DE2462110B2 - METHOD OF INCREASING THE CAPABILITY OF ENLARGEMENT OF AN OPTICAL MICROSCOPE AND MICROSCOPE FOR CARRYING OUT THE METHOD - Google Patents

Description

It is known that the recognizability of details in Objects essentially depends on the beneficial magnification of the microscope used. When using normal light, the resolution is around 280 nm, when using ultraviolet light at around 140nm, i.e. details with smaller dimensions are with the help of an optical Microscope no longer recognizable. If you use electron microscopes, you can increase the resolution significantly. However, it can be done with Such microscopes on the one hand do not achieve a contrast on the living object, and on the other hand they hold living biological objects do not exclude electron bombardment in a vacuum.

In addition, the production of specimens for an electron microscope is very complex. The preparation thicknesses should not exceed 100 nm, which is why very fine cuts must be made. Electron microscopic Working with it requires extensive, lengthy and expensive preparation methods, such as fixation, embedding of the object, production of ultramicrotome sections and contrasting of the Preparation.

If you want to microscopically observe the fine structure of a cell and its organelles, i.e. plasma membranes, endoplasmic reticulum and ribosomes, dictyosomes and Golgi apparatus, cytosomes, mitochondria, Plastids, centrioles and nucleolus, then the beneficial magnification of an optically working one is sufficient Microscope does not work, on the other hand the organelles would be alive with a purely optical view stay. If you use an electron microscope, you can see such organelles and thus the Recognize the fine structure of many cell types. The organelles die with the cell. So are at Use of an electron microscope basically only provides information about the statics, but not about the < > o Genesis and dynamics of a cell possible.

The object of the present invention is to provide a method in which the objects are alive remain and are nevertheless observable with sufficient, beneficial magnification. fts

This object is achieved according to the invention by optionally using one of the methods according to the Claims 1-4 solved.

When using one or, if applicable, several of these methods, the objects to be observed no longer come into contact with ovarian lesson beams. The total magnification compared to the purely optical microscopes increases by a factor of about 5. An electronic amplification of the modulation transfer function (MuF) is possible and useful below the spatial frequency at which the modulation transfer function of the optical device part has dropped to zero

According to the prior art, various methods of spatial frequency filtering, especially the MOF boost at higher spatial frequencies, are known, see, for example: A. Lohmann "Active contrast transfer theory", Optica Acta, 1959, pages 318-338 and Menzel, Mirande, Weingärtner "Fourier optics und Holographie «, Springer-Verlag, Vienna - New York, 1973.
These procedures are

1. Purely optical frequency filter in real time, e.g. apodization, i.e. the filter works during the Image of the lens;

2. Subsequent filtering of photographic recordings, ι. B. British Patent 97,424;

3. Photographic frequency filters, e.g. B. "Blurred Mask", Neighbor effect;

4. Frequency filter through motion effects,

5. Non-optical optical frequency filters

5.1 Electronic mask (»Logetron«),

5.2 Fluoro-Dodge,

5.3 Television methods, other than the method mentioned here, e.g. B. without taking logarithms or use of a high pass which only works in the direction of the rows

All of these methods have disadvantages, especially when the recognizability of details is more dynamic Objects to be improved:

1. Purely optical frequency filters in real time reduce the maximum available aperture and thus inevitably the information that can be obtained.

2. and 3. With subsequent filtering of photographic recordings and use of photographic ones Frequency filter, real-time observation of moving objects is not possible.

4. Frequency filters through motion effects are due to the required variation in light intensity and the necessary image shifting is very complex, and photometry is also required and thus also real-time observation of moving objects is not possible.

5. Of the non-light-optical frequency filters, the Fluoro-Dodge method is not based on the Microscopy of moving objects can be transferred.

The Logetron process could theoretically be carried out by a greatly reduced image of a fluorescent television screen on the object can be used in microscopy However, the type of objective illumination leads to failure to utilize the objective aperture to a reduction in the resolution In addition, considerable intensity problems arise me on.

