Basic Microscopy

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.

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.

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.

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.

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.

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.

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-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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 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.