DE2462111B2 - Process to improve contrast and to increase the useful magnification (resolution) of an optical microscope - Google Patents

Description

55

The invention relates to a method for contrast ver eo improvement and to increase the beneficial magnification of an optical microscope after Preamble of claim 1.

It is known that the recognizability of details in Objects depends on the one hand on the resolution of the microscope used and on the other hand the achievable contrast.

When using normal light, the resolution is

caution at 280 mn, when using ultraviolet light around 140mn, i.e. details with Smaller dimensions can no longer be recognized with the aid of an optical microscope.

A good contrast ensures the recognizability of details, especially with amplitude objects.

The contrast can be improved by using dark field, phase contrast, interference contrast, polarization or fluorescence microscopy Staining methods are preferred to biological objects. Staining is time consuming and gets in many cases damage or even death of the object.

If you use electron microscopes, you can increase the resolution significantly. It leaves However, on the one hand, no contrast can be achieved on the living object with such microscopes, and on the one hand for others the living biological objects cannot stand the electron bombardment in a vacuum.

In addition, the production of preparations for an electron microscope is very expensive. The preparation thickness should not exceed 100 nm, which is why very much fine cuts need to be made. Electron microscopic work thus requires extensive, lengthy and expensive preparation methods such as fixation, embedding of the object, manufacture of ultramicrotome sections and contrasting the specimen.

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 magnification of an optically working microscope is sufficient not off, but on the other hand the organelles would remain alive with a purely optical view. If you use an electron microscope, then you can recognize such organelles and thus the fine structure of many cell types. The organelles die with them the cell. This means that basically only statements are made when using an electron microscope possible via the statics, but not via the genesis and dynamics of a cell.

Optical devices that generate two images of an object are known. In these devices, the two images generated for other purposes, for example for a variable phase contrast and / or interference microscopy (AT-PS 29 81 07). This is not in the spirit the invention.

The object of the present invention is to provide a method in which the objects are alive remain and are still observed with sufficient resolution and in sufficient contrast.

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

As a result of the invention, when one or, if necessary, several of the methods are used, the objects to be observed no longer come into contact with electron 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 (MTF) is possible and useful below the spatial frequency at which the modulation transfer function of the optical device part has dropped to zero.

This is explained in more detail below. Various methods are known in the art

Spatial frequency filtering, especially the Λ / ÜF increase in higher spatial frequencies known, see z. E.g. A. Lohmann "Active contrast transfer theory", Optica Acta, 1959, pages 318-338 and Menzel, Mirande, Weingärtner "Fourier Optics and Holography", Springer-Verlag, Vienna-New York, 1973, namely Chapter V: "Spatial filtering", pages 111 to 125, in particular Section: “Subsequent filtering of photographic recordings”, pages 115 and 116 and section: “That inverse filter «, page 121. ι ο

These procedures are

1. purely optical frequency filters in real time, e.g. B. apodization, d. H. the filter works during the imaging of the object;

2. Subsequent filtering of photographic recordings, e.g. B. according to the British patent specification 7 97 424;

3. photographic frequency filters, e.g. B. "Unsharp mask", neighbor effect;

4. Frequency filter through motion effects;

5. non-optical frequency filters

5.1 Electronic mask (»Logetron«),

52 Fluoro dodge,

53 television methods, different from these mentioned method, e.g. B. without taking the logarithm or using a high pass, which only acts in line direction

All of these methods have disadvantages, in particular those when the detail recognizability of moving objects is 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 photographic Recording and using photographic frequency filters is a real-time observation more agile Objects not possible.

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

5. Of the non-light optical frequency filters, the fluoro-dodge method is not applicable to microscopy movable objects transferable.

The Logetron method could theoretically be carried out by a highly reduced image of a fluorescent TV screen can be used on the object in microscopy, however, for the type of Objective lighting leads to a reduction in resolution due to failure to utilize the objective aperture. In addition, there are considerable intensity problems.

