitched edges providing for example pathologists with a cleaner, smoother image that benefits analyses and diagnoses.
In confocal imaging, improved image flatness makes signal intensity measurements more reliable and independent of the location within the field of view.
The flatness of an image is typically limited by spherical and coma aberration, field curvature and peripheral darkening.
These limitations cause inconsistencies in the clarity of the image from the center out to the edges of the image. This means the same signal can appear lower at the edges of the image than the center, which may be interpreted as inconsistent staining or lower protein expression.
The new objectives from Olympus are able to improve upon image flatness by combining ultra-thin concave and convex lenses (fig. 1).
Fig 1: Compared to conventional objectives (left), the X Line UPLXAPO60XO objective (right) has improved image flatness by combining ultra-thin concave and convex lenses. Confocal image of fixed mammalian cells acquired with Olympus’ FV3000 (blue: nuclei, purple: actin, green: microtubules).
This takes place without an adverse effect on the two other key areas of optical performance: numerical aperture or chromatic correction.
This means accurate and reliable data can be acquired for true interpretations by the processing algorithms used in image analysis software.
High Numerical Aperture - Feature Every Photon
Numerical aperture (NA) is a measure of an objective’s ability to gather light, which improves the resolution and brightness of the images.
NA is particularly important for fluorescence imaging as it creates a brighter signal at the same excitation intensity, thereby avoiding excessive photobleaching and phototoxicity, and enabling longer illumination and thus longer imaging times.
To maximize numerical aperture without sacrificing image flatness or chromatic correction, X Line objectives use a precise arrangement of lenses within a three-lens group consisting of convex-concave-convex lenses (fig 2).
Fig. 2: The thin edges of the lenses inside the objective allow more extreme angles for collecting light and for moving the beam up and down more.
The convex lenses also had to be manufactured in a way that produces extremely thin edges.
By improving the NA, the new objectives make fluorescence microscopy easier and allow pathologists to see extra detail in brightfield images, such as H&E staining.
For fluorescence microscopy, users can get more signal from their samples without issues with phototoxicity or photobleaching.
The extra light that the objectives gather also means that fine, sub-micron structures, such as the cellular cytoskeleton, are brighter and easier to visualize - benefiting for example super-resolution applications (fig. 3).
Fig. 3: Maximizing numerical aperture produces brighter fluorescence images, which makes it easier to visualise fine structures, such as the cytoskeleton. Blue: nuclei, red: actin, green: microtubules.
All Colours in One Spot with Chromatic Correction
Chromatic correction determines the lens’s ability to focus different wavelengths of light on the same spot, thereby avoiding chromatic aberration (fig. 4).
Fig. 4: Chromatic correction focuses different wavelengths of light on the same spot. The ultra-thin lenses in X Line objectives produce an extra level of chromatic correction. Conventional objective (left); X Line objective (right). The excitation wavelength was 405 nm for the cyan signal and 640 nm for the purple signal.
Chromatic aberration occurs when dispersion causes spots of different colours to appear from a single beam of light. It is the result of glass having slightly different refractive indices for each wavelength of light.
Shorter wavelengths of light have higher refractive indices, meaning that convex lenses focus violet light on a spot closer to the lens than red light.
Correcting these aberrations is important for creating high-quality, multicolor, tiled fluorescence images and for co-localization analysis.
Co-localization analysis is used to determine the spatial relationship between two targets by tagging them with different fluorescent labels.
When carrying out distance measurements, chromatic aberrations are an important source of error, which means that objectives with optimal chromatic correction provide substantial improvements in accuracy when doing sub-micrometer measurements in multicolour confocal, TIRF or super-resolution images.
Good chromatic correction also benefits tiled images, which are often required in embryology to image whole specimens, such as C. elegans or zebrafish embryos.
To get around the problem of chromatic aberration, a convex lens can be combined with a concave lens to form a lens group.
As a result, blue and red light are focused on the same spot.
For wide-range chromatic correction several lens groups consisting of 2 or 3 lenses are required.
The conventional design consists of 13 lenses in 7 groups.
The ultra-thin lenses used in Olympus’ X Line objectives take this one step further.
X Line objectives consist of 15 lenses in 9 groups, which results in excellent chromatic aberration correction over a wide spectral range (400 to 1000 nm) (fig. 5).
Fig. 5: X Line objectives consist of 15 lenses arranged into 9 groups; conventional lenses consist of 13 lenses in 7 groups.
The manufacturing of conventional objectives introduced a trade-off between three critical areas of optical performance: image flatness, numerical aperture and chromatic correction.
Consequently, conventional objectives can only produce high-resolution images across a small field of view.
Olympus’ novel lens polishing technology, used to create X Line and several specialized objectives, has overcome this trade-off by producing ultra-thin lenses, which together are capable of producing high-resolution, high-quality images over a large field of view.
This technological advance has made it possible to obtain the reliable and accurate imaging data required for the processing and analysis algorithms used by imaging software.
These high-performance objectives can easily be incorporated into existing compatible microscopes to bring an immediate improvement in quantitative and qualitative imaging results across a range of clinical and research applications, including brightfield, fluorescence, confocal, super-resolution and TIRF imaging.
Contact: Jan Barghaan Olympus Europe SE & Co. KG Hamburg, Germany.