Television methods other than those described here also have disadvantages

ζ. B. a purely electronic RC high-pass filter only works in line direction. A pure difference formation between two television signals without taking the logarithm is almost ineffective for raising high spatial frequencies. In the proposed method, taking the logarithm of the television signals according to claim 11 for realizing claims 1 to 4 plays an essential role. The method according to the invention has in common that the quotient (logarithmic difference) of two images is produced
This means in the notation of Fourier optics

J ₁ [OF) _ L $ (0F) MUF ₁ (OF) J ₁ (OF) ~ L ⁺ ₂ (0F) 'MUF ₂ (OF)

with the designations:

OF - spatial frequency

/) (J ₂ ) - image modulation of the »counter« - (»nominal

ner «-) picture

L \ ⁺ (L ₂ ⁺ ) —object contrast MÜF \ (MUF ₂ ) - modulation transfer function

the "numerator" ("denominator") system,

or with the abbreviations
MOF) ZJ ₂ (OF) = J (OF); L _x ⁺ (OF) IL ₂ ⁺ (OF) = L + (OF) and MUF _x (OF) I MUF ₂ (OF) = MÜF _gestml (OF):

J (OF) = L ⁺ (OF) ■ MUF (OF).

The special case (ideal case) of a constant modulation transfer function, ie AfOF _5eramr = const, results if MUF _x (OF) = MUF ₂ (OF) . B. according to claim 4 (2 images with the same wavelengths and thus the same MTF) the case.

According to claims 1-3, the respective "fuzzy" system is always used as a "denominator system", so that because MUF ₂ (OF) <MÜF _X (OF) a MÜFg ^ m (high-pass filtering) increases with increasing spatial frequency (OF) ) results

It can thus be stated that the production of the logarithmic difference between two images as an average for the purpose of generating optical high-pass filters according to claims 1-4

To comply with the condition mentioned, the modulation transfer function of the overall system, which is the product of the modulation transfer angsfunktion for the optical and for the electronic part, can be raised to the maximum possible spatial frequency with means belonging to the state of the art. The electronic part is then e.g. B. for the special case of a constant MÜF the appropriate corrector for the optical part of the overall transmission system.

The input of the AfOF of the electronic part according to claim 1 can be carried out as follows: by means of a device, the voltage or current intensity carried by the focusing unit "" can be changed by hand or by an electronic control circuit. This allows the cross-section of the writing beam in the television pickup tube to be changed. This allows the desired modulation transmission function of the electronic part to be set, e.g. B. so that it represents the reciprocal of the MTU of the optical part within the maximum possible spatial frequency range and thus the constancy of the entire AfOF results .

MÜF _total = MÜF, + ML) F ₂ ⁺ ,

where MUF ₂ ⁺ = MMUF ₂ or in another notation according to the logarithmic difference formation (quotient)

MÜF _gcsaml = MUF _x / MUF ₂ .

According to claim 2, the matching MUF _{2 is} generated by varying the unsharpness of the unsharp image.
If the method according to claim 2 and / or claim 3 is selected, more than two picture tubes may also be useful, namely when frequency filtering of a color television picture is sought or is observed in at least 2 wavelengths.

In principle, transmitted light or incident light can be applied to the object and, in particular, mil ordinary light, the methods according to claims 1 and 4 can be applied.

However, it has proven advantageous to use ultraviolet light of a suitable wavelength. There the ultraviolet light, which has a shorter wavelength than the visible light, is a priori the resolving power is greater and the necessary magnification can be increased accordingly However, the ultraviolet light also has other significant advantages.

First, the substance-specific parts of the absorption spectra with high extinction are in the UV range and not in the visible area, where only the less substance-specific long-wave runners are less

Absorbance can be found. This makes images of living cells with UV light in the transmitted light method essential They are richer in contrast than those with visible light and they are also with suitable pre-filtering of the transmitted light substance-specific for the

different cell constituents.

Second, the fluorescence of most cell constituents capable of luminescence "can only be caused by ultraviolet" Light can be stimulated. This also gives rise to further differentiation options from the visual one Separation of the different cell constituents and thus opportunities to gain new knowledge about Genesis and dynamics, biochemistry and energetics of the cell as well as the breakdown of the metabolism aul different reaction spaces and the material transport

through cell interfaces.

