DE2605261A1 - Optical microscope resolving power enhancement - uses electronic scanning of real sharp and unsharp images to form difference image - Google Patents

Abstract

A method is used for improving the resolving power of a microscope, in which either a real, sharp optical image and an unsharp electronic image of the object are formed, or a real sharp and a real unsharp optical image, or two optical images at different optical wavelengths. The images are scanned electronically and are amplified to form a differential image. The product of the modulation transfer function with optical imaging and that with electronic amplification, as a function of the spatial frequency, is raised or held constant over a wide range of spatial frequencies. The optical imaging may be effected in ultra-violet light.

Description

Method of increasing the beneficial magnification

(Resolving power) of an optical microscope The invention relates to a method to increase the useful magnification (resolving power) an optical microscope It is known that the visibility of details in Objects essentially from the necessary magnification (resolving power) depends on the microscope used.

When using normal light, the resolution is about at 280 nm, when using ultraviolet light around 140 nm, i.e. Details with smaller dimensions are no longer possible with the help of an optical microscope recognizable.

Do you use electron microscopes? so one can have the resolving power significantly increase. On the one hand, however, it cannot be achieved with microscopes of this type Achieve contrast on the living object, and on the other hand keep the living biological Objects do not stop electron bombardment in a vacuum.

In addition, the production of specimens for an electron microscope very expensive, the specimen thicknesses should not exceed 100 nm, which is why very fine cuts must be made. Electron microscopic work sets extensive, lengthy and expensive preparation methods such as fixation, Embedding of the object, production of ultramicrotome sections and contrasting of the preparation, one wants to microscopically the fine structure of a cell and its organelles observe, i.e. plasma membranes, endoplasmic reticulum and ribosomes, dictyosomes and Golgi apparatus, cytosomes, mitochondria, plastids, centrioles and nucleolus, then the necessary magnification of an optically working microscope is not enough on the other hand, the organelles would be alive when viewed from a purely optical point of view stay. If you use an electron microscope, you can see such organelles and thus recognize the fine structure of many cell types. But the organelles also die of the cell, so are basically when using an electron microscope only statements about the statics, but not about the genesis and dynamics of a cell possible.

The object of the present invention is to provide a method in which the objects remain alive and still with sufficient beneficial magnification (Resolution) are observable.

This object is achieved according to the invention in that the object produces either a real sharp image and a blurred electronic image or that optically a real sharp image and a real blurred image be generated or that optically two images with different wavelengths of the Light are generated that these images are electrically amplified and a difference image is generated and that the product of the modulation transfer function in the optical Mapping and modulation transfer function in electrical amplification constant over a wide spatial frequency range as a function of the spatial frequency is held.

When this method is used, the objects to be observed come no longer in contact with electron beams, i.e. they remain alive because of Object purely optical images are generated. Due to the electronic influence of the optically generated images is achieved, however, that the overall magnification in Compared to optically working microscopes, it grows by a factor of about 5. One electronic amplification of the modulation transfer function (MüF) is up to that Spatial frequency possible and useful, in which the modulation transfer function of the optical device part has dropped to zero.

To comply with the conditions mentioned, you can use the stand the means belonging to the technology the modulation transfer function of the overall system, which is the product of the modulation transfer functions for the optical and the electronic part is to make one up to the maximum possible spatial frequency, in that the modulation transfer function of the electronic part is equal to the reciprocal Value of the modulation transfer function of the optical part is made.

The electronic part is then the correct correction work for the optical one Part of the overall transmission system.

In a further embodiment of the invention, however, there are still methods are shown, with which the same can be achieved, but which of the invention underlying principle of the optical imaging of the object and the following electronic amplification of the generated image take greater account.

In principle, transmitted or reflected light can be applied to the object be and in particular also with ordinary visible light, being in addition the methods mentioned at the beginning can be used to increase contrast.

However, it has proven advantageous to use ultraviolet light suitable wavelength to be used. Because the ultraviolet light has a shorter wavelength than visible light, the resolution is greater from the outset. However, the ultraviolet light also has other significant advantages.

