Microscopic imaging method using a correction factor

Abstract

A microscopic imaging method, includes illuminating a specimen with illumination radiation and capturing detection radiation along a detection axis. The detection radiation is caused by the illumination radiation, at a first time as a wide-field signal and at a second time as a composite signal. The composite signal is formed by a superposition of a confocal image and a wide-field image; extracting the confocal image by subtracting the wide-field signal from the composite signal, wherein a correction factor is used. A current correction factor is ascertained for each executed imaging and/or for each imaged specimen (1) and the confocal image is extracted using the respective current correction factor.

Claims

1. Microscopic imaging method, comprising: illuminating a specimen with illumination radiation and capturing detection radiation along a detection axis, said detection radiation having been caused by the illumination radiation, at a first time as a wide-field signal and at a second time as a composite signal, forming said composite signal by a superposition of a confocal signal and a wide-field signal, extracting the confocal signal by subtracting the wide-field signal from the composite signal, wherein a correction factor is used, ascertaining a current correction factor for each executed imaging and/or for each imaged specimen and extracting the confocal signal using the respective current correction factor, wherein, for the purposes of ascertaining the current correction factor in a correction plane chosen along the detection axis at a distance from a surface of the specimen, at least one correction wide-field image and one correction composite image are captured in each case, the image data thereof are ascertained and a current correction factor is ascertained on the basis of the ascertained image data; wherein the distance of the correction plane is chosen to be so large that no structures of the surface pass through the correction plane.

2. Method according to claim 1, wherein the distance of the correction plane is chosen from a range of four to six full widths at half maximum of the pointspread function of the objective lens used to capture the detection radiation.

3. Method according to claim 1, wherein at least one correction wide-field image and one correction composite image are captured in each case in a correction plane in front of and behind a current focal plane.

4. Method according to claim 1, wherein the current correction factor is ascertained from a mean brightnesses of a correction wide-field image and a correction composite image.

5. Method according to claim 1, wherein a current correction factor is ascertained for each pixel pair of the correction images.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The invention will be explained in more detail below on the basis of figures and exemplary embodiments. In the figures:

(2) FIG. 1 is a schematic illustration of a section through a z-stack (orthoview);

(3) FIG. 2 is a schematic illustration of a first option for determining a current correction factor;

(4) FIG. 3 is a graphical representation of a surface of a specimen with selected signal curves according to the prior art;

(5) FIG. 4 is a graphical representation of the surface of the specimen with selected signal curves according to the method according to the invention;

(6) FIG. 5 is a graph of signal curves along a z-stack of a metal surface machined with material removed according to the method of the invention;

(7) FIG. 6 is a graph of signal curves along the z-stack of the metal surface machined with material removed according to the prior art;

(8) FIG. 7 is a graph of signal curves alone a z-stack of a surface of a specimen provided with metallic lacquer according to the method of the invention;

(9) FIG. 8 is a graph of signal curves along the z-stack of the surface of the specimen provided with metallic lacquer according to the prior art;

(10) FIG. 9 is a graph of signal curves along a z-stack of a surface of a specimen made of paper according to the method of the invention; and

(11) FIG. 10 is a graph of signal curves along a z-stack of the surface of the specimen made of paper according to the prior art.

DETAILED DESCRIPTION OF THE DRAWINGS

(12) FIG. 1 shows an example of a so-called orthoview of a z-stack of images of a specimen 1. In each case, the images were calculated according to the formula
Confocal signal=(composite signal)−n*(wide-field signal).

(13) The position of the surface Ao of the specimen 1 is indicated by an arrow and by a dotted line. Moreover, there is a plot of the intensity maxima along the x-axis. The x-axis extends orthogonal to the z-axis and approximately parallel to the surface Ao.

(14) Over large sections of the x-axis, the surface Ao is characterized by strong reflections and intensity maxima connected therewith, approximately level with the same z-coordinate. The intensity maxima are found at higher z-coordinates in a section in the left-hand part of the curve Imax. This may mean that a depression or an elevation is situated in this region of the specimen 1, which corresponds to the relevant x-coordinates, depending on how the coordinate system in FIG. 1 was placed in advance.

