Water immersion objective

Last updated

In light microscopy, a water immersion objective is a specially designed objective lens used to increase the resolution of the microscope. This is achieved by immersing both the lens and the specimen in water which has a higher refractive index than air, thereby increasing the numerical aperture of the objective lens.

Objective (optics) optical element that gathers light from the object being observed and focuses the light rays to produce a real image

In optical engineering, the objective is the optical element that gathers light from the object being observed and focuses the light rays to produce a real image. Objectives can be a single lens or mirror, or combinations of several optical elements. They are used in microscopes, telescopes, cameras, slide projectors, CD players and many other optical instruments. Objectives are also called object lenses, object glasses, or objective glasses.

Optical resolution describes the ability of an imaging system to resolve detail in the object that is being imaged.

Refractive index optical characteristic of a material

In optics, the refractive index or index of refraction of a material is a dimensionless number that describes how fast light propagates through the material. It is defined as



Water immersion objectives are used not only at very large magnifications that require high resolving power, but also of moderate power as there are water immersion objectives as low as 4X. Objectives with high power magnification have short focal lengths, facilitating the use of water. The water is applied to the specimen (conventional microscope), and the stage is raised, immersing the objective in water. Sometimes with water dipping objectives, the objective is directly immersed in the solution of water which contains the specimens to look at. Electrophoretic preparations used in the case of comet assay can benefit from the use of water objectives.

The focal length of an optical system is a measure of how strongly the system converges or diverges light. For an optical system in air, it is the distance over which initially collimated (parallel) rays are brought to a focus. A system with a shorter focal length has greater optical power than one with a long focal length; that is, it bends the rays more sharply, bringing them to a focus in a shorter distance.

Comet assay

The single cell gel electrophoresis assay is an uncomplicated and sensitive technique for the detection of DNA damage at the level of the individual eukaryotic cell. It was first developed by Östling & Johansson in 1984 and later modified by Singh et al. in 1988. It has since increased in popularity as a standard technique for evaluation of DNA damage/repair, biomonitoring and genotoxicity testing. It involves the encapsulation of cells in a low-melting-point agarose suspension, lysis of the cells in neutral or alkaline (pH>13) conditions, and electrophoresis of the suspended lysed cells. The term "comet" refers to the pattern of DNA migration through the electrophoresis gel, which often resembles a comet.

The refractive index of the water (1.33) is closer to those of imaged materials or to the glass of the cover-slip, so more light will be collected/focused by this type of objective comparing to air-immersion ones, leading to a range of higher numerical apertures (NA). [1]

Correction collar

Unlike oil, water does not have the same or near identical refractive value as the cover slip glass, so a correction collar is needed to be able to variate for its thickness. Lenses without a correction collar generally are made for the use of a 0.17 mm cover slip or for use without a coverslip (dipping lens). [2]

See also

Microscopy technical field of using microscopes to view samples and objects that cannot be seen with the unaided eye

Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye. There are three well-known branches of microscopy: optical, electron, and scanning probe microscopy, along with the emerging field of X-ray microscopy.

Optical microscope microscope that uses visible light and a system of lenses to magnify images of small objects

The optical microscope, often referred to as the light microscope, is a type of microscope that commonly uses visible light and a system of lenses to magnify images of small objects. Optical microscopes are the oldest design of microscope and were possibly invented in their present compound form in the 17th century. Basic optical microscopes can be very simple, although many complex designs aim to improve resolution and sample contrast. Often used in the classroom and at home unlike the electron microscope which is used for closer viewing.

In optics, an index-matching material is a substance, usually a liquid, cement (adhesive), or gel, which has an index of refraction that closely approximates that of another object.

Related Research Articles

Microscope instrument used to see objects that are too small for the naked eye

A microscope is an instrument used to see objects that are too small to be seen by the naked eye. Microscopy is the science of investigating small objects and structures using such an instrument. Microscopic means invisible to the eye unless aided by a microscope.

Numerical aperture dimensionless number that characterizes the range of angles over which the system can accept or emit light

In optics, the numerical aperture (NA) of an optical system is a dimensionless number that characterizes the range of angles over which the system can accept or emit light. By incorporating index of refraction in its definition, NA has the property that it is constant for a beam as it goes from one material to another, provided there is no refractive power at the interface. The exact definition of the term varies slightly between different areas of optics. Numerical aperture is commonly used in microscopy to describe the acceptance cone of an objective, and in fiber optics, in which it describes the range of angles within which light that is incident on the fiber will be transmitted along it.

Transmission electron microscopy

Transmission electron microscopy is a microscopy technique in which a beam of electrons is transmitted through a specimen to form an image. The specimen is most often an ultrathin section less than 100 nm thick or a suspension on a grid. An image is formed from the interaction of the electrons with the sample as the beam is transmitted through the specimen. The image is then magnified and focused onto an imaging device, such as a fluorescent screen, a layer of photographic film, or a sensor such as a scintillator attached to a charge-coupled device.