When using ultraviolet light, however, there is a risk that chemical bonds, especially low-energy bonds, will break.
In a further embodiment of the invention, the

optimal light intensity, according to claim 6 set as follows:

First, the Objekidetai you are looking for! characteristic wavelength set, e.g. in transmitted light microscopy the wavelength of the absorption

tion maximum, to the greatest possible contrast thereby resulting Subsequently, the intensity of the illumination light at the same time control (increase) det electronic image gain reduced so as £ the increasing with decreasing light intensity quantum noise secure visibility of jeweili gen details still permits the order before wavelength selection and reduction of light intensity can also be swapped.

Both measures together lead to the achievement of the greatest possible contrast / radiation damage ratio of the property details.

If this condition is met, low-intensity illuminating light, in particular UV light, can be used to image the object. A breaking chemical bonds to a large extent is thus no longer be feared when using UV light i \ i.

In previous microscopy, compliance with these conditions is not common because the area of the ι ο Quantum noise is not achieved through dark adaptation of the observer's eye.

When studying a microscope, usually neither quantum noise nor its meaning for the lower limit of the lighting intensity recognized. Experience has shown that one only comes near the The image noise becomes visible when the viewer's eye has to be adapted to be dark. This applies, provided that there is no additional image-intensifying element in the optical beam exit, such as B. an image intensifier is available

With the usual microscopic methods, the observer's eye is adapted to light without an image intensifier worked, so that the lower limit of the required to avoid excessive image noise Illumination intensity is exceeded by several orders of magnitude.

However, this is what is aimed for for vital observations Reduction of cell damage from radiation not achieved. Because an optimization of the ratio microscopic information gain on cell damage. especially with UV microscopy, urgently is required, this is done according to the method according to the invention by using image intensifiers enables.

The real image of the object can be according to the healing field, dark field, phase contrast, interference contrast or Fluorescence method can be generated, as well as real images if several images of the object are used Implementation of the procedures are necessary.

As the wavelength becomes smaller, there is a steeply increasing, contrast-reducing scattering of the light which is usually noticeable, especially when using ultraviolet light According to the invention, this effect is suppressed in that, for. B. according to claim 1, the real image is generated on the photosensitive layer of a first television pickup tube and the photosensitive Layer of a second television pickup tube with Constant light is applied that the light-sensitive Electron beam scanning layer of the television pickup tube exposed to constant light is controlled by the television tube receiving the real picture in the current strength that this Electron beam is deliberately kept out of focus by defocusing, and that of the television tubes incoming signals are combined to form a logarithmic difference image.

The deliberate blurring of the scanning beam of the television pickup tube exposed to constant light is at the same time one of the mentioned methods according to the invention in order to increase the product of the modulation transfer functions of the optical and the electronic system. For example, the cross-section of the scanning beam can be changed with the aid of a rotary resistor. By observing the difference image, the desired Det ' ^: ' resolution can be set empirically.

The methods of claims 2 to 4 solve the problem according to the invention with the same success.

The difference image is advantageously displayed on the screen of a television monitor at the same time an improvement in diagnostic reliability in the cytological laboratory through less fatigue Work achieved

In addition, the adjustability of the contrast and brightness of the television monitor is a additional improvement of the evaluability of the microscope image is possible, so that one leads to a further Increase in information gain is coming

The methods on which the invention is based are advantageously suitable for data acquisition of the Images or parts thereof and for the storage of this data, whereby the time-lapse technique is also used can be used, for example, to study cell dynamics.

The methods on which the invention is based are particularly suitable for the cytological early diagnosis of cancer, in particular in connection with the aforementioned electronic storage and data processing the microscopic results.

