Optical: systems and elements – Deflection using a moving element – Using a periodically moving element
Reexamination Certificate
2001-05-17
2004-08-03
Robinson, Mark A. (Department: 2872)
Optical: systems and elements
Deflection using a moving element
Using a periodically moving element
C359S368000, C435S173100
Reexamination Certificate
active
06771405
ABSTRACT:
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority of a German patent application 100 24 404.1 filed May 19, 2000 which is incorporated by reference herein.
FIELD OF THE INVENTION
The present invention concerns a method and an apparatus for scanning a specimen with a light beam of a light source, preferably in confocal scanning microscopy, the light beam being deflected with a beam deflection device and the scanning operation being controlled by a control device.
BACKGROUND OF THE INVENTION
In confocal scanning microscopy, a specimen is scanned with a focused light beam; this is generally achieved by tilting two mirrors arranged in the beam path of the confocal scanning microscope. The focus of the light beam is thereby moved in the focal plane, the deflection directions of the light beam most often being arranged perpendicular to one another so that, for example, one mirror deflects the beam in the X direction and another mirror deflects the beam in the Y direction. The motion or tilting of the mirrors is usually brought about with the aid of galvanometer actuating elements.
The intensity of the light coming from the specimen is measured at definable time intervals during scanning; to generate an image, the light intensity value of a specimen point must be unequivocally allocated to the associated scan position of the light beam. For this purpose, the status data for the adjusting elements of the mirrors, i.e. the galvanometer actuating elements, is usually also continuously measured.
In many applications, especially in physiology, the specimen must be observed with a confocal scanning microscope during or shortly after an external influence. These operations are in most cases highly time-critical, so that the instant of the influence must be synchronized with the scanning operation or an image taken by the confocal scanning microscope. The purpose of the physiological applications is, for example, to apply voltage using the “patch-clamp” technique to the specimen being detected, or to inject chemicals, so as to detect and analyze the specimen's reaction to the influence, during or immediately after the influence, using confocal images.
A direct synchronization of the scanning operation of commercially available confocal scanning microscopes with an external influence on the specimen being examined has not hitherto been provided. As an alternative, an internal electrical line synchronization signal of the control device of the confocal scanning microscope is used to generate a synchronization signal usable for the purpose. This merely makes a signal available to a user at the beginning of each scan line. It is not possible with this synchronization method to ascertain directly when the light beam actually reaches the location in the relevant specimen region being externally influenced. Instead, the user must have a further logic unit count the number of lines until the light beam arrives at the corresponding line of the relevant specimen region. He or she must furthermore have a calculation made, again by an external logic unit, of the time required for the light beam to travel from the beginning of the line to the relevant specimen region. This procedure entails considerable difficulty, however, for example because the scanning speed of the light beam is generally variable and this must be taken into account by the external logic unit. A delay value must therefore be calculated as a function of the scanning speed of the light beam and the corresponding line number or local coordinate. This value must be added to the signal of the first line of an image so that the influence on the specimen can be exerted at the time when the light beam will actually reach the relevant specimen region.
SUMMARY OF THE INVENTION
It is therefore the object of the present invention to describe a method for scanning a specimen with a light beam of a light source, preferably in confocal scanning microscope, with which a specimen can be scanned, with the greatest possibly accuracy in time, in order to trigger a measurement operation during or shortly after an external influence.
The above object is achieved by a method for scanning a specimen with a light beam of a light source comprising the steps of:
deflecting the light beam with a beam deflection device;
controlling scanning operation by a control device;
providing by the control device, as a function of at least one definable scan position, at least one signal; and
influencing thereby the specimen and/or for triggering a measurement operation.
It is a further object of the present invention to provide an apparatus for scanning a specimen with a light beam of a light source, preferably in confocal scanning microscope, with which a specimen can be scanned, with the greatest possibly accuracy in time, in order to trigger a measurement operation during or shortly after an external influence.
The above object is accomplished an apparatus for scanning a specimen with a light beam of a light source, comprising:
a beam deflection device for deflecting the light beam;
a control device for controlling the scanning operation and providing at least one definable scan position and the control device makes available a signal for influencing the specimen and for triggering a measurement operation; and
a logic unit for compensation for differences in the transit time of the scanning light beam.
What has been recognized according to the present invention is firstly that synchronization of the external influence on the specimen and of a measurement operation can be simplified if a signal for influencing the specimen or for triggering the measurement operation is made available by the control device at exactly the correct time. The external logic unit thus becomes superfluous, thus advantageously making possible a direct coupling, for example, between the unit for influencing the specimen and the confocal scanning microscope. In particular, the user no longer needs to program or configure the logic unit, for which purpose the internal details of the change over time in the scanning operation of the light beam must be known, to say nothing of the considerable electronics-related and hardware-related programming knowledge needed for the purpose.
According to the present invention, the control device makes the signal available as a function of at least one definable scan position. This definable scan position could be, for example, exactly the image point that is to be scanned during or after an external influence on the specimen, so that an observation of the reaction of the specimen to the influence is possible by way of an accurately timed image. In very general terms, the definable scan position is a relevant specimen region. Since the control device controls the specimen scanning operation, the control device can be configured in such a way that it also ascertains the instant at which the light beam arrives at the definable scan position.
The local coordinates of the definable scan position could be stipulated on the basis of detected specimen data. For example, a specimen for microinjection could be prepared with a corresponding microinjection apparatus, and this preparation could be performed in a conventional transmitted-light microscopy mode. A confocal scanning microscope image of the specimen would then be detected, the designated focal plane being the specimen plane in which the tip of the microinjection needle is located. On the basis of the detected confocal scanning microscope image, the tip of the microinjection needle could be exactly stipulated as the definable scan position.
Stipulation of the local coordinates of the definable scan position could be accomplished automatically or interactively. For example, the image of the tip of the microinjection needle could be automatically recognized on the basis of its pattern, using a pattern recognition algorithm. Alternatively, the user could interactively stipulate the local coordinates of the definable scan position. It is similarly conceivable, after the automatic determina
Leica Microsystems Heidelberg GmbH
Robinson Mark A.
Simpson & Simpson PLLC
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