Observing the Unseen: Advances in Optical Microscopy Techniques

How Does Optical Microscopy Work?

Optical microscopy, like any magnifying tool, can only take you so far. Viruses, neurons, and even cells can be observed, but for many years it was thought that anything smaller than atomic-size would remain beyond the reach of optical microscopes.

To overcome this obstacle, a confocal optical scanning microscopy technique was developed.

Illumination

Many different imaging techniques require specific illumination conditions. Illumination systems in microscopes may use natural daylight or artificial light sources such as tungsten-halogen bulbs, LEDs, or lasers. Microscopes also contain a variety of devices that condition and filter image-forming waves to improve contrast as a function of spatial frequency, phase, polarization, absorption, and more.

The first component of the optical train, the lamphouse, establishes the quality and quantity of the light emitted by an illumination source. It may include an aperture diaphragm to control the amount of light emitted and a series of lenses that focus the beam at a point on the specimen, known as a field diaphragm. Field diaphragms are often used in combination with other optical components, such as filters and polarizers, to manage the transmission of light through the microscope for different applications. They are especially important in techniques like darkfield microscopy, where the direct rays of light from the microscope to the sample are blocked while oblique rays illuminate the specimen.

Condenser lens

The condenser lens is responsible for shaping and optically filtering light that will be used to illuminate a sample in the microscope. It also determines the initial magnification of the image that will be created by the objective lens.

Light passes through a condenser lens and creates an inverted cone of parallel beams from every azimuth, whose size is determined by the adjustment of the aperture diaphragm. This configuration allows for the illumination of samples in a variety of imaging modes and is why backlight illumination is preferable to direct sunlight.

Microscopes cannot eliminate the shape defects in optical lenses, but the distortions can be controlled with a variety of techniques. In phase contrast microscopy, for example, a sample’s structures are revealed by changing the wavelength phases of reflected light. This technique is possible because the waveform of optical radiation has a series of peaks and troughs that alternate with each other. This creates a contrasting pattern in the image that can be detected by cameras and microscopes.

Objective lens

The objective is the first element in the microscope that light encounters as it travels from the condenser to the specimen. The critical design characteristics of the objective set the ultimate resolution limit of a microscope.

The optical resolving power of an objective depends on the wavelength of illumination used, the refractive index of the imaging medium and its angular aperture (Figure 3). A point in a sample appears as a blurred disk surrounded by diffraction rings, called Airy discs, which are three-dimensional representations of the diffraction pattern.

To increase the angular aperture of an objective and improve the image quality, most objectives are designed to use a medium such as immersion oil or water with a matching index between the front lens element and the specimen. This allows the objective to collect ray bundles at larger angles, increasing resolution. However, this method introduces additional aberrations to the optical system and requires careful adjustment.

Eyepieces

In microscopes with simple optical systems, the eyepiece lens relays a magnified image of the specimen to the observer’s eyes or camera system. This process is known as image generation and is based on the physics of refraction. The space between the light source and the first lens entrance surface is called the object space and the region of space where projected extensions of refracted light rays concentrate is called the image space. When these image-forming rays intersect to form a real (not virtual) image, a focal point is formed at the image plane.

Modern microscope objectives are designed to produce a bundle of parallel wavefronts (leaving the rear focal plane) that are matched in terms of optical factors to a tube lens to generate the final, fully-corrected intermediate image. This eliminates the need for a separate objective-eyepiece correction assembly and allows for the use of a variety of other optical components, such as filters, prisms, beamsplitters, reflectors, and aperture diaphragms.

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