First, there are the substance-specific parts of the absorption spectra with high extinction in the UV range and not in the visible range, where only the little substance-specific long-wave extensions of low extinction can be found. This makes images of living cells with UV light in the transmitted light method essential richer in contrast than those with visible light, and they are more suitable Pre-filtering of the light shining through is substance-specific for the various Cell constituents, Second, the fluorescence can be the most luminescent Cell constituents are only stimulated by ultraviolet light and lies by itself often only in the UV area. This also gives rise to further differentiation options for the visual separation of the different cell constituents and thus possibilities to gain new knowledge about genesis and dynamics, biochemistry and energetics of the cell as well as the breakdown of the metabolism into different reaction spaces and the transport of substances through cell interfaces.

However, when using ultraviolet light there is a risk of that chemical bonds, especially low-energy bonds, break.

In a further embodiment of the invention, the intensity of the illuminating light is by reducing and simultaneously regulating the electronic image amplification with a brightly adapted observer eye kept at the limit of image noise and then the wavelength of the illuminating light and fluorescence also the wavelength of the observed fluorescent light until the the greatest contrast of the object elements to be viewed is suitably changed, i.e. the ratio of the output signal intensity to the intensity of the illumination light used and the intensity of the stimulating light are kept optimal, preferably at the limit of quantum noise. If you keep this condition, you can do low-intensity Use illuminating light, especially light, to depict the object. A breaking of chemical bonds to a large extent is therefore used in V. No longer to fear from UV light. Below output signal intensity is the light intensity to understand on the first photosensitive layer of the electronic part, this is the layer on which the light rays are converted into electronic ones Hit signals. This layer can be the receiving layer in the image intensifier or when the image intensifier is included in the television image pick-up tube, the recording layer the television picture pick-up tube.

In previous microscopy, this condition has to be complied with not common because the area of quantum noise is caused by dark adaptation of the Observer's eye is not reached.

When using a microscope, neither quantum noise is usually used as a such still recognized its importance for the lower limit of the lighting intensity.

Experience shows that only then does one come close to becoming visible of image noise when the observer's eye has to be adapted to dark. this applies if there is no additional image-enhancing feature in the optical beam path Element, such as an image intensifier is present.

With the usual microscopic procedures, no image intensifier is used worked with lightly adapted observer eye, so that the purpose of avoiding the lower limit of the lighting intensity required for excessive image noise exceeded several orders of magnitude.

This, however, results in the reduction aimed at for vital observations cell damage from radiation is not achieved. Because an optimization of the ratio microscopic gain of information on cell damage, especially with W microscopy, is urgently required, is used in the process according to the invention made possible by image intensifiers.

The real image of the object can be based on the brightfield, darkfield, Phase contrast, interference contrast or fluorescence method or by polarization or interference microscopy.

As the wavelength becomes smaller, there is a steeply increasing contrast-reducing one Scattering of light, which is particularly evident when using ultraviolet Light usually makes noticeable.

According to the invention, this effect is suppressed in that either. The Real image on the photosensitive layer of a first television pickup tube generated and the photosensitive layer of a second television tube with constant light it is applied that the electron beam scanning the photosensitive layer is intensity-modulated by the television tube receiving the real image, that this electron beam is deliberately kept out of focus by defocusing, for example by appropriately changing its cross-section and that of the television pick-up tubes incoming signals are combined to form a difference image, or that when generated two optical images one image of the object kept more or less blurred is or also by creating two optical images with different Wavelength are generated, so that due to the different diffraction of light one image appears blurred than the other.

The deliberate blurring of the scanning beam after the former The method of the television tube exposed to constant light is one of the mentioned method according to the invention to obtain the product of the modulation transfer functions of the optical and electronic system. For example, can change the cross-section of the scanning beam with the help of a rotary resistor.

By observing the difference image, the desired one can be determined empirically Set detail resolution. When choosing a different object, a different setting must be used of the electron beam (switching).

However, the resolution improvement is expedient only at use the lens with the highest aperture.

The difference image is advantageously displayed on the screen of a television monitor , also shown color television monitor. This is an improvement at the same time for example diagnostic reliability in the cytological laboratory through less fatigue Work achieved.

In addition, the adjustability of contrast and brightness of the television monitor an additional improvement in the evaluability of the microscope image possible, so that one comes to a further increase in the information gain.

That. stimulating or illuminating light can be light.