Television methods other than those described here also have disadvantages, for example a purely electronic AC high-pass filter only works in the line direction. _Μ A pure difference formation between two television signals without loganthmation is almost ineffective for raising high spatial frequencies.

In the proposed method, by loganthmizing the television signals according to claim 5 a substantial improvement can be achieved for the realization of claims 1-4.

In the case of loganthmation, the quotient (logarithmic difference) of two images is created. This means in the notation of Fourier optics

Ji (OF) L ⁺ i (OF) MUF ₁ (O F) J ₂ (OF) ~ L ⁺ ₂ (0F) 'MOF ₂ (OF)

with the designations:

OF - spatial frequency

• Λ (Λ) - image modulation of the »counter« -

("Denominator" -) image ^L X ( ^{L +} ₂ ) - object contrast

MUFi (MUF ₂ ) - modulation transfer function of the »Zähier« (»Neni; er«) system,

or with the abbreviations

J ₁ (OF) U ₂ (OF) = J (OF); LX (OF) IU ₁ (OF) = L ⁺ (OF) and MUF ₁ (OFyMUF ₂ (OF) = MUF total (OF); J (OF) = L ⁺ (OF) -MUF (OF).

The special case (ideal case) of a constant modulation transfer function, ie MÜF total = const, results if MÜF \ (OF) = MUF ₂ (OF) . B. according to claim 3 (2 images with the same wavelengths and thus the same MTF) the case.

If an image is kept blurred, the respective "blurred" system is always used as a "denominator system" so that, due to MUF ₂ (OF) K MUF ₁ (OF) , the MÜF total (high-pass filtering) increases with increasing spatial frequency (OF)

To comply with the condition mentioned, the modulation transfer function of the overall system, which is the product of the modulation transfer function for the optical and for the electronic part, can be increased to the maximum possible spatial frequency with means belonging to the prior 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.

For example, the input of the MTF of the electronic part according to claim 1 can be carried out as follows: The voltage or current strength supplied to the focusing unit can be changed manually or by an electronic control circuit by means of a device. This allows the cross-section of the writing beam in the television pickup tube to be changed. So that the desired modulation transfer function of the electronic part can be adjusted, for example, so that it represents the reciprocal of the MTF of the optical part within the maximum spatial frequency range and thus the stability of the entire Af DF gives the total MTF a hintsreinandergeschalteten of two individual systems is known to the Product of the modulation transmission functions of the two individual systems:

AfOF total = MÜF \ MUF ₂ ⁺ ,

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

MÜF total = MUF ₁ ZMUF ₂ .

According to claim 1, the matching MTF2 is generated by varying the unsharpness of the unsharp image.

Under certain circumstances, more than two picture tubes can be useful, namely when the> Frequency filtering of a color television picture is sought or observed in at least two wavelengths will.

In principle, transmitted or incident light can be applied to the object, and in particular with κι 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 has a shorter wavelength than the visible light, is a priori The resolving power is greater and the necessary magnification can be increased accordingly. That But 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 range, where only the few substance-specific long-wave extensions of low extinction can be found. This makes pictures more alive Cells with UV light in the transmitted light method have a significantly higher contrast than those with visible light, in addition, with suitable pre-filtering of the transmitted light, they are substance-specific for the different cell constituents. jo

Second, the fluorescence of most cell constituents capable of luminescence can only be caused by ultraviolet light 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 different reaction spaces and the transport of substances through cell interfaces.

When using ultraviolet light, however, there is a risk that chemical bonds, in particular low-energy bonds, will break.

In a further embodiment of the invention, the optimal light intensity, according to claim 7, set as follows:

First of all, the wavelength characteristic of the object detail you are looking for is set, e.g. at Transmitted light microscopy the wavelength of the absorption maximum, creating the greatest possible contrast Then results in the intensity of the illuminating light with simultaneous regulation (increase) of the electronic image intensification is reduced to such an extent that that increases with decreasing light intensity Quantum noise still allows the reliable recognizability of the respective details. The sequence of The choice of wavelength and reduction of the light intensity can also be interchanged.

