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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Optical Pathways in the Transmitted Light Microscope - The design of an optical microscope must ensure that the light rays are organized and precisely guided through the instrument. This interactive tutorial explores the function of the field and condenser aperture diaphragms of a transmitted light microscope.
Microscope Alignment for Köhler Illumination - Illumination of the specimen is the most important variable in achieving high-quality images in microscopy and critical photomicrography or digital imaging. This interactive tutorial explores how to establish Köhler illumination on a transmitted light microscope.
Objective Specifications - Microscope objectives are precision optical systems that feature a wide range of magnifications, numerical aperture, immersion media, specialized contrast applications, and other properties. This interactive tutorial examines the specifications found on typical objectives.
The Concept of Magnification - A simple microscope or magnifying glass (lens) produces an image of the specimen upon which the microscope or magnifying glass is focused. This interactive tutorial explores how a simple magnifying lens operates to create a virtual image of the specimen on the retina of the human eye.
Microscope Conjugate Planes - The conjugate planes critical for establishing proper illumination in the microscope are examined in this interactive tutorial. Four conjugate planes can be brought simultaneously into focus: the field diaphragm, the specimen plane, the intermediate image plane (where the reticule is positioned), and the human eye.
Fixed Tube Length Microscope Conjugate Field Planes - The geometrical relationship between image planes in the traditional fixed tube length (usually 160 millimeters) optical microscope is explored in this tutorial. In most of the imaging steps in the microscope optical train, the image is real and inverted, but a virtual image is also produced in one of the image planes.
Infinity Corrected Microscope Conjugate Field Planes - A majority of modern research microscopes are equipped with infinity-corrected objectives that no longer project the intermediate image directly into the intermediate image plane. Light emerging from these objectives is instead focused to infinity, and a second lens, known as a tube lens, forms the image at its focal plane.
Infinity Optical System Basics - Infinity-corrected microscope optical systems are designed to enable the insertion of auxiliary optical devices into the optical pathway between the objective and eyepieces without introducing spherical aberration, requiring focus corrections, or creating other image problems.
Field Iris Diaphragm Function - When the microscope is properly configured for Köhler illumination, the field diaphragm is imaged in the same conjugate plane as the specimen, and in fact, all of the image-forming conjugate planes are simultaneously imaged into each other and can collectively be observed while examining a specimen in the eyepieces.
Numerical Aperture and Light Cone Geometry - The light-gathering ability of a microscope objective is expressed in terms of the numerical aperture, which is a measure of the number of highly diffracted image-forming light rays captured by the objective. This interactive tutorial explores the effect of numerical aperture on light cone geometry.
Airy Disk Formation - When an image is formed in the focused image plane of an optical microscope, every point in the specimen is represented by an Airy diffraction pattern having a finite spread. This interactive tutorial explores the origin of Airy diffraction patterns formed by the rear aperture of the microscope objective and observed at the intermediate image plane.
Spatial Frequency and Image Resolution - When a line grating is imaged in the microscope, a series of conoscopic images representing the condenser iris opening can be seen at the objective rear focal plane. This tutorial explores the relationship between the distance separating these iris opening images and the periodic spacing (spatial frequency) of lines in the grating.
Conoscopic Images of Periodic Structures - This tutorial explores the reciprocal relationship between line spacings in a periodic grid (simulating a specimen) and the separation of the conoscopic image at the objective aperture plane. When the line grating has broad periodic spacings, several images of the condenser iris aperture appear in the objective rear focal plane.
Numerical Aperture and Image Resolution - The image formed by an objective at the intermediate image plane of a microscope is a diffraction pattern produced by spherical waves exiting the rear aperture and converging on the focal point. This tutorial explores the effects of objective numerical aperture on the size of Airy disk patterns.
Fundamental Aspects of Airy Disk Patterns - This tutorial explores how Airy disk pattern size changes with objective numerical aperture and the wavelength of illumination. It also simulates the close approach of two Airy patterns as they approach the Rayleigh criterion for determining the ability to resolve two closely spaced objects in the microscope.
