Homepage - Microscopy from the very beginning
Transmitted light - bright field
In practical microscopy, you do not always have nicely stained samples which are easy to view in simple brightfield. Unstained samples, such as bacteria or living cell cultures, absorb practically no light and are barely or not at all visible in brightfield, even in a well-aligned microscope.
It is now taken for granted that everyday microscopy always provides perfect images. However, this has not always been the case. For several centuries, the construction of microscopes and the appropriate optics was purely a matter of craftsmanship.
Photomicrography.
If you want to or have to document microscope images, the easiest way is to hold a standard camera to an eyepiece and to release the exposure. This will actually work, since the design of the camera is identical to that of the eye.
The Objectives.
There are hundreds of problems which can be solved by using the light microscope. In addition, the microscope also has to meet a number of demands made by the users regarding performance and purchase prices.
Illumination in the transmitted light
The design of a microscope must ensure that the light rays are precisely guided through the microscope. Only this will make it possible to obtain a bright image even with illuminators of a low wattage.
IIn spite of all technical progress, the eye as the visual organ – in combination with the brain behind it – is the most efficient image-processing system available to date. All the appliances provided by technology are no match for the eye as regards speed and resolution.
IIn spite of all technical progress, the eye as the visual organ – in combination with the brain behind it – is the most efficient image-processing system available to date. All the appliances provided by technology are no match for the eye as regards speed and resolution.
It is now taken for granted that everyday microscopy always provides perfect images. However, this has not always been the case. For several centuries, the construction of microscopes and the appropriate optics was purely a matter of craftsmanship.
Photomicrography.
If you want to or have to document microscope images, the easiest way is to hold a standard camera to an eyepiece and to release the exposure. This will actually work, since the design of the camera is identical to that of the eye.
Basic adjustments in the transmitted light
The procedure we choose will always lead to success. What we have just learned in theory will now be put into practice – following Köhler's principles. Everything that applies to the Axiolab microscope is also applicable to any other microscope model, provided it is fully equipped for Köhler illumination.
The Objectives.
There are hundreds of problems which can be solved by using the light microscope. In addition, the microscope also has to meet a number of demands made by the users regarding performance and purchase prices.
Confocal fluorescence microscopy
This method is only possible with very special microscopes, i.e. the confocal microscopes. This method will be mentioned briefly here because it considerably enriches microscopy.
Digital camera solutions are being relied upon more and more frequently – the high sensitivity of camera sensors and the quick availability of the images are only two reasons for this new trend.
For images of size: the eyepieces
Eyepieces (or oculars, from the Latin “oculus” = the eye) are the magnifiers with which you view the intermediate image in the microscope, produced by the objective and the tube lens.
Fluorescence
Illumination in reflected light
Anyone working with metal, ceramics or other technical samples will use a reflected-light microscope in most cases, since such samples are opaque to light and normally allow only the surface to be examined.
Illumination in the transmitted light
The design of a microscope must ensure that the light rays are precisely guided through the microscope. Only this will make it possible to obtain a bright image even with illuminators of a low wattage.
In the 19th century, the precise natural sciences experienced an enormous upswing. Even in the twenties and thirties, the science of light and the theory of optical imaging were placed on a sound foundation.
What does “useful magnification” mean?
“A lot helps a lot” – however, this does not apply to the selection of the “useful” magnification. By this we mean that you should not try to increase the overall magnification of a microscope by using eyepieces providing a high additional magnification (e.g. 16x) or other optical “after-burners” if the objective does not supply enough pixels at a low numerical aperture.
If one lens is not sufficient, several lenses can be arranged one behind the other. The magnifying effect is thus multiplied, allowing magnifications of up to 2000x. The classic microscope magnifies in two steps: The objective produces a magnified image of the object in the so-called intermediate image plane, and the eyepiece or ocular (Latin: oculus = eye) magnifies the intermediate image in the same way as a magnifier.
The Objectives.
There are hundreds of problems which can be solved by using the light microscope. In addition, the microscope also has to meet a number of demands made by the users regarding performance and purchase prices.
