Phasenkontrast [Document] English Translation

[A combination of my knowledge of the German language and Google's. Click images to get a bigger size]
 
Die Bilder sind nicht in allen Einzelheiten für die Ausführung der Geräte maßgebend. Für wissen- schaftliche Veröffentlichungen stellen wir Druckstöcke der Bilder oder Verkleinerungen davon — soweit sie vorhanden sind — gern zur Verfügung. Die Wiedergabe von Bildern oder Text ohne unsere Genehmigung ist nicht gestattet. Das Recht der Übersetzung ist vorbehalten.VEB CARL ZEISS JENA

Abteilung für Mikroskopie Drahtwort: Zeisswerk Jena Fernsprecher 3541

COMMENT:
Phase contrast is a standard feature in modern microscopes and "Prof. Zernike, Groningen, received the Nobel Prize in Physics for his phase contrast method". See the "Afterword" in ths article.
The prize for a modern standard microscope, with the same functionality, is around 1700€
END COMMENT.

Is is the task of microscopy to make the smallest objects and object structures as visible as possible to the eye. For objects that differ from their surroundings in their absorption (so-called amplitude objects), as Abbe has shown, this is always possible if the aperture of the objective is large enough to resolve the object structures. This subheading includes: B. colored histological sections and smears or scattering preparations of diatoms in air. It is different with such objects, which differ from the surroundings only by a different refractive index (so-called phase objects), such as unstained frozen sections or coverslip preparations from living bacteria or from infusory bloating.

The latter remain invisible in the normal bright field image, since their brightness does not differ from the surroundings. This is where the phase contrast method specified by the Dutch physicist Zernike 1) in 1932 and theoretically founded by him begins, which in all its points is based on the consistent application of the Abbe theory of image formation in a microscope to phase objects and first in the Jena Zeisswerk by A. Köhler and W Loos has been introduced into microscopic practice. With the help of this method, the refractive index in the phase object that deviates from the environment is converted into a brightness that deviates from the environment in the image of the phase object. To gain a deeper understanding of the process, the essence of Abbe's theory must first be explained.

Their physical basis is Huygens' principle of light propagation and the diffraction of light derived from it. Assuming a point of light in the front condenser focal plane, the object plane is struck by a parallel light beam and any inhomogeneity in it caused by that of the surroundings [1) see epilogue 3E] deviating absorption or refractive index, creates a Fraunhofer diffraction pattern of the light source in the rear focal plane of the lens. Since their extension is inversely related to the size of the object, it is spread apart for a sufficiently small object, while the diffraction figure created by the sufficiently large limitation of the illuminated field contracts with the geometric image of the light source (Figure 1). 

This corresponds to the direct light unaffected by the object. Abbe describes the entire diffraction phenomenon in the rear focal plane as the primary intermediate image. The actual intermediate image in the image plane (according to Abbe: secondary intermediate image) is then created by superimposing (interference) the light excitation resulting from the two diffraction figures mentioned above. Abbe has also experimentally proven that the diffraction phenomenon in the rear lens focal plane is essential for image formation. He used an amplitude grating (ordinary line grating) as an object for his experiments and was able to show that a suitable intervention in the rear focal plane can give an image that is not object-like. Zernike has used this knowledge to visualize phase objects. The phase contrast method is therefore an object-unlike image in the Abbe's sense. The essence of the process can best be clarified using vector notation.

A vector is a directed quantity, i. 2. It is only clearly defined when the amount and direction are specified. It can be represented by an arrow and broken down into components; it is identified with a German letter. Examples from physics are the velocity v and the force K.4

If you idealize the wave trains emitted by a light source as infinitely extended sine waves, you can represent them in a known manner with the help of an arrow rotating at constant speed, which we want to call the light vector (Figure 2).

Figure 2. Representation of a sine wave 300390/1aT

The state of vibration can therefore be represented at any point and at any time by a light vector. The intensity is then given by the square of the amount

The parallel light beam originating from a point light source in the front focal plane of the condenser corresponds to a plane wave and therefore has the same phase position in the entire object plane; this can be indicated with arrows of the same direction (Figure 1). Provided that no specimen is initially placed, only diffraction at the light beam limit will occur. Since the diameter of the light beam used is always large compared to the light wavelength, the diffraction figure has only a very small extent; the light source is imaged in the rear lens focal plane.

f there is now a small inhomogeneity in the object plane, the direction and length of the light vector have changed behind this point in relation to the homogeneous environment, depending on 5 | r | = ao = Amplitudeφ = phaser angle a = ao · sin φ = Deflection image 3 300 388 / aT light vectors in the object level
whether it is a place with a different refractive index or different absorption. Generally both will be the case. This modified vector r can, as indicated in Figure 3, becomposed of a vector r_u , which corresponds to he undisturbed light, and an additional vector r_z,caused by the interference.

