DE2301800B2 - Process for expanding the depth of field in optical and electron microscopic imaging - Google Patents

Description

Integrating the intensity across different object planes has serious disadvantages, which are its Prevent practical application: The integrated image consists of the overlay more or less fuzzy images, including a sharp image of an object detail for a short period of time. The blurred images superimposed on the sharp image reduce the quality of the image thus generated. The contrast at high spatial frequencies becomes low, and small object details are only imperfect seven again. The additional information about before Object planes that are not reproduced sharply are at the expense of the previously sharply reproduced object details been won.

The object of the invention is to expand the depth of field with negligible losses in image quality. This object is achieved according to the invention by a two-stage process in which in one first step by superimposing different defocused images first a modified image of the Object is generated in which all object levels with Almost the same transfer function is shown, and that in the second step the one obtained in this way Image is subjected to high-pass filtering, which compensates for the resulting drop in contrast at high spatial frequencies.

The procedure is based on knowledge. that one transmitted from a system of poor quality Image can be improved if a) the transfer process by an equation according to

Ö = Ö D

55 can be described and if b) the

tragunesfunktion D is known. See also »Opt. Acta «5, 1958. p. 256. In (2) are Ö. Ö ' the

The invention relates to a method for expanding spatial frequency spectra of the object or the image, serting the SehuiTentiefbereich.es in the optical The object 0 can then be from the image spectrum

iind electron microscopic illustration. The 60 dream with
Depth of field Ir is reached at the con-. Λ <

ventioncllcn mapping from the required resolvable distance Iv between two object points

O =

Ir <

Iv-

ID

65 determine optically by Fourier transformation, as in »Opl. Spectra «. 1971, p. 31. Since when an object is shown in depth / the wavelength of the object stretched during the image, the transfer function for each

Object level is a different one. a re-tightening according to (3) cannot initially be applied here (See also H. H. H ο ρ k i η s in "Proc. Roy Soc". A 231 [1955]. p. 91).

In the first step of the process to be patented Therefore, a modified image of the object is first generated with the details of each object plane can be mapped with approximately the same transfer function, and this image is displayed in the The second procedural step is subjected to a coherent spatial frequency filtering.

Further explanations are given on the basis of the figures. It shows

Fig. 1 schematically shows the principle of integration by moving the object along the optical axis.

2a to 2c the transfer function for the

- With regard to the depth - the edge point of the object at different object depths la, the integration area ± «, 10. 20 and 50 optical units.

Like F i g. 1 shows, the object with the depth 2 Iz is moved symmetrically to the plane of the sharp image by the distance Iz ₁ . The object is illuminated incoherently and the intensity is integrated in the image plane. So that the image scale becomes independent of the defocusing, the beam path must be telecentric on the object side.

If a linear receiver is used for integration, the transfer function of this process can be calculated from the integral over all transfer functions depending on the defocusing:

For object point 2 (located in the middle with respect to the depth dimension of the object), the transfer function of the integrated image

-ι

D ₂ = NJ

D (Z) Ref.

(N = normalization factor)

The transfer function

D ₁ = N- JD (z) dz.

When there are no aberrations. applies to the other edge point 3 D ₃ = D ₂ . It is therefore sufficient to make the transfer functions for object points 1 and 2 the same (or as similar as possible) so that points of all object planes are mapped with the same or a similar transfer function. Obviously, this goal is achieved when the integration range ± z, is very much larger than the depth extension -t Iz of the object. In order to achieve a high signal-to-noise ratio, however, the shift in the direction of the optical axis should remain as small as possible. The calculation shows that the difference between the transfer functions D _{1 and D 2 is} negligible if the integration area is about twice as large as the object depth (see Much O. H auster in "Opt. Comm." 6 [1972]).

P. 38). This can be seen from the diagrams in FIGS. 2a._2b, 2c. The transfer function D ₁ for the Ra:; d point at different object depths Iz is plotted against the standardized spatial frequency S in them.

Displacements on the optical axis are given in optical units: The quantities z, and Iz are linked with u, and I «by equation (6):

Ό]

(/ = - k- z- sin ² u .

Here k = 2.-Τ / Λ and sin ii is the aperture of the imaging system. The greater the object depth becomes relative to the integration area, the more the transfer function of the edge plane deviates from that of the middle object plane (Ia = O) . However, as the diagrams show, the deviation is negligible if the object depth is not significantly greater than half the integration area: There is no discernible difference between the image quality of different object planes.

The goal of the first step has thus been achieved. A modified image of the object was produced, whereby all object levels are mapped with the same transfer function.

The incoherently integrated image generated as described is subjected in the second step to a coherent spatial frequency filtering according to (3). The filter must increase the contrast at high spatial frequencies, which was reduced by the first step according to FIGS. 2a to 2c. This succeeds if the frequency response of the resharpening filter is proportional to the reciprocal transfer function 1 / D ₁ * 1 / D ₂ . Since the transfer function of the integrated image depends on the integration area, the resharpening filter must be matched to the required depth of field or to the aperture of the imaging system.

Advantages of the procedure

With the invention is an expansion of the depth of field possible with, in principle, not reduced image quality and resolution. The degree the expansion that goes beyond the conventional mapping depends essentially only on the Noise from the storage material used for integration. In preliminary tests with the help of photographic Integration, a 25-fold expansion of the focus delivery area was achieved. Another increase seems possible.