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

If you adhere to this condition, you can use low-intensity illuminating light, in particular UV light, use to map the object. A breaking of chemical bonds to a large extent is thus at Use of UV light is no longer to be feared.

In previous microscopy, compliance with these conditions is not common because the area of the Quantum noise is not achieved, for example, by 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 detected. Experience has shown that you only come 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 does not achieve the reduction in cell damage caused by radiation that is aimed for in vital observations. Since an optimization of the ratio of microscopic information gain to cell damage, especially in UV microscopy, is urgent 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 brightfield, darkfield, 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 particularly evident when using ultraviolet light usually makes noticeable. This effect is suppressed in that, for. B. according to Claim 4 generates the real image on the light-sensitive layer of a first television pickup tube and the light-sensitive layer of a second television tube is exposed to constant light becomes that the electron beam scanning the light-sensitive layer of the television pick-up tube exposed to the same light from which the real image is scanned receiving television tube is controlled in the amperage that this electron beam through Defocusing is deliberately kept out of focus, and that the signals coming from the television pick-up tubes are combined to form a logarithmic difference image.

The deliberate blurring of the scanning emitter of the television pickup tube exposed to the same light is at the same time one of the methods mentioned to to raise the product of the modulation transfer functions of the optical and electronic systems. For example, the cross-section of the scanning beam can be changed with the aid of a rotary resistor. The desired detail resolution can be set empirically by observing the difference image.

The methods of claims 1 to 4 solve those on which the invention is based with the same success Task.

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 is achieved through less fatigue-free work

In addition, the adjustability of the contrast and brightness of the television monitor is a additional improvement of the evaluability of the

Microscope image possible, so that one comes to a further increase in the information gain.

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 device in various variants for carrying out the method. It shows

F i g. 1 the optical device part, partly in section.

F i g. 2 is a block diagram of the electronic part in a first embodiment,

F i g. 3 a modified circuit diagram,

F i g. 4 a modified circuit diagram,

F i g. 5 shows a modified exemplary embodiment for the optical device part.

According to Fig. 1, the object 1 is either illuminated with the aid of a light source 2 in transmitted light or optionally with the help of a light source 3 in incident light. The imaging lens 4 is with the help of a dovetail guide 5 inserted into the device so that it can be replaced. The lens 4 is a partially transparent mirror 6 connected downstream mirrors 7 and 8 direct the transmitted by the partially transparent mirror Rays of light into a projection lens 9 that form a real image of the object on the light-sensitive layer a television pickup tube 11 generated (Fig.2). The reflected on the mirror 6 rays are with the help a mirror 12 and a projection lens 13 on the photosensitive layer 14 of a television pickup tube 15 steered and generate a real image of the object there. In the partial beam paths they are Elements 16 and 17 which change the quality of light are used, for example different color filters Use of natural light or polarizers with polarized fluorescent light or a demodulation device, like a Pockels cell to utilize possibly different fluorescence decay times when irradiated with ultraviolet light In the latter case one becomes the ultraviolet light used modulate time-dependent. The elements mentioned are advantageous on a slide or the like arranged to be optionally inserted into the microscope.

According to FIG. 2 acts on the television pick-up tubes 11 and 15, a deflection system 31, so that the photosensitive layers are generated synchronously and correctly scanned in terms of point and line. the Signals coming from the television pickup tube 11 are input to an amplifier 32 which Outputs image signals to a logarithmizer 33. The logarithmizer 33 outputs its signals to a differentiating institution 34.

The signals coming from the television pick-up tube 15 are fed to an amplifier 35 its signals to a logarithmizer 36 also sends its signals to the subtraction device 34 are fed. A synchronous generator 37 controls the synchronization of the signals. Since the light qualities for each pixel on the photosensitive layers 10 and 14 of the television pickup tube are different, get into the device 34 for difference formation signals of different intensities, so that Let signal differences form for each scanned point.