  

Light vectors in the object plane Fig. 3 300 388 /aT

This additional vector thus corresponds to the light diffracted from the small object. So we now have the image of the light source in the focal point in the rear lens focal point, caused by the undisturbed light, and - depending on the size of the

object - a more or less extensive diffraction figure of the light source, which is associated with the additional vector, and is derived from the latter (Photo 4). Since all rays that contribute to the imaging of the small object have the same optical path length, the vectors r_u and r_z in the image plane are composed in the same way as the corresponding vectors r and r_z immediately behind the object.

Figure 4. Phase contrast Microscopic image of a phase object 300391/1 a T

The fact that the eye only perceives amplitude differences but no phase differences follows from the fact that a pure phase object remains invisible in the normal microscopic image, because in this case | r | = | r_u | or | r '| = | r'_u | , If the vector r'_u or r'_z could be rotated so that both were oriented in the same or opposite directions, a larger or smaller resulting vector would be obtained at the location of the image, that is greater or smaller amplitude and thus greater or smaller brightness than in the
surroundings. This would have made the phase object visible in the image. Zernike has shown that this is possible by consistently applying the Abbe theory to non-absorbing objects. Since only small objects are of interest in microscopy, the diffraction figure they produce always extends considerably, so that with a sufficiently small light source, its image can be seen in the rear focal plane covers the diffraction figure mentioned only slightly and you can largely influence both separately. In the descriptive explanation, we want to restrict ourselves to phase objects with very small phase changes. Then the additional vector stands almost vertically on the undisturbed vector, so that you only have to attach a plate in the geometric image of the light source that rotates the phase of the latter by ± 90 ° (Figure 4).

Bild 5. Phasenkontrastmikroskopische Abbildung eines Amplitudenobjektes 300 389/a

Figure 5. Phase contrast microscopic image of an amplitude object 300 389/a

 

The positive contrast is shown in the illustration (the phase change caused by the phase plate

is - 90 °), places with a higher refractive index appear darker than the surroundings. In the case of small phase changes, the additional vector is significantly smaller I than the undisturbed vector, so that the amount of the resulting and undisturbed vector, ie | r '| and | r'_u |, and thus only slightly differentiate the brightness in the image of the object from that of the surroundings. For this reason, the phase plate is given an absorbing effect at the same time and the amount of the resulting vector can thus be reduced to zero, that is. reach complete darkness in the image of the object. However, one has to accept that small amplitude objects disappear in the picture or appear even brighter than the surroundings; this is evident from the illustration in Figure 5, which is analogous to that in Figure 4.

Basically, phase and amplitude objects with maximum contrast cannot be imaged at the same time. With the previous restriction to small relative phase changes in the object, a phase change of 90 ° caused by the phase plate has proven to be the most advantageous, since in this case the additional vector in 

the object stands almost perpendicular to the undisturbed vector (Figure 4). This no longer applies to larger relative phase changes in the object. Here, other phase changes in the phase plate deviating from 90 ° are the cheapest, and its most favorable permeability then also depends on the object. Strictly speaking, a phase plate of a specific permeability and phase change would be required to achieve optimal contrast for each object. This requirement led to the variable phase contrast proposed by some authors. A suitable combination of polarizers with a birefringent crystal plate can be used to continuously change the phase change and permeability, but in practice this proposal, which is good in itself, is of little importance for the following reasons:

 

1. It has been shown that in 90% to 95% of all cases you can get by with a fixed phase plate of 90 ° phase change with a permeability of 25% for each lens. This fact can be justified by the fact that in phase contrast imaging only objects with a very small relative phase change are of interest, since others usually already absorb so strongly that they can also be observed quite well in the bright field.

2.The microscope is extremely sensitive to interference in the beam path (this is precisely the basis of the phase contrast method). One must therefore always endeavor to introduce as few additional, optically effective agents into the beam path and to manufacture them with extreme precision. With the device mentioned to achieve variable phase contrast this can hardly be realized, so that an image deterioration can be expected in any case, especially with strong lenses.

Another complication arises in practical use. Since the light source and thus also the phase plate must have a certain extent in order to achieve sufficient image brightness, part of the diffraction figure originating from the object is always influenced by the phase plate. This leads to structures in the image of a phase object that are not present in the object itself. Bright courtyards are created around the phase object and brightening inside. So you have to give the aperture diaphragm and thus also the phase plate such a shape that the disruptive influence is as small as possible with the largest possible luminous area. These conditions are best met by the annulus. It is therefore not surprising that all replicas of our Jena phase contrast device that have appeared on the market so far use the ring diaphragm and the ring-shaped phase plate. In addition, it is easy to see from the explanation given that the disruptive influence becomes smaller and smaller as the object becomes smaller and the phase ring narrows.

