Microscopy

In the inverted microscope shown, the sample sits on the stage in the center of the microscope. It is viewed through the eyepieces on the right and the objective below the sample.

The illumination is a critical part of the image formation. For transmitted light the sample is illuminated from above through the condenser. For fluorescence imaging the epi-fluorescence illumination is used. The light source is mounted on the left side of the microscope and the sample is illuminated through the objective.

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Seminars: Foundations of Microscopy

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Optical Microscopy Primer: Microscope Optical Components

To view the magnified sample by eye, it is imaged and magnified by the objective (19) and the eyepiece (2). The tube lens (7) can be considered part of the objective. Modern microscopes use a tube lens to create an infinity space between objective and tube lens.

Within the infinity space additional optical components can be added, like reflector cubes (9) for fluorescence imaging, polarizers and DIC prism.

Additional mirrors and prisms are used to direct the light to different ports if the sample is imaged using a camera or additional optical equipment is attached (e.g. confocal microscope).

In an inverted light microscope, relay optics transfer the image to the eyepieces and accommodate for the longer distance between objective and eyepieces compared to an upright microscope.

In front of the eyepieces the single optical path is split into the two paths for the two eyepieces.

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Imaging
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Seminars: Foundations of Microscopy

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Optical Microscopy Primer: Microscope Objectives
Optical Microscopy Primer: Infinity-Corrected Optical System
Optical Microscopy Primer: Eyepieces (Oculars)

Almost all modern light microscopes use a tube lens to create an infinity space. To focus the image the sample is brought into the focal point of the objective.

Light rays created in one spot of the focal plane of a lens are parallel after passing the lens. They will converge in infinity, thus the name infinity space (the maximum size of the space between objective and tube lens is restricted).

To form an image, the parallel beams have to be captured by the tube lens. The plane where the intermitted image is formed in the microscope is called the conjugated plane in respect to the sample plane. The objective and the tube lens together provide the first magnification step of the microscope (typical values are between 5x and 100x).

The eyepiece adds an additional magnification step. The intermittent image formed by objective and tube lens is projected in between the lens and the focal plane of the eyepiece. Thus the eyepiece forms a magnified virtual image. When looking at the image it appears to reside inside the microscope. To see it one needs the additional lens of the eye. A screen or camera without additional optics placed after the eyepiece would not be able to capture the image.

The retina of the eye forms another conjugated plane in respect to the sample and the intermitted image.

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Imaging
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Seminars: Foundations of Microscopy

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Optical Microscopy Primer: Infinity-Corrected Optical System

In microscopes with infinity optics the sample is located in front of the objective front lens, in the focal plane of the whole lens assembly. In this case the focal plane coincides with the working distance of the objective. The back focal plane of the objective is typically indicated by an aperture at the back end of the objective.

The tube lens is inside the microscope body. Normally users can not access it. Sometimes it can be exchanged to add additional magnification or correction for special equipment like confocal microscopes.

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Imaging
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Seminars: Foundations of Microscopy

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Optical Microscopy Primer: Microscope Objectives

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.

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Imaging
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Optical Microscopy Primer: Eyepieces (Oculars)

For transmitted light illumination the sample is located between light source and objective. Typical imaging modalities are bright-field illumination, DIC (Normarski) and phase contrast.

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View Presentation: Transmitted Light Microscopy
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Optical Microscopy Primer: Microscope Illumination

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.

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Imaging
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Seminars: Foundations of Microscopy

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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

This Excel spreadsheet will help you to calculate the optical resolution in all three dimensions for wide-field and confocal microscopes. You can download the sheet or view it in your web browser (as long your browser supports Microsoft Excel).

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Imaging
Image Center
Seminars: Foundations of Microscopy

View Presentation: Image Formation
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View Presentation: The Objective
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Web Links:
Optical Microscopy Primer: The Concept of Magnification
Optical Microscopy Primer: Numerical Aperture and Resolution
Institut Pasteur - Plate-Forme Imagerie Dynamique (PFID): Microscope Resolution Calculator

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Imaging
Image Center
Seminars: Foundations of Microscopy

View Presentation: Transmitted Light Microscopy
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Microscopy Primer: Köhler Illumination

Literature:
Microscopy from the very beginning (Carl Zeiss)Download PDF
Change a Mercury Burner (Carl Zeiss)Download PDF
Install & Align Your Mercury Bulb (Carl Zeiss)Download PDF
Properly Set-Up Köhler Illumination (Carl Zeiss)Download PDF