According to the second method, in which two real images are cast on the light sensitive layers of two television pick-up tubes are created, the product becomes the modulation transfer function of the optical and electronic system thereby kept constant or at least improved in this respect by adding a the pictures with known optical means are more or less blurred at night. at this method is used to keep the product of the modulation transfer functions constant the modulation transfer function of an optical part of the device respectively appropriate blurring changed.

The third method, which provides for two real images of the object with different wavelength = to generate, becomes the product of the modulation transfer function the optical system and the electronic system thereby kept constant, that suitable for the image of the object Wavelengths (filter) be chosen so that the product of the modulation transfer function of the optical and electronic system raised in a desired manner at higher spatial frequencies The method on which the invention is based is advantageously suitable for data acquisition of the images or parts thereof as well as for storage and further mathematical processing by process computers.

The reasons for this are: by the formation of the difference between the images, the the strong contrasting that was carried out and the creation of the logarithmic Image signal and the possibility of fading out parts of the image area in the electronic The (television) part of the overall facility is already becoming essential computing operations anticipated, which in a computer a high expenditure of storage space and Would cause data flow rate.

The arithmetic operations still to be performed for various purposes can thus be carried out by much smaller computers than would otherwise be required will. The microscope provides, especially because of its additional electronic parts, like logarithmizer, integrator, in the extended television section, i.e. when one is connected digitally working computer the analog computer part of an analog and digital computer formed hybrid computer. This hybrid computer in turn can be used as a process computer work and essential parts of the measurement process, such as screening the specimen via stage control, fine adjustment of the focus, wavelength selection that for the spectral decomposition of illuminating light and the light to be registered provided facilities, control and dosing with automatic suction of cell suspensions or the automation of the setting process to keep a constant over the broadest possible spatial frequency range MüF.

The time-lapse technique can also be used, for example for Investigation of cell dynamics.

The method on which the invention is based is also suitable for the cytological early diagnosis of cancer in large groups of people, especially in Connection with the aforementioned electronic storage and data processing of the microscopic results, the drawing shows examples of implementation the method according to the invention shown.

1 shows the optical device part, partially in section; Fig. 2 is a block diagram of the electronic part; 3 shows a modified embodiment for the optical device part; Fig. 4 shows a modified embodiment for the optical device part; Fig. 5 shows a modified embodiment for the optical Device part; 6 shows a graphic illustration to explain the mode of operation.

According to Fig. 1, the object 1 is either with the aid of a light source 2 illuminated in transmitted light or optionally with the help of a light source 3 in incident light. The imaging lens 4 is, for example, with the aid of a dovetail guide 5 inserted into the device so that it can be replaced. The lens 4 is a mirror 7 connected downstream, which directs the light rays into a projection lens 9, the a real image of the object on the photosensitive layer 40 of a television pickup tube 30 is generated (Fig. 2). A light source 12 illuminates with the aid of a projector 13 the light-sensitive layer of a television tube 41 with constant light.

Fig. 2 shows the electronic part of the device.

A deflection system 31 acts on the television pick-up tubes 30 and 41, so that the light-sensitive layers are synchronously correct in terms of point and line are scanned.

The signals coming from the television pickup tube 30 are converted into a An amplifier 32 is inputted, which outputs image signals to a logarithmizer 33. The logarithmizer sends its signals to a device 34 which forms the difference.

The signals coming from the television pickup tube 41 become one Amplifier 35 supplied, which gives its signals to a logarithmizer 36, the Signals are also fed to the subtraction device 34.

The amplification of the amplifier 35 can be manual or electronic Regulation for the purpose of influencing the MüF can be changed. A synchronous generator 37 controls the synchronization of the signals. As the light qualities for each pixel on the photosensitive layers of the television pickup tubes are different, signals enter the device 34 for forming the difference different intensities, so that signal differences were sampled for each Make a point. In addition, the electron beam is the pickup 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 of the Charge image of the television pickup tube 30 can be subtracted.

By means of a device 44, that of the focusing device 43 applied voltage or current intensity by hand or by an electronic control circuit be changed. This allows the cross section of the writing beam to be changed in the television pickup tube 41. This enables the desired modulation transfer function of the electronic part can be set, for example, so that they have the reciprocal value the MuF of the optical part within the maximum possible spatial frequency range and thus the constancy of the entire MüF results.