The image output signals are input to a television monitor 38 so that a screen appears on the television monitor A high-contrast difference image of the images generated on the light-sensitive layers 10 and 14 appears. So that the signals generating the difference image with

If the scanning signals in the television pick-up tubes coincide, the synchronous generator 37 acts on a corresponding addition stage 39 assigned to the television monitor.
3 shows a modified device. A real image of the object is generated on the photosensitive layer 40 of the television pickup tube 30. The television pickup tube 41 is exposed to constant light. Since the signals here cover unequal path lengths, the logarithmizer 33 is shown in FIG. 3 a delay line 42 upstream, so that the subtraction of the individual signals from one another can again take place in the device 34 for forming the difference. The amplifier 32 controls the electron beam of the television pickup tube 41. In addition, the electron beam of this pickup tube is kept out of focus by defocusing, so that a blurred charge image is obtained on the back of the television pickup tube 32, which can be subtracted from the charge image of the television pickup tube 40.

F i g. 4 shows the design as a densitometer. Measurement images of the object are generated on the light-sensitive layers 50 and 51 of the television pick-up tube 52 and 53 and scanned as in the previous examples Gate 54, which is controlled by a window generator 55 at the same time. The signals supplied by the goal can, for example, be registered and stored in an electrical data processing device or can also be input into a writing device or the like, which prints them out
The television pick-up tubes are firmly connected to the optical device part in order to avoid relative shifts of the images on the light-sensitive layers if the device is shaken.

The television pickup tubes may be color television pickup tubes and the monitor is correspondingly a color television set

If UV light is used, the lenses of FIG. 1 from quartz. But it is also possible to use a mirror optics according to Figure 5. That UV light is fed to a condenser consisting of mirrors 21 and 22 with the aid of a mirror 20, with the help of which the object 1 is illuminated. The lens also consists of mirrors 23 and 24. The Objective creates an intermediate image in plane 25. To the Objective 23 and 24 can be the image-splitting mirror 6

bu of FIG. 1 follow a In the present case, however, only one image of the object is to be generated. The real image generated in the intermediate image plane 25 is imaged with the aid of a projection lens consisting of the mirrors 26 and 27 in the intermediate image plane 28, in which the light-sensitive layer of the television pickup tube 30 is arranged

In addition 5 sheets of drawings

Claims (24)