The resharpening filter has a real frequency response without zeroing. This is a decisive advantage for practical use: It is mil can be implemented optically or electronically with little effort. This also includes the filter at higher Spatial frequencies has a linearly increasing frequency response.

Since the condition "object depth equals half the integration area" does not have to be strictly adhered to Fs. F i g. 2). For practical use, one set of fewer filters is sufficient to filter objects with different Process depths.

The following methods are described for practical implementation:

1. Application to the light microscope im
off-line procedure

The first step, the generation of the integrated image, is carried out by evenly focussing through the lines by a distance about twice as large as the object depth symmetrically to the Mittenebenc des Object. During focusing, for example, by motor drive of the fine drive on the microscope a photo plate exposed in the intermediate image plane. The plate is reverse developed with x = -2 and subjected to a coherent filtering in the second step. In the focal plane of the Fourier transform lens the filter arrangement creates the spectrum of the integrated image. A Brought high-pass filter, its amplitude transparency proportional

(Si means integral sine)

is. if constant contrast for all spatial frequencies should be achieved. The filter characteristic depends on the shift «during the recording. A sufficient approximation is a frequency-linear high-pass filter, which also has a constant component according to (7) lets through.

Should a transfer function through the filtering proportional to I - .v / 2 are achieved, the amplitude transparency of the filter becomes proportional

The present results are due to the closed display option for a square Specified aperture. They differ from the numerical values for the round pupil only insignificantly.

The filter can be implemented for example by a photo plate that is attached to a rotating stencil like this was exposed that the amplitude transparency / proportional to (7) or (8 (becomes. Phase disturbances are compensated by immersion. In the initial image plane the filter arrangement then produces the filtered image with an increased depth of field.

2. Apply in real time

For a real-time process. in which the photographicc If processing is not required, the specimen is mounted on an object table that faces dor optical axis executes a triangular shape: the oscillation amplitude is equal to twice the object depth. The oscillation takes place symmetrically to the central plane of the object.

ίο The integrated image is on the cathode of a Television pick-up tube (e.g. Image-Orthicon) generated when the oscillation frequency of the stage is equal to the scanning frequency of the camera. The video signal contains the information about the inte'S grated picture. In the second step, the video signal is subjected to suitable high-pass filtering. Of the The frequency response of the filter was described under 1. A picture of the object can be displayed on a monitor can be observed with an increased depth of field.

With this method, the filtering is only complete in the row direction. Contours that are not perpendicular running towards the direction of the line, the more incompletely the filtering is carried out, the more they move in the direction of the line ^ 5 run. As for the picture impression, this disadvantage does not weigh heavily, a second filtering perpendicular to the first, which is possible in principle, can be dispensed with.

The advantage of the electronic method is that the frequency response of the electronic filter is easy to change. Therefore, when changing lenses or changing the depth of field, the Adjustment possible quickly. Real-time processing means that the filter can also search for the best visual image impression can be set.

3. Echetime process with phototropic materia!

The object is moved to 2 with the help of a vibrating table. The integrated image is

bleaching of a previously blackened photochromic material generated in the intermediate image plane of the microscope. The information is read out and the filtering is carried out according to FIG. 1 by means of coherent filtering at a wavelength that is not blackening or bleaching Has an effect on the photochromic recording material.

1 sheet of drawings

Claims (9)

Patent claims: 1. Method of enlarging the depth of field beyond the conventional one Figure given limit addition, characterized in that in a first First, a modified image is created by superimposing various defocused images of the object is generated in which all object levels with almost the same transfer function are mapped, and that in the second step the image obtained in this way is high-pass filtering is subjected, which compensates for the resulting drop in contrast at high spatial frequencies. 2. The method according to claim 1, characterized in that a photographic one for the overlay Recording material is used and the sharpening by coherent optical Filtering takes place. 3. The method according to claim 1, characterized in that that the coherent filtering with different filters depending on the one to be processed Object depth is made. 25 - 4. The method according to claim I, characterized in that that the superimposition takes place with the help of a television tube while the object moved on a vibrating table with a suitable frequency in the direction of the optical axis and that the filtering is achieved in the second step by suitable filtering of the video signal will. 5. The method according to claim 1, characterized in that that the overlay takes place with the help of a photochromic storage material, which in the / wide step the object of the coherent optical filtering becomes. 6. Process according to claims 1 to 5, characterized in that in the case of an electron microscope Illustration the depth of field is enlarged. 7. The method according to claims 1 to 5, characterized in that with electron microscopic Figure the focusing tolerance is increased. 8. The method according to claims 1 to 5, characterized in that the depth of field is enlarged in the case of bladder chamber exposures. 9. Process according to claims 1 to 5, characterized in that the depth of field is enlarged in X-ray slices. used radiation. Should the resolution be doubled, so I .ν are halved, it follows from (I). that the Depth range Ir. in which the image is sharp, is halved to a quarter. According to the invention the depth of field is significantly beyond the limit of conventional imaging given by (I) expanded. This increases the demand for high lateral resolution and, at the same time, a high range of focus fulfilled This is general with the Abbilduim with a high aperture, especially of particular interest in microscopic imaging. It is from DT-PS 10 29 591 and from the magazine "Applied Optics". Year 1971. p. 208. that by scanning the object in the depth direction, an expansion of the depth of field can be achieved. The different levels of the object are periodically focused. If this happens quickly enough, the images that emerge one after the other are superimposed: the observer recognizes object details in an extended depth range.