Oil Immersion and Refractive Index - One way of increasing the optical resolving power of the microscope is to use immersion liquids between the front lens of the objective and the cover slip. This tutorial explores how changes in the refractive index of the imaging medium can affect how light rays are captured by the objective, which has an arbitrarily fixed angular aperture of 65 degrees.
Condenser Numerical Aperture - The size and numerical aperture of the light cone emitted by a substage condenser is determined by adjustment of the aperture diaphragm. This interactive tutorial examines how changing the aperture iris diaphragm opening size alters the size and angle of the light cone.
Condenser Aperture Diaphragm Function - The size and numerical aperture of the light cone produced by the condenser is determined by adjustment of the aperture diaphragm. Appropriate use of the adjustable aperture iris diaphragm (incorporated into the condenser or just below it) is of significant importance in securing correct illumination, contrast, and depth of field.
Condenser Light Cones - It is critical that the condenser light cone be properly adjusted to optimize the intensity and angle of light entering the objective front lens. Each time the objective is changed, a corresponding adjustment must be performed on the condenser to provide the proper light cone to match the numerical aperture of the new objective.
Coverslip Thickness Correction - High magnification dry objectives are often subject to aberration artifacts due to variations in cover glass thickness and dispersion. This tutorial demonstrates how internal lens elements in such an objective may be adjusted to correct for these fluctuations.
Focus Depth and Spherical Aberration - Explore the three-dimensional aspects of spherical aberration that is generated when imaging deep into specimens using the meridional section of a point spread function with this interactive tutorial. Spherical aberration is a significant problem when imaging specimens in aqueous media.
Inverted Microscope Lightpaths - Microscopes featuring an inverted-style frame are designed primarily for live-cell imaging applications and are capable of producing fluorescence illumination through an episcopic and optical pathway. This interactive tutorial explores illumination pathways in the Zeiss Axio Observer research-level inverted tissue culture microscope.
Reflected Light Microscope Optical Pathways - 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.
Basic Principles in Optical Microscopy - A list of the best textbooks that provide a general knowledge of the principles and practice of optical microscopy, as well as an introduction to contrast-enhancing techniques. The volumes listed here are useful in both the classroom and the research laboratory.
Microscope Optical Systems - The microscope optical train typically consists of an illuminator (including the light source and collector lens), a condenser, specimen, objective, eyepiece, and detector, which is either some form of camera or the observer's eye. These components are often supplemented with contrast-enhancing optical elements.
Specimen Contrast - Control of image contrast in a microscope optical system is dependent upon several factors, primarily the setting of aperture diaphragms, degree of aberration in the optical system, the optical contrast system employed, the type of specimen, and the optical detector.
Phase Contrast Microscopy - Phase contrast was introduced in the 1930's for testing of telescope mirrors, and was adapted by the Carl Zeiss laboratories into a commercial microscope for the first time several years later. The technique provides an excellent method of improving contrast in unstained biological specimens.
Differential Interference Contrast (DIC) Microscopy - Differential interference contrast converts gradients in specimen optical path length into amplitude differences that can be visualized as improved contrast in the resulting image. Images produced in DIC microscopy have a distinctive shadow-cast appearance
Fluorescence Microscopy - The application of fluorescence illumination and detection in optical microscopy has ushered in a wide range of advanced applications for live-cell imaging and in vivo observations. The articles tabulated in this section discuss the basic aspects of fluorescence, microscope configuration, fluorescent probes, software, light sources, detectors, objectives, filter sets, and a variety of other pertinent topics.
Polarized Light Microscopy - The polarized light microscope is designed to observe specimens that are visible due to their birefringent character. Polarizing microscopes must be equipped with a polarizer, positioned in the light path before the specimen, and an analyzer placed in the optical pathway between the objective rear aperture and the observation tubes or camera port.
Microscope Ergonomics - 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. Microscope operators often must assume an unusual and challenging position.