Microscopy

This graphic represents the common arrangement for an inverted microscope. Samples are placed on the stage located above the objective and viewed through the eyepieces on the right. For transmitted light imaging, light from a bulb mounted on top of the microscope is projected onto the sample by the condenser. For fluorescence imaging, an epi-fluorescence light source, mounted on the back of the microscope, illuminates the sample from below through the objective.

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Microscopy Center Internal
Foundations of Microscopy Seminars
View Presentation: Building Blocks of a Microscope
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Objectives: Molecular Expressions
Infinity-Corrected Optical Systems: Molecular Expressions
Eyepieces: Molecular Expressions

In front of the eyepieces a single optical path is split to direct light to both eyes. The sample's image is magnified by the objective (19) and the eyepiece (2). Modern microscopes use a tube lens (7) to compensate for the infinity space created directly behind an infinity corrected objective, which allows for the formation of an image. Within the infinity space, additional optical components can be added. For example, reflector cubes (9) for fluorescence imaging, polarizers, and DIC prisms. Further discussion of the tube lens and infinity optics can be found in the following sections on this page. Because an inverted light microscope has a longer distance between the objective and eyepieces, relay optics are used to accomodate this difference by transferring images to the eyepieces.

Additional mirrors and prisms can be used to direct light to different ports on the microscope. This allows for viewing the sample with different detection schemes, such as multiple cameras or a confocal attachment, on the same system.

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Microscopy Center Internal
Foundations of Microscopy Seminars
View Presentation: Building Blocks of a Microscope
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Objectives: Molecular Expressions
Infinity-Corrected Optical Systems: Molecular Expressions
Eyepieces: Molecular Expressions

Image formation of a specimen is achieved by bringing the sample into the focal point of the objective front lens. Because light rays originating from a single spot of the focal plane are parallel after passing the objective, they converge in infinity. Thus, the space between the objective and tube lens is referred to as the "infinity space". The advantage of using an infinity-corrected optical system is that additional components can be introduced into this space without effecting focus or abberation correction.

A tube lens is used in almost all modern light microscopes to recombine the parallel beams in the infinity space and produce an intermediate image in a plane conjugate to the sample. To capture this image an additional lens, such as the human eye, is needed. The visualized image, referred to as a virtual image, appears to reside within the microscope as an inverted and magnified representation of the sample.

Stowers Links:
Microscopy Center Internal
Foundations of Microscopy Seminars
View Presentation: Building Blocks of a Microscope
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Objectives: Molecular Expressions
Infinity-Corrected Optical Systems: Molecular Expressions
Eyepieces: Molecular Expressions

As mentioned above, an infinity-corrected optical system relies on the tube lens to form an image from parallel light rays that exit the back aperature of the objective. Because the tube lens is typically not accessible for adjustment, its focal length and distance from the eyepieces can be considered fixed. This means that the creation of an intermediate image is highly dependent on correct placement of the tube lens in relationship to the objective.

Depending on the manufacturer, correction for optical abberations are made in the objective, tube lens, or both.

Stowers Links:
Microscopy Center Internal
Foundations of Microscopy Seminars
View Presentation: Building Blocks of a Microscope
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Objectives: Molecular Expressions
Infinity-Corrected Optical Systems: Molecular Expressions
Tube Lens Focal Length: Microscopy U

The eyepiece magnifies the intermediate image formed by the objective and the tube lens. A typical magnification of an eyepiece is 10 times.

The overall magnification of the microscope is the magnification of the objective - tube lens combination times the magnification of the eyepiece. E.g. when using a 63x objective together with a 10x eyepiece the overall magnification would be 630x. Thus a 10µm cell would appear as if it is 6.3mm in size.

The intermediate image formed by objective and tube lens is projected into the space between the eyepiece lens and its focal plane. Thus the eyepiece forms a virtual image that appears to be 25cm away from the observer.

The physical location of the intermediate image is typically marked by an aperture inside the eyepiece. Because this position is a conjugate plane to the sample plane, cross-hairs or micrometer scales mounted here appear in focus with the sample.

Stowers Links:
Microscopy Center Internal
Foundations of Microscopy Seminars
View Presentation: Building Blocks of a Microscope
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Optical Microscopy Primer: Eyepieces (Oculars)

For transmitted light illumination the sample is placed between the light source, typically a tungste-halogen bulb, and objective. Light from the bulb is passed through the field stop and focused by the microscope's condenser onto the sample where it is altered in a manner dependent on the refractive properties of the specimen. Light scattered from the sample is then collected by the objective and optically relayed through the tube lens to a desired detector. For transmitted light imaging it is important to always properly adjust the microscope for Köhler Illumination which is discussed in further detail on this page.

Several methods can be used to enhance the contrast of images acquired with transmitted light. These methods require the placement of extra other optical components into the light path. Examples of these methods include Differential Interference Contrast (DIC), Phase Contrast, and Darkfield microscopy.

Stowers Links:
Microscopy Center Internal
Foundations of Microscopy Seminar
View Presentation: Building Blocks of a Microscope
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View Presentation: Transmitted Light Microscopy
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Microscope Illumination: Molecular Expressions
Transmitted Light Illumination: Molecular Expressions

The resolution of an optical microscope is limited by the numerical aperture (N.A.) of the objective used, the illumination, the wavelength of the light and the contrast method.

Using a magnification beyond the resolution limit of a microscope creates empty magnification and should be avoided.

Cameras and other detectors should match the resolution of the optical system.

Stowers Links:
Microscopy Center Internal
Foundations of Microscopy Seminar
View Presentation: Image Formation
Download Presentation: Image Formation
View Presentation: The Objective
Download Presentation: The Objective

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Optical Microscopy Primer: The Concept of Magnification
Optical Microscopy Primer: Numerical Aperture and Resolution
Institut Pasteur - Plate-Forme Imagerie Dynamique (PFID): Microscope Resolution Calculator

Köhler Illumination is the recommended way to adjust the condenser of a modern bright field microscope. It creates a uniformly illuminated field of view and optimizes the balance between contrast and resolution by placing the lamp filament and sample in separate conjugate planes.  Because the filament and field stop diaphragm are physically fixed in opposite planes, simultaneously focusing on the sample and field stop ensures that filament will not introduce any unwanted artifacts in the resulting image.  The condenser centering screws are then used to center the field stop aperture with the image to prevent an illumination gradient from creating shadows that disrupt the field of view.  The procedure for configuring a microscope for Köhler Illumination is detailed in the steps below.  

1. Focus on the sample
2. Close the field stop diaphragm until you can see the edges
3. Adjust the height of the condenser until the edges of the field stop   diaphragm are in focus
4. With the diaphragm still visible, center the image using the condenser centering screws
5. Open the field stop diaphragm until it is just outside the field of view
6. Adjust the numerical aperture to 75% of the numerical aperture of the objective
7. When changing objectives small adjustments to all of the steps listed above are required to properly maintain Köhler Illumination

Stowers Links:
Microscopy Center Internal
Foundations of Microscopy Seminars
View Presentation: Transmitted Light Microscopy
Download Presentation: Transmitted Light Microscopy

Web Links:
Köhler Illumination: Molecular Expressions
Microscopy from the Very Beginning: Zeiss

Literature:
Setting-Up Köhler Illumination: Zeiss