The entire MüF of one of two individual systems connected in series is the product of the modulation transfer functions of the two individual systems: MÜF total = MüF1 MüF2.

The image output signals are shown in a television monitor 38, which is also a color television set, input via a delogarithmizer 45, which if necessary, e.g. at too great differences in brightness in the Image, can be bridged by a switch 46, so that on its screen a high-contrast differential image of the images produced on the photosensitive layers Images appears.

So that the signals generating the difference image with the scanning signals run the same in the television pick-up tubes, the synchronous generator 37 acts on one corresponding addition stage 39 assigned to the television monitor.

Since the signals from the television pick-up tubes 30 and 41 are unequal Cover distances, the logarithmizer 33 is preceded by a delay line 42, so that in the device 34 for difference formation always exactly the subtraction of individual signals from each other. The amplifier 32 controls the beam current strength of the electron beam of the television pickup tube 41.

This is available at the output of the difference-forming device 34 standing logarithmized video signal is particularly suitable for the quantitative determination of the amount of substance.

For this purpose, the border of the in Question coming area proportions set, with the window generator too doses right times the integrator 48 samples. That at the output of the integrator 48 available summed signal is the amount of substance to be determined of the image detail proportionally.

The one picked out by the window generator 47 for processing The area proportion of the object is displayed on the monitor 38 in relation to the rest of the object shown brightened.

The intensity control of the monitor 38 by the window generator is used for this purpose 47.

The television tubes are firmly connected to the optical part of the device, relative shifts in the images on the light-sensitive layers in the event of a Avoid shaking the device.

If UV light is used, the lenses of FIG. 1 will be made of quartz form. However, it is also possible to use mirror optics according to FIG. 3. The UV light becomes one of the mirrors 21 and 22 with the aid of a mirror 20 Condenser supplied, with the help of which the object 1 is illuminated. As a condenser A special brightfield-darkfield mixing condenser is advantageous for the purpose of widening the MuF of the optical part into account. This condenser delivers from the field the zeroth diffraction order, from the dark field the first diffraction order. In are the front focal plane of such a condenser, as not explained in detail has been, for example, different ring diaphragms can be used for the brightfield-darkfield illumination to separate. The ring diaphragms bring the desired distance between the dark field and brightfield.

The lens also consists of mirrors 23 and 24.

The objective produces an intermediate image in plane 25.

The real image generated in the intermediate image plane 25 is shown with the aid of a projective consisting of the mirrors 26 and 27 into the intermediate image plane 28, in which the photosensitive layer of the television pickup tube 30 is arranged.

According to FIG. 4, the object is on the photosensitive layers the television pick-up tubes 30 and 41. each generate a real image of the object. Of the The optical imaging beam path is used for this purpose with the aid of a partially transparent mirror 6 shared.

The fuzzy storage layer scan of the television pickup tube, which was applied with constant light, according to the embodiment according to FIGS. 1 and 2, is replaced according to FIG. 4 by the fact that on the photosensitive layer the tube 41 produces a blurred image of the object.

The size of the blurring can be adjusted with means known in optics be.

A corresponding effect can be achieved if you turn to 16 and 17 color filters of different colors or other optical elements for spectral Decomposition of the light in the partial beam paths and on the light-sensitive Layers of the television pickup tubes produce sharp real images.

As shown in FIG. 5, the filters 16 and 17 also be arranged at 16 'and 17', i.e. between the projectives 9 and 13 and the TV tubes. If you arrange the filters at 16 'and 17', you can the image shifts that usually occur when changing the filters on the television pick-up tubes keep it limited.

Fig. 6 shows a graph to explain the mode of operation the invention. It is here the modulation transfer functions solely due to diffraction a circular diaphragm, for example the aperture diaphragm of the objective of a microscope shown. Curve 1 applies to a smaller wavelength # 1 during the curve 2 corresponds to a larger wavelength # 2, (# 2 @ # 1) of the light used.