The Objectives.
There are hundreds of problems which can be solved by using the light microscope. In addition, the microscope also has to meet a number of demands made by the users regarding performance and purchase prices.
Resolution and Numerical Aperture
White light consists of electromagnetic waves, the period lengths of which total 400 to 700 nm.
Are the objective and the specimen clean?
A fingerprint on the front lens of an air objective alone may be sufficient to affect the high-contrast reproduction of a specimen since scattered light is produced. The same applies to immersion objectives soiled with residues of resin or emulsions (e.g. oil and water). Such cases require careful cleaning using a soft cloth and pure alcohol.
Reflected light methods
Transmitted light methods
Videomicroscopy
About 30 years ago, researchers tried to replace the heavy, slightly whirring 16 mm cine cameras by TV cameras – then also rather heavy. The movement of tiny organisms could now be shown “live” to many spectators.
View by the eyepiece
If you are a “beginner” in microscopy, you may tend to tense up when viewing. You think you have to set your eyes for near vision because, after all, you want to view something small. This is not the correct attitude in microscopy and may cause you strain in the long run – and you do not even know the cause.
It is no exaggeration to say that almost the entire art of microscopy – if specimen preparation is not taken into account – consists in the correct use of the luminous field and aperture diaphragms.
Function: to display color, this technique uses a separate CCD chip for each primary color.
4D – the wavelengths: cell or tissue components can be marked selectively with different fluorescence dyes
5D – the time: if living cells are to be observed over a long period of time, single images can be repeated at set points in time.
6D – different specimen spots at the same time: fully motorized stages permit the fast positioning of the microscope in all three spatial axes.
Auflicht - Fluoreszenzmikroskopie
In der Fluoreszenzmikroskopie werden die Präparate mit speziellen Reagenzien behandelt. Deren einzelne Moleküle sind in der Lage, Licht für eine extrem kurze Zeit - üblich sind milliardstel Sekunden - aufzunehmen und dann wieder abzustrahlen.
Adaptation at the microscope.
If camera and microscope are perfectly matched to each other, the benefits of digital microscopy become evident. In the following, we will inform you of the major criteria for an ideal digital micrograph.
Auflösung - resolution
Now hold the paper strip between the sample and the objective. Fully open the aperture diaphragm of the condenser.
The sturdy design of our microscopes will even excuse incorrect treatment
Auflicht - Fluoreszenzmikroskopie
In der Fluoreszenzmikroskopie werden die Präparate mit speziellen Reagenzien behandelt. Deren einzelne Moleküle sind in der Lage, Licht für eine extrem kurze Zeit - üblich sind milliardstel Sekunden - aufzunehmen und dann wieder abzustrahlen.
The resolving power of the microscope is judged by the limit up to which two small objects are still recognized as being separate.
Black-and-white cameras.
Although digital color photos feature a high information content and are very appealing, the use of black-and-white cameras is nevertheless recommended in many cases. Unlike the color sensors, gray level display of an object does not require a color filter mask to be inserted between the light source and the sensor pixel array.
Transmitted light - bright field
In practical microscopy, you do not always have nicely stained samples which are easy to view in simple brightfield. Unstained samples, such as bacteria or living cell cultures, absorb practically no light and are barely or not at all visible in brightfield, even in a well-aligned microscope.
Reflected light - Brightfield
For images of size: the eyepieces
Eyepieces (or oculars, from the Latin “oculus” = the eye) are the magnifiers with which you view the intermediate image in the microscope, produced by the objective and the tube lens.
CCD sensors are available in different sizes and with different basic resolution parameters.
An analog/digital converter integrated in the camera converts the output voltage of the output amplifier into a digital number.
Our microscope image is now slowly taking shape.
Insert the eyepiece again and look into the microscope.
A further important reason for the existence of diaphragms and filters in the microscope is that, strictly speaking, the illumination should be reset after every change of objectives for two reasons.
