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Kind code of ref document: Ref legal event code: Country of ref document: Year of fee payment: The system has an image sensor , readout electronics and a controller 35 for configuring at least one gain so that a boundary charge value is a small first charge value, binary numbers are received by the readout electronics representing an image detected by the image sensor and the gain is readjusted so the boundary charge value is higher than for the preceding setting if at least one binary number represents a charge value above a maximum value in a repeating process.

An independent claim is also included for a method of recording a series of fluorescence images using a camera system. Die Erfindung betrifft ein Mikroskopiesystem und ein Aufnahmeverfahren zur Sichtbarmachung einer Fluoreszenz. The invention relates to a microscopy system and a recording method for visualizing a fluorescence.

Fluorescent dyes are used in medicine and biology for various purposes, such as for the visualization of specific tissues, tissue structures, tissue functions, etc. This one to be examined tissue sample or to be examined patients fluorescent dye or a precursor of such a fluorescent dye is administered. Der Farbstoff reichert sich in bestimmten Gewebearten bzw.

Gewebearten sichtbar gemacht und von einem Beobachter lokalisiert werden. The dye accumulates in particular types of tissue or tissue structures of the tissue sample or the patient, and by observing the fluorescence light, this tissue structures or tissue types can be visualized and localized by an observer.

For this purpose, special optical aids continue to be used to make the weak may fluorescent light for the observer clearly visible. Information in this document is fully incorporated here. With this known system it is possible to observe the resulting fluorescence after the administration of the fluorescent dye or its precursor, and receive corresponding images to archive and reproduce as a film. A problem with the conventional system is that in some tests the captured images is too low quality, such as a low brightness or a weak contrast to show a significant fluorescence, and occasionally in the captured images is not noticeable.

A test must be repeated with a higher concentration of fluorescent dye and other observation conditions then optionally to achieve a desired goal. Es ist eine Aufgabe der vorliegenden Erfindung, ein Mikroskopiesystem und ein Aufnahmeverfahren zur Sichtbarmachung einer Fluoreszenz vorzuschlagen, bei welchem eine auftretende Fluoreszenz mit besserer Wahrnehmbarkeit darstellbar ist. It is an object of the present invention to provide a microscopy system and a receiving method for visualizing a fluorescent propose in which a fluorescence occurring with better visibility can be displayed.

According to embodiments of the invention, for this purpose, an image sensor and readout electronics for the image sensor is used. The image sensor comprises a plurality of pixels in the accumulation of heat generated by incident radiation load, and the readout electronics convert the accumulated charge in the pixels in binary numbers.

Hierbei ist ein Gain der Ausleseelektronik einstellbar. Here, a gain of the readout electronics is adjustable. Zu Beginn einer Untersuchung wird der Gain auf einen geeigneten Maximalwert eingestellt.

At the beginning of an investigation, the gain is set to an appropriate maximum value. If it is found during the investigation that one or more brightness levels of an acquired image exceed a suitably chosen maximum brightness value, the gain is reduced. By adjusting the gain before the measurement to a maximum value ensures that a particularly weak fluorescence in the context of methods available leads to a significant image signal.

Nach dem Applizieren des Fluoreszenzfarbstoffes bzw. After the application of the fluorescent dye or its precursor, the image brightness of the fluorescent image is continuously increasing.

Thus, an optimal recording images of increasingly stronger fluorescence phenomenon, which is already high-contrast images are taken at the beginning of the fluorescence phenomenon, where the detectable fluorescence intensities are low is carried out by the continuous reduction of the gain. In general, the binary numbers each representing the assigned to the individual pixels of the image sensor detected brightness values.

In a binary numbers generated from the displayed image, such areas then appear light, which pixel regions of the image sensor correspond to which incident radiation much, while pixel areas of the image sensor, to which little radiation is incident, then appear dark in the displayed image.

However, it is also possible that the binary code the amount of charges generated in the pixels differently. For example, it is possible that the binary numbers encode the brightness inverse, so that pixel regions of high incident radiation intensity in the corresponding areas of the displayed image produced will appear dark, while appear light in the displayed image areas are assigned to which pixel regions of the image sensor, to which little radiation is incident.

The readout electronics convert the accumulated amount of charge in a group of pixels in the plurality of binary numbers so as to ensure that each binary number has an associated amount of charge range, the pairwise different binary numbers associated amount of charge ranges do not overlap with each other substantially.