The drawing shows a microscope for carrying out the method according to the invention shows

F i g. 1 the optical device part, partly in section F i g. 2 a block diagram of the electronic part,

F i g. 3 another exemplary embodiment, shown schematically for the optical device part,

4 shows another embodiment for the optical device part, shown schematically

According to FIG. J the object 1 is developed with the help a light source 2 illuminated in transmitted light or optionally with the help of a light source 3 in incident light Das Imaging lens 4 is inserted into the device with the aid of a dovetail guide 5 * "o that it can be exchanged. The lens 4 is followed by a mirror 7 which the light beams in Projective 9 directs a real image of the object onto the light-sensitive layer 40 of a television pickup tube 30 generated (Fig. 2). A light source 12 illuminates the light-sensitive layer of a television tube 41 with the aid of a projector 13 Constant light

F i g. 2 shows the electronic part of the device.

On the television pick-up tubes 30 and 41, a deflection system 31 acts so that the light-sensitive Layers are scanned correctly in terms of point and line synchronously. The image signal from the pickup tube 30 controls the current intensity of the light-sensitive layer of the tube 41 to which the same is applied scanning electron beam. The one from the television tube 30 incoming signals are also input into an amplifier 32, the Bildaus Gangsstgnale to a logarithmter 33 gives The logarithmizer gives its signals in a difference-forming Establishment 34.

The signals coming from the television pickup tube 41 are fed to an amplifier 35, dei its signals to a logarithmic egg 36 are better Signals are also fed to the subtraction device 34. A synchronous generator 37 control the synchronization of the signals. Since the light qualities ffii every pixel on the light-sensitive layers of the television pick-up tubes are different, ge long into the device 34 for differential bikhinj signals of different intensities so that sid Forms signal differences for each sampled point

permit. In addition, the electron beam is the Pick-up tube 41 kept out of focus by defocusing, so that on the back of the storage layer of the television pickup tube 41 a blurred charge image is obtained which is derived from the charge image of the Television pickup tube 30 can be subtracted.

The image output signals are input to a television monitor 38 so that a screen appears on the television monitor 38 'High contrast difference image of the images produced on the light-sensitive layers appears so that the the signals generating the difference image are synchronized with the scanning signals in the television pick-up tubes, the synchronous generator 37 acts on a corresponding addition stage 39 assigned to the television monitor.

Since the signals coming from the television pick-up tubes 30 and 41 have unequal transit times, is the logarithmizer 33 a delay line 42 upstream, so that in the device 34 for The subtraction of the individual signals from one another can always take place exactly.

The television pick-up tubes are firmly connected to the optical device part in order to avoid relative displacements of the images on the light-sensitive layers to be avoided if the device is jolted.

If UV light is used, the lenses of FIG. 1 from quartz. But it is also possible according to FIG. 3 to use a mirror optic. The UV light is with the help of a mirror 20 one of the Mirrors 21 and 22 supplied to existing condenser. with the help of which the object 1 is illuminated. That 5 lens also consists of mirrors 23 and 24. The Objective creates an intermediate image in plane 25. The real image generated in the intermediate image plane 25 becomes imaged in the intermediate image plane 28 with the aid of a projective consisting of the mirrors 26 and 27, in

ίο which the photosensitive layer 40 of the television pickup tube 30 is arranged

According to FIG. 4 is from the object on the photosensitive layers of the television pickup tubes 30 and 41 each creates a real image of the object. The optical one

■ 5 imaging beam path is for this purpose with the help of a partially transparent mirror divided.

The fuzzy scanning of the constant light image according to FIGS. 1 and 2 is according to FIG. 4 replaced by the fact that on the light-sensitive layer of the tube 41 eir blurred image of the object is generated.

A corresponding effect can be achieved if one differentiates between 16 and 17 (FIG. 4) color filters light color or polarization filter or light module gates in the partial beam paths and on the light-sensitive layers of the television recording tubes produce sharp and real images

For this purpose 4 sheets of drawings

Claims (19)