Color imaging
Since the described method does not provide any information about the color of the pixel concerned, but only about its degree of brightness, various techniques are used to obtain a color image.
Comparison between cameras using film and digital cameras
This is where Köhler illumination really begins: we narrow the luminous-field diaphragm and move the condenser carefully up and down via the condenser drive until we see a sharp image of the luminous-field diaphragm, or at least a piece of it in the edge.
The height of the condenser can be adjusted via the condenser drive.
Now you almost have a good microscope image. Only the contrast remains to be improved.
In the reflected-light microscope, the condenser function is mainly performed by the objective
Transmitted light - darkfield
Fine structures can often not be seen in front of a bright background. This situation changes if the structures are illuminated from the side and viewed in front of as dark a background as possible. The structures then really seem to light up.
Reflected light - Darkfield (DF)
This method is ideal for the inspection of surfaces.
Design and function of a CCD sensor
A CCD (charge-coupled device) sensor as used in digital cameras consists of a rectangular surface made of silicon semiconductor material, on which thousands of small, light-sensitive sensor elements (pixels) are arranged in regular patterns of rows and columns.
The ultimate in sophistication: Differential Interference Contrast (DIC) in transmitted light.
This highly efficient contrasting technique is based on the Pol contrast technique as far as the components used are concerned.
Reflected light - Differential Interference Contrast (DIC)
As an extension of polarization contrast, this method also allows the visualization of minute elevation differences in surfaces.
This accessory for the demanding user contains two reticles which are laterally displaced with respect to each other by a sensitive micrometer screw.
Microscopes are used for an average of 15 years or longer.
The number of levels between the charge which is only just detectable and the full charge of the electronic image sensor is called dynamic range and is specified in decibels.
In TV cameras, the brightness information of an image is changed to an electric signal which can be influenced by means of amplifiers.
Reflected light - fluorescence microscopy
In fluorescence microscopy, the specimens are treated with special reagents. Their individual molecules are able to absorb light for an extremely short time – usually billionths of a second – and then to emit it again.
Optimum use of the brightness differences contained in the image and an ideal signal-to-noise ratio require the brightness range which can be recorded by the electronic sensor to be controlled as well as possible.
Eyeglass wearers requiring simple lenses with a spherical power can use the microscope with or without their glasses, provided that the diopter setting of the (“foc”) eyepiece is sufficient.
Features of digital cameras
Resolution: the resolving power of the microscope is judged by the limit up to which two small objects are still recognized as being separate. Referred to the CCD sensor, resolution is defined by the number of pixels available.
Function: the color filters are not positioned on the pixel structure, but on a filter wheel in front of the recording sensor.
Reflected light - fluorescence microscopy
In fluorescence microscopy, the specimens are treated with special reagents. Their individual molecules are able to absorb light for an extremely short time – usually billionths of a second – and then to emit it again.
Reflected light - fluorescence microscopy
In fluorescence microscopy, the specimens are treated with special reagents. Their individual molecules are able to absorb light for an extremely short time – usually billionths of a second – and then to emit it again.
Reflected light - fluorescence microscopy
In fluorescence microscopy, the specimens are treated with special reagents. Their individual molecules are able to absorb light for an extremely short time – usually billionths of a second – and then to emit it again.
How an electronic image is created.
When analog film material is exposed, a chemical reaction blackens the silver halogenides on the film surface. A color image is then created through chemical interaction with various dyes during development of the film.
The parfocal length, i.e. the distance between the specimen plane and the screw-on surface of the objective nosepiece, is always 45 mm.
As in chemical microphotography, correct exposure conditions are of major importance in digital image recording as well.
Increasing Resolution
Increasing Resolution
Light should now already be discernable in the eyepieces. If it is very bright, reduce the brightness until you find it comfortable to work with.
Microscope eyepieces from Carl Zeiss have usually been designed for eyeglass wearers.
The enterprise continued to evolve. Ernst Abbe became an equal partner of Zeiss: intelligence became the inherent capital of the young company.