Here, the charge quantities from the converted binary numbers in amount of charge areas are smaller than a threshold charge amount. A accumulated in a group of pixels charge amount which is greater than the limit amount of charge is converted to a specific binary number which can also represent an overflow. For example, the charge quantities in eight-bit binary numbers can be converted from 0 to , and then the binary number represents the overflow or a charge amount which is greater than the limit amount of charge.

Die Grenzladungsmenge ist durch den einstellbaren Gain bestimmt. The limiting amount of charge is determined by the adjustable gain.

The image sensor may be, for example, a CCD sensor, wherein in the individual pixels accumulated charges are shifted out in the row direction serially from the sensor, the charge amounts of each line is serially converted into voltages, these voltages are amplified, and the amplified voltages by an analog -digital converter to be converted into binary numbers. Amplifying the voltages is done with a voltage amplifier whose gain can be adjustable.

Ebenso kann ein Gain des Analog-Digital-Wandlers einstellbar sein. Similarly, a gain of the analog-to-digital converter may be adjustable. Here, too, each one gain can be adjusted both in the conversion of the amounts of charge to voltages as well as a reinforcement of these tensions and also in a conversion of the increased tensions in binary numbers. According to one embodiment of the invention will be received binary numbers representing a detected by the image sensor image stored in a recording medium.

According to an exemplary embodiment, this storing in the recording medium is carried out only from a starting time, so that a plurality of images which were detected before the start time point can not be recorded. Alternatively, an embodiment provides that all the detected images are stored in the recording medium and that in addition a mark is stored in the recording medium, which indicates a recorded image which has been recorded at a time near the start time.

Der Startzeitpunkt kann auf verschiedene Weisen ermittelt werden. The starting time can be determined in various ways. According to one embodiment of the invention is provided to determine the start timing depending on a contrast of the captured images.

Zu Beginn der Aufnahme, wenn sich noch kein Fluoreszenzfarbstoff in dem untersuchten Objekt angereicht hat, wird ein Kontrast des aufgenommenen Fluoreszenzbildes nicht vorhanden bzw. At the beginning of the recording, if no fluorescent dye has enriched in the object being examined, a contrast of the captured fluorescence image will be absent or minimal.

With increasing accumulation of the fluorescent dye in the object, the contrast increases and will exceed a lower threshold value. The time at which the lower threshold value is exceeded, can then be chosen as the start time.

The group of pixels whose accumulated charge amount is converted into a binary number, may comprise a single pixel.

However, it is also possible that the group of pixels includes several pixels. According to one embodiment of the invention, for this purpose, an image sensor such as a CCD sensor may be used, which already leads together when linewise readout of pixel charge amounts of each of four adjacent pixels and converts it into a respective binary number. According to another embodiment, a plurality of binary numbers, each representing charge quantities of one or more pixels are averaged, so that, merged from a plurality of binary numbers thus represent an intermediate result of the conversion to a final binary number.

In this way, among other things, a data reduction can be achieved as well as a displayable image having as compared to the pixel resolution of the image sensor a reduced resolution, which, however, it is reduced to a visible image noise with respect and ultimately results in a more pleasing image. Als Fluoreszenzfarbstoff kann ein beliebiger Fluoreszenzfarbstoff bzw. As a fluorescent dye, any fluorescent dye or precursor thereof may be used.

Examples include indocyanine green ICG , the excitation wavelength is in a range from about nm to about nm and the fluorescence wavelength in a range from about nm to about nm. Another example of a fluorescent dye precursor is aminolevulinic acid 5-ALA , which leads to a fluorescent substance whose excitation wavelength is in a range from about nm to about nm and the fluorescence wavelength in a range from about nm to about nm.

Embodiments of the invention are explained below with reference to drawings. In einer Objektebene des Objektivs 5 ist ein zu untersuchendes Objekt 9 angeordnet. A schematically illustrated in Figure 1. The microscopy system 1 comprises a microscopy optics 3 having an objective lens 5 having an optical axis 7. In an object plane of the lens 5, an object to be inspected 9 is disposed.

From the object 9 outgoing light is converted by the objective lens 5 in a parallel beam 11, in which two at a distance from the optical axis 7 arranged zoom systems 12, 13 are arranged, and 14 and 15 to pick out a respective partial beam from the parallel beam 11 and perform 16 and 17 in Figure 1, not shown, deflecting prisms oculars into which a viewer perceives with his left eye 18 and right eye 19 insight in order to perceive a magnified representation of the object 9 as an image.

