Contrastingly, in an infinite conjugate or infinity-corrected optical system, light originating from an infinite distant source is focused on a small spot. Within an objective, this spot serves as the object under examination, while infinity points toward the eyepiece or camera sensor (refer to Figure 12). This sophisticated design incorporates an additional tube lens between the object and eyepiece, enabling the production of an image. Despite its complexity compared to finite conjugate designs, the infinite conjugate system allows the integration of optical components like filters, polarizers, and beamsplitters into the optical path. This feature facilitates advanced image analysis and extrapolation in complex systems.

Darkfield microscopy is ideal when you want to reveal edges or contours, it is perfect for observing microorganisms such as diatoms, unstained bacteria, bones, fibers, hair, protozoa and tissue, among others. Non-biological samples such as crystals, colloidal particles, powders, ceramics and thin sections of polymers can also be observed.

Dark fieldscattering spectroscopy

Microscope objectives employ two primary imaging modes: finite conjugate imaging and infinite conjugate imaging. Infinite conjugate objectives project images to infinity and necessitate the use of a tube lens for imaging assistance. The parallel light path between the infinite conjugate lens and the tube lens facilitates the incorporation of optical components, such as splitters and polarizers, without compromising imaging quality. This flexibility has made infinite conjugate lenses a mainstream choice in the market.

Dark-field microscopy has certain disadvantages compared to light-field microscopy, especially in the visualization of the sample. With the darkfield microscope it is necessary to prepare the sample correctly so that it is not too thick, since this way nothing could be appreciated, on the other hand, the resolution is lower and internal structures cannot be observed.

In darkfield microscopy, there are two types of illumination on which it is based, trans-illumination and epi-illumination, the use of these depends on what you want to highlight, for example, if you want to observe thin particles or if it is necessary to appreciate the edge of what is observed in the sample. Both types are explained below:

Darkfield microscopy is a type of optical microscope used in certain types of laboratories. It has the same characteristics as any composite microscope, except that it undergoes modification in the condenser before light reaches the sample. This type of microscope allows the contrast level to be increased, so it is possible to observe certain characteristics that with light-field microscopy would be impossible.

They can also visualize live suspended samples, for example, certain microorganisms, so that the cell wall or capsule can be seen. In bioassays, they are especially useful because you can observe the behavior of the blood cells, without the need to add any additional reagents.

High magnification means a short focal length, and short focal length can achieve large NA. Increasing NA is a common method to improve the resolution of objective lenses. As a small aberration optical system, a high NA means that more light is collected, but the light must be concentrated in a smaller area. With high NA as the premise, it is not easy to correct the on-axis and paraxial aberrations, and it is even more difficult to achieve long working distances at the same time.

Resolution is expressed as a function of wavelength (λ) and numerical aperture (NA), with NA calculated as the product of the refractive index (n) and the sine of the half-angle (θ) of light from the object. Achieving high magnification often involves a trade-off, as increasing NA for better resolution concurrently poses challenges in correcting on-axis and paraxial aberrations and achieving long working distances. For an aberration-corrected objective that reaches the diffraction limit, the resolution is usually expressed as follows:

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However, this does not make it a bad choice, since everything depends on the type of sample to be analyzed, for example, if you need to observe living microorganisms without damaging them or if you only want to see the outline of certain organisms to identify them, this equipment becomes a very useful tool.

They are especially useful when samples need to be evaluated without causing damage or staining. Some bacteria, for example, need certain dyes, to be observed under the light field microscope, a variant is the use of dark field microscopes, which allow to detail certain characteristics, this makes them very useful equipment to analyze certain types of clinical samples.

Apart from operational flexibility, long working distances offer advantages such as collision avoidance between the sample and the objective, suitability for samples of varying thickness, and reduced risks of debris contamination and lens damage during processing. In specific scenarios like atom trapping and crystal growth observation, long working distance objectives become indispensable.

HAADF-STEM

In the optical configuration of a finite conjugate system, light emanating from a light source, not positioned at infinity, converges to a specific spot (see Figure 11). In the context of a microscope, the image of the examined object undergoes magnification and is projected onto the eyepiece or camera sensor. The system’s particular distance is defined by either the DIN or JIS standard, with all finite conjugate microscopes adhering to one of these two standards. This design is prevalent in basic microscopes and finds application in scenarios where cost-effectiveness and simplified design are primary considerations.

The evolution of microscope objectives, with a historical backdrop, underscores the ongoing importance of design and development. The demand for specialized non-standard lenses in scientific research and increasing requirements in various fields drive the development of objectives with better flat-field characteristics, chromatic aberration correction, and, notably, long working distances.

However, achieving long working distances often involves employing a reverse telephoto design structure, which increases lens diameter and complexity. This imposes higher requirements on optical design and lens processing capabilities.

Despite the challenges posed by achieving long working distances, the ongoing advancements in optical design and lens processing capabilities continue to push the boundaries of microscope objective performance, ensuring clear imaging and enhanced resolution for a wide range of applications.

Dark fieldmicroscopy

Working distance, denoting the distance between the object and the lens’s front end, emerges as a crucial parameter in microscope objective selection. Longer working distances afford greater flexibility in applications. For instance, in optical fiber fusion, a high-magnification objective with a long working distance improves alignment precision by providing ample space for the optical fiber.

For infinite conjugate microscope objectives, magnification is determined by the focal length of the objective and the tube lens. High-magnification objectives, typically exceeding 50X, are associated with enhanced resolution. Resolution, indicating the minimum resolvable distance between two points, is a critical factor in applications requiring detailed imaging, such as optical fiber alignment. The magnification is calculated as follows:

Where n is the refractive index of the medium between the lens and the object, and θ is the half-angle of the light from the object. The medium between a conventional lens and an object is air, called a dry lens, and the theoretical limit of NA is 1. If you want to break the theoretical limit, you need to change the medium to water or oil. Long working distance microscope objectives are usually dry lenses.

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Phase contrast

Microscope objectives are essential components in optical microscopes, serving to magnify and capture images of observed objects. They find applications in diverse fields such as biomedical research, precision detection, and semiconductor processing. Additionally, microscope objectives are employed independently in scientific research and industrial production, for activities such as atom capture and laser processing.

Where λ is the band and NA is the numerical aperture of the lens, which is the most commonly used aperture representation method for microscopic objective lenses:

For instance, the introduction of a filter between the objective and tube lens permits the observation of specific wavelengths of light or the blocking of unwanted wavelengths that might disrupt the setup. Fluorescence microscopy commonly employs this design. Another advantage of the infinite conjugate configuration is its capability to adjust magnification according to specific application requirements. The objective magnification is determined by the ratio of the tube lens focal length (fTube Lens) to the objective focal length (fObjective). By altering the tube lens focal length, typically a 200mm achromatic lens, the objective magnification can be customized. If an objective follows an infinite conjugate design, the objective body will bear an infinity symbol.

In conclusion, infinite conjugate long working distance microscope objectives play a pivotal role in various scientific and industrial applications by providing high-resolution imaging capabilities. Their evolution highlights the constant need for advanced design and development to meet diverse research and production demands. Long working distances offer operational flexibility, collision avoidance, and suitability for various sample thicknesses, making them indispensable in critical scenarios like optical fiber alignment, atom trapping, and crystal growth observation.