Microscope
ray gear
As you have seen, eyepieces have been designed in such a way that the intermediate image of the microscope is located inside them.
Köhler
Open the luminous-field diaphragm as far as it will go. The spot of light on the paper is now at its maximum diameter.
For images of size: the eyepieces
Eyepieces (or oculars, from the Latin “oculus” = the eye) are the magnifiers with which you view the intermediate image in the microscope, produced by the objective and the tube lens.
Function: this technique uses a color CCD with a conventional Bayer mask. Through precise position changes of the CCD by the size of the pixels (a few µm)
The problems just mentioned will not occur with microscope cameras in the MC series from Carl Zeiss.
New recording dimensions through software-controlled camera systems
The easy control of digital camera systems via image recording software installed on the connected PC combined with a motorized microscope permits entirely new image types which can be easily produced at the push of a button.
The numerical aperture of objectives increases with the magnification, up to about the 40x objective.
Objectives for reflected light can be recognized by the “Epi” inscription.
The entire optics are computed in such a way that aperture angles of the light cones are correctly set together with the aperture diaphragm
The overall magnification of the microscope is easy to calculate
Transmitted light - phase contrast
This method described by the Dutchman Frits Zernike in 1934 not only earned its discoverer the Nobel prize for physics in 1953, but also revolutionized biomedical basic research of living – i.e. unstained – cells.
Transmitted light - phase contrast
This method described by the Dutchman Frits Zernike in 1934 not only earned its discoverer the Nobel prize for physics in 1953, but also revolutionized biomedical basic research of living – i.e. unstained – cells.
Transmitted light - polarization contrast
In this method, polarized light is used; it consists of light waves which all feature the same direction of vibration, i.e. which are linearly polarized. This very “ordered” light is generated by polarizers which filter out a privileged plane from the statistical confusion of vibration directions prevailing in natural light.
Reflected light - polarization contrast (POL)
Suitable for surfaces with structures which change the state of polarization during reflection, e.g. structure grains in samples of ore.
If you do not need glasses or wear your eyeglasses for microscopy, always use one of the eyepieces without focusing (or a foc. eyepiece in the “0” position).
Electronic image sensors can be extremely sensitive. If the CCD stechnology is combined with efficient residual light amplifiers, even single photons can be detected.
Even if you are good with your hands, we must strongly advise you against performing your own repairs on the microscope.
The marking techniques in modern fluorescence microscopy are becoming increasingly specific.
Like the microscopist looking through the eyepiece, the camera also only sees an image of the microscopic specimen.
Resolution and Numerical Aperture
White light consists of electromagnetic waves, the period lengths of which total 400 to 700 nm.
Now look into the microscope and carefully move the stage, including the sample, up and down until you see the details as sharply as possible
When a CCD sensor is used, the quality of the output signal is not only influenced by the charge produced by incident light, but also by parasitic currents (dark noise).
An SLR camera can be attached to the camera connector mentioned above via an adapter including a built-in objective.
To help him in this endeavor, Carl Zeiss found the 26-year-old, highly talented physicist and mathematician Dr. Ernst Abbe.
A remedy has been known for such cases for centuries: the use of a “magnifying glass” which – when put between the eye and the object – makes everything appear larger
For videomicroscopy, adapters with a fixed (reducing) factor are normally used.
If you want to vary the image section on the monitor or the video print, the TV zoom adapter is the method to choose.
Unlike the dynamic range of a CCD, the useful brightness range describes the ratio of minimum and maximum brightness required by the sensor to produce an interpretable signal
Transmitted light - VAREL contrast
A new contrasting technique, in which phase contrast and inclined unilateral illumination are mixed, has been developed for the examination of living cells in culture vessels.
TV images from the microscope sometimes appear to be enormous on the monitor.
Since we want to have a closer look at the fine capillaries of the stalk of a plant, we cut a wafer-thin slice from the stalk, place it on a microscope slide (glass plate) and protect the sensitive object using a cover slip.
The progress made in the development of objectives led to fields of view larger than anything achieved before.