Fig. 6. Köhlersches lighting principle 300 387/1aT

These critical considerations can in no way reduce the importance of the phase contrast method; on the contrary, only when you know the effect of the method can you be largely protected against misdiagnosis based on the images obtained.

In this context, it should be particularly pointed out that with less known objects it is absolutely necessary to compare the bright field image with each phase contrast image. The many scientific publications of recent times testify to the success of the phase contrast method.

Image 7 300 385 / a Annular phase plate in the focal plane of the lens on the image side

 

The Köhler lighting principle (Figure 6) is used to carry out the process in practice. First of all, this achieves the uniform illumination of a sharply delimited part of the object plane and also the required mapping of the aperture diaphragm into the rear lens focal plane, in the following way:

The light source is imaged with the help of a collector lens in the front condenser focal plane (aperture diaphragm plane) and together with the aperture diaphragm by condenser + objective in the rear lens focal plane. The phase plate arranged here is designed so that it just covers the image after adjustment of the aperture diaphragm. To limit the illuminated object field, a light field diaphragm designed as an iris is attached directly behind the collector lens and imaged in the object plane with the aid of the condenser.

Bild 10. Ringblendenzentrierung300 384/a

Fig. 10. Ring aperture centering 300 384/a

To set the Köhler principle, place a conventional transmitted-light specimen on the microscope table, focus on the specimen after the lighting has been roughly set up, and then adjust the height of the condenser until the light field diaphragm appears sharp at the same time as the specimen. Then it is opened until the field of view is just illuminated.

n order not to impair the centering accuracy of the lenses, which is already increased in Zeiss devices, through arbitrarily operated centering, the centering option for the ring diaphragm images was placed in the condenser. With the Zeiss phase condenser, this is done with the tried and tested three-point centering, with the Lumipan with the eccentric device of the aperture diaphragm.

Since phase plates (Fig. 7) of different dimensions are required for the lenses used for phase contrast observation due to their different apertures, aperture diaphragms of different dimensions also had to be provided. Two solutions have been found for this: firstly, the diaphragm turret (Fig. 8), which carries the various diaphragms in an approximately aperture diaphragm plane, and secondly, the variable imaging using a pancratic system under the condenser, as the "Lumipan" ( Fig. 9) was the first microscope to be able to achieve a perfect phase contrast image by precisely adjusting the ring diaphragm image (Fig. 10) .For this it is necessary that the ring diaphragm that matches the lens used is always switched on.

 

The auxiliary microscope belonging to each phase contrast device (Figure 11) is used to observe the centering, with which the ring diaphragm image and the phase ring in the rear focal plane of the lens can be viewed (Figure 10).

Although the phase plates we use can be used for the entire visible spectral range, that is, with the help of our phase contrast device, phase structures can be made visible in objects of any color, it is advisable to observe a limited spectral range to emphasize the last subtleties to use. Since green light is the most pleasant for the eye, each phase contrast device is given a light filter with the maximum permeability at 550 mμ.

A prerequisite for working successfully with the phase contrast device is a microscope light with a collector and iris diaphragm like the types D and E manufactured by us as well as a microscope with a height-adjustable, interchangeable condenser like our L-tripods. At the Lumipan research microscope, the demand for an optically flawless luminaire with the built-in lighting has been met; the interchangeability of the condenser is not applicable with regard to the pancratic system.

The following parts therefore form the Zeiss phase contrast device for normal microscopes (Figure 13):

1. Phase condenser, a dry condenser not specified, 0.65, with aperture turret including 4 ring apertures and 2 free passages

2. Phase objectives Achromat Ph 10 / 0.30; Ph 20 / 0.40; Ph 40 / 0.65; Ph 90 / 1.25 H. I, marked as such by a red-inlaid Ph

3. Separate microscope for alignment

4. Yellow-Green filter

The condenser contains an iris diaphragm, so that when a free passage of the diaphragm turret is switched on, it can be observed like any dry condenser in the bright field and is to be used in polarized light and for luminescence microscopy. The four lenses are correct according to achromatic lenses and can also be used for ordinary bright field observation.

In the future, the phase condenser with individual centering (Fig. 14) will replace the phase condenser shown in Fig. 13. It has the essential advantage over the current design that the ring diaphragms can be individually centered for the respective Ph objective; In addition, the position of the aperture diaphragm can be easily read from a division.

The phase contrast device for the "Lumipan" (see Fig. 9) contains a ring diaphragm instead of the unnecessary phase condenser, which is inserted into the colored glass holder above the aperture diaphragm. Otherwise, the two equipment are completely identical.

