Microscopy
Optical microscopes are versatile measurement devices. They are becoming increasingly precise and provide object or structural images of samples that are often smaller than the resolution of the human eye.
Modern microscopy has changed the world in many ways. Modern medicine would be almost unthinkable without the microscopic magnification of cells and other objects.
Microscopy has also improved the bond between research and industry. Microscopes are widely applied in the field of materials research, for example, to analyse the structure and properties of materials. This allows the development of new materials and technologies and enhances productivity.
Each specific area of application requires certain types of microscopes. Monocular, binocular and trinocular microscopes are just a small selection. It is also important to know the right illumination for different microscope models and to achieve perfect images.
Contents
Planachromatic objectives
Planachromatic objectives have the advantage over simpler objectives in that they also correct for field curvature. This results in a sharp image across the entire field of view. Thanks to their consistently sharp images, these objectives are particularly well-suited for microphotography and for examining objects in detail.
Numerical aperture
The numerical aperture (NA) describes the light intensity and resolving power of an objective. It is calculated using half the objective’s angle of aperture (α) and the refractive index of the medium (n) between the front lens and the cover glass (usually air or oil). NA = n – sin α. The higher the numerical aperture value, the better an objective can achieve resolution in the specimen.
condenser
Put simply, a condenser provides homogeneous illumination of the object and ensures that as much light as possible reaches the object from the light source. To achieve the objective’s maximum resolution, the condenser used should have a numerical aperture that is greater than or equal to that of the objective. Contrast, depth of field, and resolution are controlled by the Aperture diaphragm. It is located below the condenser.
Microscope functioning
Stereo microscopes display a separate image of the object in each eye. Both eyes view the specimen from slightly different angles, creating a stereo effect. The human brain combines these two separate images into a unified image with a certain level of depth. This creates an almost three-dimensional appearance.
The large zoom range and wide depth of field make these microscopes ideal for gemstone examination. They are used to observing internal properties and check external characteristics such as damages or the quality of cut. Another area of application is quality control for electronics, precision engineering, and plastic products.
The binocular microscope produces an image with only one objective and makes it visible to both eyes, but it does not allow spatial viewing of the object. Binocular microscopes are ideal for biological studies, especially microscopy of low-contrast objects, e.g. observation of microorganisms or red blood cells, or blood analysis according to Enderlein. It is also used to identify different cell structures or to perform water analysis (hydrobiology) in waters and wastewater treatment plants.
Click here to learn all about image quality and objectives
Optimal magnification with first-class eyepieces and objectives
An eyepiece is closest to the observer’s eye. While the eyepiece is part of what makes up the total magnification of the microscope, it does not have any influence on the resolution. Usually eyepieces have a magnification of 5x, 10x, 15x, 16x, 20x.
The objective provides the magnified, laterally inverted real intermediate image. When selecting objectives, it is often unclear what the terms magnification, Magnification of the objective, field number and resolution mean and what role they play.
Ocular and field of view
In order to be able to look at the image depicted by the objectives in the Magnification of the objective, the image is magnified using an eyepiece. This corresponds to the function of a magnifying glass. In addition, eyepieces are also decisive for the field of view. This is the area of the sample visible under the microscope.
So wird der Wert errechnet: 10 (Sehfelzahl) : 40 (Maßstabszahl) = 0,25 mm. This means that the diameter of the field of view – with the eyepieces and objectives used – is 0.25 mm. The area of the field of view is then around 0.05 mm².
The field of view enables a rough measurement of the observed objects. Wide-field eyepieces are used for a large field of view, because these provide a larger field of view.
Objectives and total magnification
The image quality of a microscope depends mainly on the quality of the objectives. While eyepieces provide magnification, objectives do not magnify the image every time. Rather, objectives image the object at a scale. For example, an objective with a magnification of the objective of 40 produces an image of the objective with a magnification of 40:1.
The value is calculated as follows: 10x (eyepiece)∗ 40x (objective) = 400x microscope magnification.
The special plan-achromatic objectives have the advantage over simpler objectives that the image field curvature is also eliminated. This results in a sharp image across the entire field of view.
The resolution, the magnification of the objective, the numerical aperture (NA) and the condenser
The resolution of a microscope is determined by the objectives used. The resolution means how large the minimum distance between two structures must be in order to recognize these structures separately. The numerical aperture (NA) is decisive here. This results from the aperture angle of the objectives and the refractive index of the surrounding medium.
Also decisive for the resolution is the wavelength (λ) of the light that is used for microscopy. If a white light source is used, a wavelength of λ = 550 nm is used, as this is the value for the highest eye sensitivity.