On the abscissa is the spatial frequency (line pairs / length unit) OF (l / mm) plotted on the ordinate the modulation transfer functions (MüF) (Output (image) contrast / input (object) contrast), which used to be the contrast transfer function was designated. The points GF1 and GF2 on the abscissa mean the limit frequencies, above which the MuF for the two wavelengths 1 and # 2 becomes zero.

If, for example, a harmonic elementary lattice is used as an object the modulation L (OF) is selected, you get the modulation I (OF) in the picture as follows: I (OF) = L (OF) MüF (OF).

This equation applies to all linear systems and all object modulations. For linear chains (e.g. chain arrangements of n optical and electronic Systems) one obtains the modulation transfer function MüF overall as the product of Modulation transfer functions MüF1, MüF2, ..., MüFn: Mtotal = MüF1. MüF2 ... MüFn.

The MuF of an opening is equal to the normalized autocorrelation integral the amplitude opacity.

For a circular aperture with the radius a one obtains up to a number of factors the course of the MüF if you move two circles of the same size against each other and the area of the two circles forms the common area. So it becomes understandable that the MüF for the shift # = O (OF = 0) is maximum and for shifts i > 2a becomes zero. The upper limit frequency GF is determined by the shift 2a. The following applies: GF = 2a / 5 B. Here, B is the image distance; furthermore, G should be the object distance, M = B / G denotes the magnification, a / B = AB denotes the aperture on the image side.

For a magnification M = B / G * 1, the spatial frequencies are OFgild And OF0bject (related to image and object) different. Object / OF picture applies = B / G = M. This means that the object-related limit frequency GFobject = 2a / 2 G = (Aobject is the object-side aperture: A0bjekt = a / G).

The equation for the object-related cutoff frequency is identical to the resolution limit dg = l / GFobject # / 2Aobject for objects illuminated from all sides, as derived from the elementary theory of the microscope (cf.

for example: Menzel / Mirande '/ Weingätner: Fourier optics and holography, 2.9-a. The Duffieux integral; Springer, Vienna-New York 1973).

The present invention consists in the quotient of two Generate images as the difference between the logarithms in two wavelengths # 1 and # 2.

It applies to the image modulation I (OF) with the object modulation L + (OF) System # 1: I (OF) # 1 = L + (OF) # 1. MüF (OF) # 1 System # 2: I. (OF) # 2 = L + (OF) # 2. MüF (OF) # 2.

Dividing these two equations gives: L + (OF) # 1 MüF (OF) # 1 I (OF) = I (OF) @ 1 / I (OF) # 2 = --------------. --------------.

L + (OF) # 2 MüF (OF) # 2 I (OF) # 1 1 OF is independent of the spatial frequency, namely N2 is a constant for each spatial frequency.

The image modulation, which depends on the spatial frequency OF, is -I (OF) thus equal to the quotient of the modulation transfer functions, which is a curve 3 is shown in the figure except for the constant factor and with increasing Spatial frequency increases. Otherwise higher spatial frequencies are generated by such a system, so smaller structures, preferred in the picture. By adding an electronic System, for example according to the embodiment of FIG. 2, the total MUF can be changed even further, for example an undesirably strong increase be made flatter again at high spatial frequencies.

Claims (1)