The hundreds of years of scientific work that have been carried out with the phase contrast microscope clearly shows the importance of this method. It is essentially based on the following points:

The phase contrast method allows the observation of living or surviving material without the dubious aids previously required for this purpose. Since living objects often have a refractive index very similar to their surrounding medium and are usually not very impressively colored, the bright field observation in this case resulted in extremely low-contrast images. The attempt was made to increase the contrast by closing the aperture diaphragm, adjusting the focus beyond the best, or using vital stains. All of these methods have considerable disadvantages: closing the aperture diaphragm limits the lighting aperture and thus significantly reduces the resolution, setting outside the best focus logically results in blurred images, and vital coloring represents an ultimately uncontrollable intervention in the life of the object.

In the dark field observation that is frequently used, one only sees the outer contours of a phase object and cannot therefore infer its inner structure based on the dark field image.

The phase contrast method enables the living observation of the object in the natural, surrounding medium. With somewhat careful treatment, the observation material can be cultivated further and so z. B. Study development processes in life. Such works are e.g. B. possible with bacterial, fungal and tissue cultures as well as smears, smears, [‘Klatsch-’ / test-] and squeeze preparations of various objects.

Hand and frozen sections of unfixed tissue are also well suited for phase contrast observation. It is therefore understandable that the phase-contrast microscopic examinations come mainly from the fields of biology, medicine and microbiology.

For example, cell division processes were examined and filmed several times, sperm examinations were carried out, and mitochrondria and Golgi apparatuses were observed.

Of particular interest has been the phase contrast microscopic examination of malignant tumors. Several papers dealt with the application of the phase contrast microscope in normal and pathological histology; it was found that the phase contrast method may show more details within colored complexes than the brightfield observation, even in colored specimens.

Much work has also been published on phase contrast observations on living bacteria, and more recently the phase contrast microscopic examination of fresh blood has proven to be promising.

In entomology, the phase contrast method has been used to observe and systematically determine the smallest objects such as mites, mallophages and the like. a., proven. In these transparent objects, the phase contrast microscope shows the finest hair and bristles as well as systematically important chitin structures more clearly than any other device.

Paleontology uses the phase contrast microscope e.g. B. for pollen analysis, and in the study of the structure of coal, its application has led to new results as well as in fiber and textile research.

he phase contrast method has also been used to examine surfaces, in particular by examining the polishing of optical surfaces on glasses, taking collodion or lacquer prints from opaque objects and observing them in transmitted light. The phase contrast method has also been used in the field of mineralogical as well as ore and metal microscopy, where investigations are carried out on thin sections and with the help of varnish prints.

This short, by no means exhaustive summary of the possible uses of the phase contrast microscope shows that over the course of 13 years this device has become an indispensable tool in microscopy. A compilation of publications in the field of phase contrast microscopy that have become known to us is given in our publication CZ 30-L304a-l "Directory of documents on phase contrast microscopy".

Afterword

In November 1953, Prof. Zernike, Groningen, received the Nobel Prize in Physics for his phase contrast method. Since then, various notes on this topic have been published, which do not always correctly depict the facts in their historical development. Therefore, it seems appropriate to us, based on the documents available to us, to reproduce the development of the method and the devices necessary for its implementation true to history. The theoretical foundations of the phase contrast method were already communicated to us by Prof. Zernike in 1932 and 1933 or in Jena from Inventor demonstrated by means of several experiments on suitable objects. In 1933 and 1934, Prof. Zernike published fundamental works on it in various magazines. (Handelingen van het XXIVe Ned. Natuur- u. Geneskundig Congr. 18 to 20 April te Wagenlingen [1933]; Physica 1934; Z. f. Physik 1934 etc.) The patent for this process (DRP 636 Kl. 24 H Gr 610) was issued to Carl Zeiss, Jena, in May 1935. There was a relatively long time between this point in time and the practical implementation of the process on a microscope or until a salable device was sold in 1941. However, it meant years of hard work for the scientific staff at Carl Zeiss, Jena, who were involved at the time, during which some setbacks had to be overcome. We consider ourselves entitled to, in this context, especially the name of our now deceased employee, Prof. Dr. Dr. H. c. August Köhler, who pioneered this area and shared his experience and practical results in 1941 with Dr. Loos published in the journal "Die Naturwissenschaften". It is now generally known that the phase contrast device for microscopy is now produced again in the known quality, and in an improved form, in the VEB Carl Zeiss Jena. Of course, we are also at the birthplace of the practical Realization of the phase contrast process for its further development is constantly striving.

January 15, 1954

Translation from German: Google Translate with some help from

per.funke@gmail.com

The translation method has it’s pitfalls. The original originates from

http://www.smartrepair.ch/forum/Phasenkontrast.pdf

Errors, ambiguities etc. may be resolved by correlating with this document, the "original". If the link ends up in the big 404-bucket I can supply the PDF.

Just for sake of interest, look through the"Afterword".