Patent claims: 1. Process for enhancing contrast and / or for increasing the useful magnification of a optical microscope in which at least two real images of the object are optical generated by different amplitude, wavelength, polarization, or phase Difference in duration from one another, thereby characterized in that the images are electronically scanned and amplified and that the Difference of the image signals is reassembled into a visible image that the modulation transfer function of the overall transmission system, consisting of the optical imaging and the electronic amplification, at high Spatial frequencies is raised over a wide spatial frequency range that one of the images is conscious is kept out of focus and that the signals coming from the television pick-up tubes are too be composed of a difference image. 2. Process for contrast enhancement and / or to increase the useful magnification of an optical microscope in the case of the object at the same time at least two real images are optically generated, which are distinguished by different Differentiate amplitude, wavelength, polarization, phase or duration from one another, characterized in that the images are scanned electronically and that the modulation transmission function of the overall transmission system, consisting of the optical imaging and the electronic amplification, at high spatial frequencies over a wide spatial frequency range ben is that the images are generated with light of different wavelengths and that of the Signals coming from television pick-up tubes are combined to form a difference image. 3. Process for enhancing contrast and / or for increasing the useful magnification of a Optical microscope in which two real images of the object are optically generated at the same time different amplitude, wavelength, polarization, phase or duration differ from each other, characterized in that the images are electronically scanned and be reinforced that the modulation transfer function of the overall transmission system, consisting of the optical imaging and the electronic amplification, raised at high spatial frequencies over a wide spatial frequency range, so that the images with light are the same Wavelength are generated and that the signals coming from the television pick-up tubes be composed of a difference image. 4. Process for enhancing contrast and / or for increasing the useful magnification of a optical microscope, in which at least one real image is optically generated from the object, the is electronically sampled and amplified, characterized in that the modulation transmission function of the overall transmission system. consisting of the optical image and the electronic amplification, at high spatial frequencies over a wide spatial frequency range, is increased by placing the real image on the light-sensitive layer of a first television pickup tube is generated and the photosensitive layer of a second television tube with constant light it is applied that the electron beam νου scanning the light-sensitive layer of the television tube to which equivalency is applied the real picture receiving television tube is controlled in the current strength that this Electron beam is deliberately kept out of focus by defocusing and that the Signals coming from television pick-up tubes are combined to form a difference image. 5. The method according to any one of claims 1 to 4, characterized in that the signals to one logarithmic difference image. 6. The method according to any one of claims 1 to 4, characterized in that for the light-optical Illustration of the object using ultraviolet light. 7th Method according to one of Claims 1 to 4, characterized in that in order to achieve the greatest contrast in relation to the radiation exposure of the object elements to be viewed Intensity of the illuminating light with simultaneous regulation of the electronic image intensification up to the quantum noise limit is reduced and then the wavelength of the illuminating light is suitably changed. 8. The method according to any one of claims 1 to 4, characterized in that the real images of the Object can be generated according to the brightfield, darkfield, phase contrast, interference contrast or fluorescence method. 9. The method according to any one of claims 1 to 3, characterized in that the light-optically generated images on the light-sensitive layers two television tubes, e.g. B. Vidikons are projected. 10. The method according to any one of claims 1 to 4, characterized in that the difference image is displayed on the screen of a television monitor will. 11. The method according to any one of claims 1 to 4, characterized by its use for densitometry. 12. \ ^r experience according to one of claims 1 to 4, characterized in that for the application of the fluorescence method, the stimulating light is modulated over time and the resulting fluorescent light is demodulated with a simultaneous phase shift. 13. The method according to claim 12, characterized in that the stimulating light is UV light is used. 14. The method according to any one of claims 1 to 4, characterized in that the electronically generated image or a part thereof is recorded in terms of data and, if necessary, stored. 15. The method according to any one of claims 1 to 4, characterized by the storage of individual Pictures or parts of pictures and their reproduction in time-lapse mode. 16. microscope for performing the method according to claim 5, characterized in that the electronic amplifier a logarithmizer (33), a comparator (34) for the difference formation of the Contains signals and a monitor for visualizing the difference signals. 17. Microscope for carrying out the procedure according to claim), characterized in that to generate the images of the object on the light-sensitive layers of two TV recording tubes in the optical imaging beam path partially transparent mirror (6) is provided and that the optical elements in one partial beam path image the image produced by Omen blurred by defocusing. 18. microscope for performing the method ig according to claim 1, 2, 3 or 4, characterized in that in the generating the images of the object Imaging beam paths means for adjusting filters for influencing the spectral composition of the light are provided. 19. Microscope for carrying out the procedure according to claim 1, 2, 3 or 4, characterized by an optional transmitted or reflected light illumination for the object (1), as well as exchangeable Lenses (4). 20. Microscope for performing the method according to claim 1,2,3 or 4, characterized in that a in the light-optical part of the microscope or two real intermediate images are created, which with the help of a projection lens onto the photosensitive ones Layers of the television pickup tubes are projected. 21. A microscope for carrying out the method according to claim 1, 2, 3 or 4, characterized in that the objective and the projective as Mirror optics are formed (Fig. 5). 22. microscope for performing the method according to claim 4, characterized in that for To generate a logarithmic difference image, a logarithmizer (33) is provided to which a J5 Delay line (42) is upstream 23. Microscope for performing the method according to claim 1, 2, 3 or 4, characterized by a gate controlled by a window generator, to which the image output signal is fed after the subtraction, which the image signal of an am Window generator adjustable 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 scribe, feeds 24. Microscope for performing the method according to claim 1, 2, 3 or 4, characterized by one is the image scanning beams in the television pickup tube and the imaging beams in the Monitor synchronizing device (37).