Labeling of Objectives
Leica Microsystems objectives are coded and labeled differently according to type. The coding and labeling provides a short and compact overview for the identification of the objective and for the main optical performances and applications of the objectives.
You can find information about the assignment of the optical systems, e.g. like “HC” and “∞” for infinity corrected optics. Regarding the other indications, please see below in detail.
The Leica Microsystems HC system (Harmonic Compound System) comprises the optical components that have been matched to each other for optimal image generation and are involved in the correction of the optical aberrations: objectives, eyepieces, tube lenses, adapter for camera and TV.
For the correction of certain optical aberrations, the microscope is considered as a whole system. Spherical aberrations, coma and axial chromatic aberrations are best corrected at the place where they originate, i.e. in the particular component.
Lateral chromatic aberrations and astigmatism are corrected in parallel in the objective, the tube lens and the eyepiece. The optimal image result is therefore achieved through the interplay of corrections.
HC = The objective is included in the HC system.
HCX = The objective is also compatible with optics of the past (Delta optics 1991-1997)
The HC system ensures
- balanced optical and mechanical fitting dimensions,
- balanced alignment of all optical system components,
- balanced, reliable technical solutions,
- superlative optical performance with progressive manufacturing technology.
Magnification of the Objective
Each objective is labeled with its magnification, for instance 5x or 100x. However, the magnification of the objective alone does not determine the overall magnification of the microscope. This results from the objective magnification multiplied by the eyepiece magnification (for tube lens 1x).
40x objective x 10x eyepieces = 400x overall magnification
It should be noted, however, that the higher the objective magnification, the lower the visible object field.
The numerical aperture (NA or A) of the objective is a key parameter for the optical image and determines the resolving power of the objective and the brightness of the image. It is defined by the sine of the half aperture angle a of the lens and the refractive index n of the immersion medium. According to this definition, the larger the numerical aperture, the more narrow the focal spot and hence the higher the resolving power.
The objectives are labeled with their magnification, followed by the particular NA value, for instance 10x/0.40 or 63x/1.40. The numerical aperture of the objective can be changed by using iris diaphragm objectives.
The term 'numerical aperture’ is explained in detail in the Leica Science Lab article: "Beware of "Empty" Magnification"
By using iris diaphragm objectives the numerical aperture of the objective can be changed. This is especially useful for widefield microscopy. If the iris diaphragm is closed, the numerical aperture and resolution are reduced but the depth of field is increased. If the iris diaphragm is opened again, the numerical aperture is increased, the resolution is increased, but the depth of field is decreased. An objective can also be used as a darkfield objective by narrowing the aperture.
Iris diaphragm objectives are labeled with the range within which the numerical aperture can be adjusted, for instance 1.4 – 0.7.
The physical relationship between aperture, resolving power and depth of field is shown in the graph. A small aperture produces low resolution but large depth of field. A high aperture means better resolution but less depth of field.
Linear correlation between aperture and resolution (green), respectively exponential correlation between aperture and depth of field (red)
Collecting Light: The Importance of Numerical Aperture in Microscopy
Numerical aperture (abbreviated as ‘NA’) is an important consideration when trying to distinguish detail in a specimen viewed down the microscope. NA is a number without units and is related to the angles of light which are collected by a lens.
Beware of "Empty" Magnification
In the simplest case, an optical microscope consists of one lens close to the specimen (objective) and one lens close to the eye (eyepiece). The microscope magnification is the product of the factors of both microscope lenses. A 40x objective and a 10x eyepiece, for example, provide a 400x magnification.
Immersion Objectives: Using Oil, Glycerol, or Water to Overcome some of the Limits of Resolution
To examine specimens at high magnifications using the microscope, there are a number of factors which need to be taken into consideration. These include resolution, numerical aperture (NA), the working distance of objectives and the refractive index of the medium through which the image is collected by the front lens of an objective.
The performance of high-resolution objectives is optimal when the refractive indices of the specimen and all intermediate optical media match the values for which the objective is designed. Changes in coverglass thickness and temperature as well as inhomogeneous, thick specimens introduce refractive index mismatches. This causes deterioration of the point-spread function, geometric distortion, and chromatic aberration. These effects limit penetration depth, contrast, and intensity of the microscope images.
Immersion oil traditionally has a refractive index close to standard crown glass. Oil immersion objectives are designed for the refractive index of this oil. They are optimal when working close to the coverglass or with samples embedded in a medium with a refractive index close to that of immersion oil. For samples with refractive indices deviating from this value special objectives are offered. Most common are water immersion objectives, and glycerol immersion objectives. Water and glycerol immersion objectives are very sensitive to changes in coverglass – introducing a changing thickness of a medium with refractive index mismatch – temperature, and deviations of the immersion medium or the sample itself. Therefore, water and glycerol immersion objectives with a higher NA have a correction collar to compensate for those differences.
The correction collar axially moves the central lens group and can be used to restore optimal image resolution and brightness. As manual adjustment of the correction collar requires time and experience and can disturb the sample, Leica offers water immersion objectives with a motorized correction collar.
CORR = Objective with correction collar
The free working distance (FWD) is an important application related feature of the objective. It is the distance between objective front end and coverslip surface facing the front lens (see Fig. ) The focus of the objective is exactly on the opposite side of the coverslip. I.e. The FWD is the distance you can focus into a sample, i.e. you can image inside the sample.
The accessible sample area is often restricted by collisions of the objective with the sample holder, the edges of a multiwell plate or additional equipment, e.g. for electrophysiology or intravital imaging. Objectives with an extra-long free working distance allow to image also the edges of such samples without restrictions.
Deep tissue imaging by multiphoton excitation or in cleared tissues also requires large free working distance objectives to fully benefit from the optical advantages of those techniques. Here, the need for working distances of more than one milimeter are not uncommon. However, the numerical aperture of the objective still needs to be as large as possible to provide meaningful high-resolution images.
Leica Microsystems offers a range of objectives with exceptionally large free working distance for dry or water immersion. The water immersion objectives with extra-long working distances are also available with a large access angle and an inert ceramic front with minimal electric and thermal conductivity for electrophysiology.
L = Objective with extra-long free working distance