(15) An option for ascertaining the current correction factor n is illustrated schematically in FIG. 2. Plotted along the z-axis are the values of the amplitude of the wide-field signal WF and of the composite signal CI (composite image) in exemplary fashion. A maximum (peak) of the amplitude values of the composite signal CI can be identified in a focus plane. By way of example, this intensity maximum Imax denotes the surface Ao of the specimen 1 (see FIG. 1, for example). The amplitude values xWF of the wide-field signal WF and the amplitude values xCI of the composite signal CI are ascertained in a correction plane with the z-coordinate zn. The current correction factor n can be ascertained from the ratio of amplitude values xWF and amplitude values xCI.

(16) FIG. 3 schematically shows the measurement results of a specimen 1, a groove-shaped depression 2 running through its surface Ao. The image data calculated by a method according to the prior art show high intensity peaks Imax along the edges of the depression 2. As a result, an elevated edge of the depression 2 is depicted in an image resulting therefrom, even though such an elevation is not even present in actual fact.

(17) By contrast, the intensity peaks Imax along the edges of the depression 2 are substantially lower if the image data were ascertained by means of the method according to the invention (FIG. 4). The depression 2 is depicted without an elevated edge, or with only a very small elevated edge, which corresponds to the actual topography of the specimen 1.

(18) In the further FIGS. 5 to 10, the image data obtained by means of the method according to the invention are plotted in each case as height values along the z-axis against the x-axis. Instead of along the x-axis, the height values could also be plotted along the y-axis that extends orthogonal to the x-axis and to the z-axis (not shown).

(19) FIG. 5 shows image data of the topography of a specimen 1 made of a metallic substance. The specimen 1 was machined by milling. The individual tracks of the milling tool can be identified by the periodic sequence of amplitude values around the zero.

(20) The image data of the same specimen 1 show intensity peaks Imax in the regions of the periodic changes in direction, which do not reproduce the actual profile of the surface Ao (FIG. 6). Already the fact that the intensity peaks Imax are extremely narrow indicates the presence of imaging aberrations instead of actual surface forms.

(21) The same can be seen in FIGS. 7 and 8. When using the method according to the invention, a surface Ao of the specimen 1 provided with metallic lacquer is identified, and depicted, with a topography with a spread of approximately 1.5 to 2 μm about the zero position (FIG. 7). By contrast, the topography of the specimen 1 created by means of a method according to the prior art has a spread of approximately 5 to more than 10 μm (FIG. 8).

(22) Very similar results are obtained in the case of a specimen 1 made of paper. FIG. 9 shows the comparatively rough surface Ao of the specimen 1 with a spread of approximately 10 μm about the zero position.

(23) By contrast, the incorrectly occurring intensity peaks when using a method according to the prior art lead to a spread of 30 to 40 μm about the zero position.

(24) This exemplary embodiment shows that specimens 1 made of paper or with a fluorescent (autofluorescent) surface Ao can be examined in respect of their topography with the aid of the method according to the invention. By contrast, methods according to the prior art do not lead to a satisfactory result or require additional steps in order to reduce the intensity peaks.

(25) While the invention has been illustrated and described in connection with currently preferred embodiments shown and described in detail, it is not intended to be limited to the details shown since various modifications and structural changes may be made without departing in any way from the spirit of the present invention. The embodiments were chosen and described in order to best explain the principles of the invention and practical application to thereby enable a person skilled in the art to best utilize the invention and various embodiments with various modifications as are suited to the particular use contemplated.

REFERENCE SIGNS

(26) 1 Specimen 2 Depression Ao Surface (of the specimen 1) CI Composite signal Imax Intensity maximum, intensity peak WF Wide-field signal xCI Amplitude value (of the composite signal CI) xWF Amplitude value (of the wide-field signal WF) zn Position (of the correction plane)