Patent claims: 1. A method for increasing the useful magnification of an optical microscope in which at least one real image is generated from the object optically, which is electronically scanned and is amplified, characterized in that the modulation transfer function of the overall transmission system, consisting of the opti- ι ο see image and the electronic amplification at high spatial frequencies is increased over a wide spatial frequency range by the Real image is generated on the light-sensitive layer of a first television pick-up tube and the light-sensitive layer of a second television tube is exposed to constant light that which scans the light-sensitive layer of the television pick-up tube exposed to constant light Electron beam from the television tube receiving the real picture in the current strength is controlled so that this electron beam is deliberately kept out of focus by defocusing, and that the signals coming from the television pick-up tubes form a logarithmic difference image be put together. 2. A method of increasing the useful magnification of an optical microscope in which at least two real images are generated light-optically from the object, characterized in that the images are electronically scanned and amplified that the modulation transfer function of the overall transmission system, consisting of optical imaging and electronic amplification is raised at high spatial frequencies over a wide spatial frequency range that one of the images is deliberately kept out of focus and that those coming from the television pick-up tubes Signals are combined to form a logarithmic difference image. 3. Method for increasing the useful magnification of a light-optical microscope, at at least two real images of the object are generated optically, characterized in that these images are electronically scanned and amplified that the modulation transfer function of the overall transmission system, consisting of optical imaging and electronic amplification is raised at high spatial frequencies over a wide spatial frequency range that the images are produced with light of different wavelengths and that of the television pick-up tubes incoming signals are combined to form a logarithmic difference image. 4. Method for increasing the useful magnification of a light-optical microscope, at because two real images are produced optically from the object, characterized in that these Images electronically scanned and amplified and combined to form a logarithmic difference image that the modulation transfer function of the overall transmission system, consisting from optical imaging and electronic amplification at high spatial frequencies is raised over a wide spatial frequency range that the images with light are the same Wavelength are generated, with the same modulation transfer function of the overall transmission system to generate the two real images, light with at least one other parameter is used. 5. The method according to claims 1-4, characterized in that for the optical imaging ultraviolet light is used on the object. 6. The method according to claims 1-4, characterized in that to achieve the largest Contrast in relation to the radiation load of the object elements to be observed the intensity of the illuminating light is reduced with simultaneous regulation of the electronic image intensification and then the wavelength of the illumination light is appropriately changed. 7. The method according to claims 1-4, characterized in that the real images of the object generated by the brightfield, darkfield, phase contrast, interferential contrast or fluorescence method will. 8. The method according to claims 1-4, characterized characterized in that the difference image is displayed on the screen of a television monitor. 9. The method according to claims 1-4, characterized in that the electronically generated image or a part of it is recorded in terms of data and, if necessary, stored. 10. The method according to claims 1-4, characterized by the storage of individual Pictures or parts of pictures and their reproduction in time-lapse mode. 11. Microscope for carrying out the procedure according to claims 1-4, characterized in that the electronic amplifier has a logarithmizer (33), a comparator (34) for forming the difference between the signals and a monitor for contains the visualization of the difference signals 12. microscope for performing the method according to claim 2, characterized in that for Creation of the images of the object on the light-sensitive layers of two television tubes a partially transparent mirror (6) is provided in the optical imaging beam path and that the optical elements in a partial beam path the image they generate by defocusing image out of focus. 13. Microscope for performing the method according to claims 1-4, characterized in that that in the imaging beam paths generating the images of the object means for adjusting Color filters are provided. 14. Microscope for carrying out the method according to claims 1-4, characterized by an optional transmitted or reflected light illumination for the object (1) as well as exchangeable ones Lenses (4). 15. Microscope for carrying out the method according to claims 1-4, characterized in that that the light-optical microscope generates one or two real intermediate images, which are made with the help of a projector projected onto the photosensitive layers of the television pick-up tubes. 16. Microscope for performing the method according to claims 1-4, characterized in that that the optical elements (21, 22, 23, 24, 26, 27), in particular the projectives (26, 27) by mirrors are formed (Fig. 3). 17. A microscope according to claim 11, characterized in that the logarithmizer (33) has a Delay line (42) is upstream 18. Microscope for carrying out the method according to claims 1-4, characterized by a gate controlled by a window generator to which the image output signal is sent after the ditference formation is supplied, which the image signal is adjustable on the window generator and additionally on the screen of the television monitor (38) marked part of the overall picture of a computer system or a registration device, e.g. B. a writer or the like, supplies. 19. Microscope for carrying out the method according to claims 1-4, characterized by one of the biloabasting rays in the television pickup tube and means (37) synchronizing the imaging beams in the monitor.