Angular resolution or spatial resolution describes the ability of any image-forming device such as an optical or radio telescope, a microscope, a camera, or an eye, to distinguish small details of an object, thereby making it a major determinant of image resolution. In physics and geosciences, the term spatial resolution refers to the precision of a measurement with respect to space.

Microscope slide

A microscope slide is a thin flat piece of glass, typically 75 by 26 mm and about 1 mm thick, used to hold objects for examination under a microscope. Typically the object is mounted (secured) on the slide, and then both are inserted together in the microscope for viewing. This arrangement allows several slide-mounted objects to be quickly inserted and removed from the microscope, labeled, transported, and stored in appropriate slide cases or folders etc.

Ernst Abbe German physicist

Ernst Karl Abbe HonFRMS was a German physicist, optical scientist, entrepreneur, and social reformer. Together with Otto Schott and Carl Zeiss, he laid the foundation of modern optics. Abbe developed numerous optical instruments. He was a co-owner of Carl Zeiss AG, a German manufacturer of research microscopes, astronomical telescopes, planetariums and other optical systems.

Magnification process of enlarging something only in appearance, not in physical size

Magnification is the process of enlarging the apparent size, not physical size, of something. This enlargement is quantified by a calculated number also called "magnification". When this number is less than one, it refers to a reduction in size, sometimes called minification or de-magnification.

Eyepiece type of lens attached to a variety of optical devices such as telescopes and microscopes; usually the lens that is closest to the eye when someone looks through the device, placed near the focal point of the objective to magnify the image

An eyepiece, or ocular lens, is a type of lens that is attached to a variety of optical devices such as telescopes and microscopes. It is so named because it is usually the lens that is closest to the eye when someone looks through the device. The objective lens or mirror collects light and brings it to focus creating an image. The eyepiece is placed near the focal point of the objective to magnify this image. The amount of magnification depends on the focal length of the eyepiece.

A solid immersion lens (SIL) has higher magnification and higher numerical aperture than common lenses by filling the object space with a high-refractive-index solid material. SIL was originally developed for enhancing the spatial resolution of optical microscopy. There are two types of SIL:

Bright-field microscopy

Bright-field microscopy is the simplest of all the optical microscopy illumination techniques. Sample illumination is transmitted white light, and contrast in the sample is caused by attenuation of the transmitted light in dense areas of the sample. Bright-field microscopy is the simplest of a range of techniques used for illumination of samples in light microscopes, and its simplicity makes it a popular technique. The typical appearance of a bright-field microscopy image is a dark sample on a bright background, hence the name.

Oil immersion

In light microscopy, oil immersion is a technique used to increase the resolving power of a microscope. This is achieved by immersing both the objective lens and the specimen in a transparent oil of high refractive index, thereby increasing the numerical aperture of the objective lens.

Stereo microscope

The stereo, stereoscopic or dissecting microscope is an optical microscope variant designed for low magnification observation of a sample, typically using light reflected from the surface of an object rather than transmitted through it. The instrument uses two separate optical paths with two objectives and eyepieces to provide slightly different viewing angles to the left and right eyes. This arrangement produces a three-dimensional visualization of the sample being examined. Stereomicroscopy overlaps macrophotography for recording and examining solid samples with complex surface topography, where a three-dimensional view is needed for analyzing the detail.

Optical sectioning

Optical sectioning is the process by which a suitably designed microscope can produce clear images of focal planes deep within a thick sample. This is used to reduce the need for thin sectioning using instruments such as the microtome. Many different techniques for optical sectioning are used and several microscopy techniques are specifically designed to improve the quality of optical sectioning.

PSF Lab microscope software

PSF Lab is a software program that allows the calculation of the illumination point spread function (PSF) of a confocal microscope under various imaging conditions. The calculation of the electric field vectors is based on a rigorous, vectorial model that takes polarization effects in the near-focus region and high numerical aperture microscope objectives into account.

A condenser is an optical lens which renders a divergent beam from a point source into a parallel or converging beam to illuminate an object.

Live cell imaging

Live cell imaging is the study of living cells using time-lapse microscopy. It is used by scientists to obtain a better understanding of biological function through the study of cellular dynamics. Live cell imaging was pioneered in first decade of the 20th century. One of the first time-lapse microcinematographic films of cells ever made was made by Julius Ries, showing the fertilization and development of the sea urchin egg. Since then, several microscopy methods have been developed which allow researchers to study living cells in greater detail with less effort. A newer type of imaging utilizing quantum dots have been used as they are shown to be more stable. The development of holotomographic microscopy has disregarded phototoxicity and other staining-derived disadvantages by implementing digital staining based on cells’ refractive index.