Claims 1. A method for increasing the beneficial magnification (Resolving power) of an optical microscope, characterized in that from Object optically either a real, sharp and a fuzzy electronic one Image is generated or that optically a real, sharp image and a real, fuzzy one Image are generated or that optically two images with different wavelengths of the light that these images are electronically scanned and amplified and a difference image is generated and that the product of the modulation transfer function in optical imaging and the modulation transfer function in electronic Gain as a function of the spatial frequency over a wide spatial frequency range is increased and / or kept constant. 2. The method according to claim 1, characterized in that for the light-optical imaging of the object ultraviolet light is used. 5. The method according to claim 1 and / or 2, characterized in that the intensity of the illuminating light by reducing it while regulating it the electronic image enhancement with a brightly adapted observer eye at the border of the image noise and then the wavelength of the illuminating light suitable until the greatest contrast of the object elements to be viewed is achieved will be changed. 4. The method according to claim 1, characterized in that the real Image of the object according to the light field, dark field, phase contrast, interference contrast, Fluorescence method or polarization or interference microscopy is generated. 5. The method according to claim 1, characterized in that the real Image generated on the photosensitive layer of a first television pickup tube and the photosensitive layer of a second television tube with constant light acted upon so that the light-sensitive layer of the acted upon with constant light Electron beam scanning the television pickup tube from the one receiving the real image TV tube (current strength) is controlled or intensity-modulated, that this electron beam is deliberately kept out of focus by defocusing, and that the signals coming from the television pick-up tubes result in a difference picture be put together. 6. The method according to claim 5, characterized in that the size the defocusing of the electron beam can be regulated. 7. The method according to claim 1, characterized in that the from Object optically generated two real images on the light-sensitive layers two television tubes, and that the blurring of one picture is adjustable using known optical means. 8. The method according to claim 1, characterized in that in the Generation of the images of different wavelengths at least one of the wavelengths is freely selectable or adjustable. 9. The method according to claim 7 or 8, characterized in that that creates images on the photosensitive layers of two television pick-up tubes and which scans the photosensitive layer of a television pickup tube Electron beam is defocused to the desired extent. 10. The method according to claim 1, characterized in that the difference image displayed on the screen of a television monitor, also color television monitor will. 11. The method according to claim 1, characterized in that the electronically Enhanced image or parts of it captured in terms of data and possibly stored will. 12. The method according to claim 1, characterized by the storage individual images or parts of images and their playback in time-lapse mode. 13. Microscope for performing the method according to claim 5, characterized through a light-optical microscope to generate the real image of the object (1) on the photosensitive layer of a first television pickup tube (30) and by a second television tube (41), which is exposed to constant light is as well as the fact that the the photosensitive Layer this Tube scanning electron beam in its beam current from the other television pickup tube (30) is controlled so that the charge image on the back of the storage layer the television pickup tube (41) by defocusing the electron beam consciously That the television pickup tube amplifier for amplification is kept out of focus of the signals, a comparator (34) for forming the difference between the signals and a monitor for visualization. the differential signals are connected downstream. 14. Microscope according to claim 13, characterized in that the gain the connected downstream of the television tube (41) exposed to constant light Amplifier (35) is adjustable. 15. Microscope according to claim 13, characterized in that on the television pick-up tube (41) subjected to constant light a focusing device (43) acts, which is connected to a control device (44). 16. Microscope for performing the method according to claim 7, characterized characterized in that for generating the two images of the object on the photosensitive Layers of two television tubes in the optical imaging beam path, a partially transparent one Mirror (6) is provided and that the optical elements are in one partial beam path blur the image they create. 17. Microscope for performing the method according to claim 8, characterized marked that in the, the two images of the object generating Imaging beam paths different color filters, interference filters or others optical components, which allow a spectral decomposition of the light, can be arranged are. 18. Microscope according to claim 5, 7 or 8, characterized by a optionally transmitted or reflected light illumination for the object (1) as well as exchangeable ones Objectives (4) 19. Microscope according to claim 5, 7 or 8, characterized in that the light-optical microscope generates one or two real intermediate images, which with With the help of a projective on the light-sensitive layers of the television recording tubes projected. 20. Microscope according to claim 5, 7 or 8, characterized in that the optical elements (21, 22, 23, 24, 26, 27), in particular the projectives (26, 27) are formed by mirrors. 21. Microscope according to claim 5, 7 or 8, characterized by the Use of a light-dark field mixing condenser 22.Microscope according to claim 5, 7 or 8, characterized in that logarithmizers are connected downstream of the amplifiers and a delay line (42) is upstream of a logarithmizer. 23. Microscope according to claim 5, 7 or 8, characterized in that the image output signal after the subtraction by a window generator (55) controlled gate is fed, which the image signal of a window generator adjustable and additionally marked on the screen of the television monitor (38) Part of the overall picture of a computer system or a registry device, for Example, a writer or the like supplies. 24. Microscope according to claim 23, characterized in that the window generator (47) an integrator (48) is connected downstream. 25. Microscope according to claim 5, 7 or 8, characterized by a the imaging beams in the television pickup tube and the imaging beams Device synchronizing beams in the monitor (37). 26. Microscope according to claim 5, 7 or 8, characterized by a the television monitor (38) upstream delogarithmizer (45), which is controlled by a switch (46) or the like can be bridged.