Resources

Basic Concepts in Microscopy

Basic Concepts in Microscopy

Modern compound microscopes operate using a dual stage magnifying design that incorporates a primary imaging lens, the objective, coupled to a secondary visualizing lens system known as the eyepiece or ocular mounted at the opposite ends of a body tube. The objective is responsible for primary image formation at varying magnifications, while the eyepiece is used to observe the image created by the objective. Advanced microscopes feature infinity optical systems that project a parallel bundle of wavefronts from the objective rear aperture to a tube or telan lens, which in turn focuses the image at the intermediate image plane in the eyepieces. The microscopist is able to observe a greatly enlarged virtual image of the specimen by peering through the eyepieces. Magnification is determined by multiplying the individual values of the objective and eyepiece. Resolution and contrast in optical microscopy are derived through a number of optical strategies and is strongly coupled to the types of reagents used to prepare the specimen. This section discusses the basic concepts necessary for a complete understanding of microscopy, including objectives, eyepieces, condensers, magnification, numerical aperture, resolution, contrast, and optical aberrations, along with a wide spectrum of additional considerations.

  • Basic Knowledge
    Basic Microscopy

    Introduction to Microscopy

    Microscopes are specialized optical instruments designed to produce magnified images of specimens that are too small to be seen with the naked eye. In addition to complex designs featuring objectives and condensers, microscopes also consists of very simple single-lens instruments that are often hand-held, such as a common magnifying glass.

    more
    Basic Microscopy

    How the Microscope Forms Images

    Optical microscopes belong to a class of instruments that are said to be diffraction limited, meaning that resolution is determined in part by the number of diffraction orders created by the specimen that can be successfully captured by the objective and imaged by the optical system.

    more
    Basic Microscopy

    Numerical Aperture and Resolution

    The numerical aperture of a microscope objective is the measure of its ability to gather light and to resolve fine specimen detail while working at a fixed object (or specimen) distance. Resolution is determined by the number of diffracted wavefront orders captured by the objective.

    more
    Basic Microscopy

    The Point Spread Function

    The ideal point spread function (PSF) is the three-dimensional diffraction pattern of light emitted from an infinitely small point source in the specimen and transmitted to the image plane of a microscope (or other diffraction-limited optical instrument) through a high numerical aperture (NA) objective or lens system.

    more
    Basic Microscopy

    Illumination and the Microscope Optical Train

    The design of an optical microscope must ensure that the light rays are organized and precisely guided through the instrument. Illumination of the specimen is the most important controllable variable in achieving high-quality images in microscopy and digital imaging.

    more
    Basic Microscopy

    Köhler Illumination

    Illumination of the specimen is the most important variable in achieving high-quality images in microscopy and critical photomicrography. Köhler illumination was first introduced in 1893 by August Köhler of the Carl Zeiss corporation as a method of providing the optimum specimen illumination.

    more
  • Basic Techniques
    Basic Microscopy

    Microscope Optical Systems

    Microscope objectives are perhaps the most important components of an optical microscope because they are responsible for primary image formation and play a central role in determining the quality of images that the microscope is capable of producing. Other components include the illumination collector, condenser, and eyepieces.

    more
    Basic Microscopy

    Microscope Objectives

    The most important component of an optical microscope is the microscope objective. Objectives are responsible for primary image formation and play a central role in establishing the quality of images that the microscope is capable of producing.

    more
    Basic Microscopy

    Enhancing Contrast in Optical Microscopy

    The contrast-enhancing techniques for transmitted light microscopy described in this section represent a variety of methods in sample preparation as well as optical tricks that generate intensity changes which are useful for observation and imaging.

    more
    Basic Microscopy

    Fluorescence Microscopy

    Because of the highly sensitive emission profiles, spatial resolution, and high specificity with regards to signal-to-noise and contrast, fluorescence microscopy is rapidly becoming an important tool in genetics and cell biology, and remains at the forefront of biomedical research with the continuous introduction of new techniques.

    more
    Basic Microscopy

    Reflected Light Microscopy

    Reflected light microscopy is often referred to as incident light, epi-illumination, or metallurgical microscopy, and is the method of choice for fluorescence and for imaging specimens that remain opaque even when ground to a thickness of 30 micrometers using conventional contrast-enhancing techniques.

    more
    Basic Microscopy

    Contrast Modes in Reflected Light Microscopy

    In its standard configuration, a typical reflected light microscope is readily equipped to examine amplitude (absorption) specimens using brightfield incident light. Through the addition of auxiliary components, a variety of contrast-enhancing mechanisms can be introduced.

    more
    Basic Microscopy

    Understanding the Digital Image

    This discussion is intended to aid in understanding the basics of light detection, the fundamental properties of digital images, and the criteria relevant to selecting a suitable detector for specific applications.

    more
  • Daily Use
    Basic Microscopy

    Practical Use of the Microscope

    If certain simple guidelines are followed, it will be a short matter of time before a beginner is able to obtain an image of high quality. In fact, you may be surprised at how easy it is to set up the microscope correctly so that it will produce beautiful, sharp images.

    more
    Basic Microscopy

    Microscopy in Everyday Use

    Although conventional microscope design has not necessarily been a problem for short-term use, long-term sessions have in the past created problems for scientists and technicians who used the instruments. Ergonomics is concerned with finding a better fit between people microscopes.

    more
    Basic Microscopy

    Care and Maintenance of the Microscope

    Microscopes often represent a significant investment of funds and are sophisticated optical instruments that require periodic maintenance and cleaning to guarantee successful microscopy and perfect images.

    more
  • History of Microscopy
    Basic Microscopy

    Microscopy: Historical Perspective

    For many centuries, the construction of microscopes and the underpinning optical systems was entirely an issue of exterior cosmetic craftsmanship, with the design of optical components lagging seriously behind advances in the fabrication of microscope bodies and frames.

    more
We use cookies on this site. Cookies are small text files that are stored on your computer by websites. Cookies are widely used and help to optimize the pages that you view. By using this site, you agree to their use. more