Objective and magnification of the objective
Several objectives with different magnifications of the objective are generally used for microscopy. These are usually arranged in increasing Magnification of the objective on the revolving nosepiece. As the Magnification of the objective rises, the numerical aperture usually increases, which reduces the focal length. This means that the distance between the objective and the objective decreases as the magnification of the objective increases.
We recommend only using objectives from one manufacturer for microscopy. These are usually parfocally aligned. This means that the focus is virtually maintained when changing objectives and the object hardly needs to be focused.
Reach maximum resolution
To enable the maximum resolution of the objectives, it is necessary that the condenser used has a larger or equal numerical aperture. If, on the other hand, the condenser has a smaller numerical aperture, the resolution (d) accordingly must be calculated. (However, if the condenser has a larger numerical aperture, this does not have to be taken into account in the calculation).
Calculate the value (d = λ / (NA condenser + NA objectives): An objectives 40x NA 0.65 and a condenser NA 1.25 are used. This results in: d = 550 / (2 – 0.65) = 423 nm
The result means that in order to be able to distinguish two structures with the objectives used, they must be separated by at least 423 nm.
The Right Illumination
Illumination is essential for observing the objects. Simply put, there are two types of illumination:
- Transmitted light illumination: In this setup, the light source and the observation optic are located on opposite sides of the object. This allows light to pass through the object or specimen.
- Incident light illumination: In this type of illumination, the object is illuminated from the same side as the observation optic. This enables the examination of objects that are not translucent or it can be used for gemstone examinations.
Using the phase contrast method
The phase contrast device is frequently used with our binocular microscopes. Phase contrast is an optical imaging technique that is used for very thin objects or specimens. These samples show only a very few details or contrasts under bright field observation. Such samples are analysed mainly in histology, forensics and environmental analysis. The phase contrast device is used to highlight phase differences. Because the human eye cannot recognise the phase or phase differences.
Construction: The phase contrast device consists of a special objective and condenser, each of which features a so-called phase ring. These phase rings can be superimposed by positioning the condenser. To ensure that they are matching, the objective lens and the condenser should be from the same manufacturer. The phase rings allow to visualise the phase differences, but it is to be noted that the light intensity is reduced. For this reason, a light source with high light intensity is necessary. See the video “How to use” for a demonstration of the process in practice.
See video phase contrast method
Differences between brightfield, dark-field, and Phase contrast
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Light sources for microscopy
Various light sources are used for microscopy. Halogen lamps or LEDs are generally used. For simple microscopes, the ambient light can be focussed with the help of mirrors. With freely positionable lighting sources, such as ring lights or cold light sources, additional illumination can be provided from the side.
Benefit: High light efficiency, low cost, ideal for transmitted light microscopy
Click hereLED illumination
Advantage: High light intensity, suitable for heat-sensitive samples, ideal for side illumination.
Click hereGooseneck light guide illumination
Benefit: Precise, flexible illumination, very little infrared radiation, ideal for thermosensitive samples and gemmology
Click here
Typical areas of application
Microscopes are used in many industries
The application area takes on an important part when buying a microscope. After all, what really matters is that the microscope you purchase is suitable for your area of application. Our microscopes are designed for medical and biological applications in laboratories, industry, and research. They are also used for diagnostic purposes, quality control, materials testing, and education. We also offer a metallurgical incident light microscope, for example, for identifying and analyzing steel joints and other metals, or for quality assessment. We also have many high-quality gemstone microscopes for gemmological examinations.
Find out more: In our table we have created an overview of our microscopes with the areas of application we recommend.
microscopes areas of application
Cleaning tips for optical components
The accessible optical surfaces (front lenses, back lenses of the eyepiece, front lenses of the condenser) normally should be cleaned with a mild cleaning agent.
- Suitable cleaning agents are optical cleaning tissue or a white linen cloth (both lint-free).
- You can also use a wooden stick wrapped in medical cotton wool.
- Light moistening with distilled water may be helpful for cleaning. Always clean in a circular motion from the centre to the edges. Lint and dust can be removed with a blower, which is available in camera shops.
- For persistent dirt or grease, you can occasionally use medical benzine. It has the benefit of being easily evaporated and will therefore not be absorbed into gaps or joints. The coating is undamaged due to the short exposure time of the agent.
- The supplied dust cover prevents the instrument from becoming dusty when not in use.
How to Use
Working with KRÜSS Optronic microscopes – tips from the professionals
We offer high-quality microscopes that are known for their excellent optical quality. We offer customisable laboratory microscopes. It is possible to connect a camera to a trinocular microscope for image and film recording. In addition, other models are suitable for a wide range of applications in teaching, research, and training, as well as in the fields of biology, histology, forensic science, and materials testing.
Using KRÜSS Optronic microscopes
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