In the partial beams 15, a partially transparent mirror 21 is arranged to couple a portion of light as a beam 23, which is divided by another beam splitter 25 into beams 27 and The images taken by the camera 32 are transmitted as image data via a data line 33 to a control 35th. The controller 35 transfers the captured by the cameras 32, 43 images in turn as the image data on a line 47 to a head-mounted display device "head mounted display" 49, which is so carried by a user of the microscopy system 1 as a pair of spectacles on the head that in the display device 49 built-in screens, which are designated in Figure 1 schematically by 51 and 52, can be viewed by the user with its left or its right eye.

Thus, the user who does not have the opportunity to also receive, directly into the eyepieces 16 to take 17 insight, on the display device 49 a stereoscopic impression of the object 9, by images, which represent images of the object 9 in visible light. The beam 29 is transferred via a camera adapter optical system 53 onto a light detection surface of a camera 55 such that it can accommodate a fluorescence of the object.

For this purpose, a filter is in the beam 29 in front of the camera 55 is arranged 57 which is tuned to the wavelength of fluorescence of the fluorescent dye used. In the example explained here, the fluorescence of the fluorescent dye ICG is to be observed at about nm.

Entsprechend weist der Filter 57 eine Transmissionscharakteristik auf, wonach nur Licht oberhalb eines Schwellenwerts von etwa nm transmittiert wird und Licht unterhalb des Schwellenwerts von nm nicht transmittiert wird. Accordingly, the filter 57 has a transmittance characteristic whereby only light above a threshold value of about nm is transmitted and light is not transmitted is below the threshold of nm.

Thus, the camera can record images of the object 55 9, which represent a distribution of the dye in the object 9, provided that the fluorescence of the dye is excited by a method described below illumination system 63 of the microscope system. The controller 35 transfers the captured by the camera 55 images in turn as the image data via a line 67 to an LCD display 69, which in turn represents the image data as an image, which is brought to superimpose over a collimation lens 70 and a coupling mirror 68 with the sub-beam 15 so that the image of the display 69 is also of the eye 19 of the user in superposition with the direct optical image of the object 9 perceptible.

Some 69, the LCD display the image captured by the camera 55 in the infrared intensity image, for example, in green color. Here, green is selected as the color for the representation, among other reasons, because the object 9 as human tissue in the visible range, the color green only contains a very small scale. The controller 35 further processes the data transmitted to the screen 51 of the display device 49 image data such that the screen 51 is picked up by the camera 55 with infrared light images in superposition with the images, the camera takes 32 with visible light so that the user 49 carries the display on the head with his right eye also receives a superimposed representation of the captured visible light images and captured with infrared light images.

Then, the user also receives a stereoscopic impression of the object 9 in infrared light. The image data recorded by such an additional camera can be transmitted to the screen 52 of the display 49 then also, so that the display supporting user gets a stereoscopic impression of the distribution of the fluorescent dye in the tissue region. By the glass fiber bundle 77 the light is transported to the vicinity of the objective lens 5 enters there from to an outlet end 78 of the fiber optic bundle 77 and is then collimated by a collimating optics 79 to an antibody directed to the examined object 9 illuminating light beam 81st Anstatt der Halogenlampe kann auch jegliche andere Art von Lampe eingesetzt werden, beispielsweise eine Xenonlampe.

Instead of the halogen lamp, any other type of lamp can be used, for example, a xenon lamp. The illumination system 63 further comprises a filter plate 83, which is composed of two adjacent filters 84 and 85 and which is in a direction indicated by an arrow 88 in Figure 1 in the direction back and herverlagerbar by means of a controlled by the controller 35, the drive 87 so that the filter is disposed in the beam 74 in a first position 84 of the plate 83 and is disposed in a second position of the plate 83 of the filter 85 in the beam 74th.

A transmission characteristic of the filter 84 is configured such that the filter 84 visible light and light up to a sharp edge at about nm and does not transmit well transmitted right above the edge at nm light substantially.

Der Filter 84 wird im Strahlengang 74 angeordnet, wenn die Fluoreszenz des Farbstoffs in dem untersuchten Gewebebereich 9 beobachtet werden soll. The filter 84 is arranged in the beam path 74 when the fluorescence of the dye is to be observed in the examined tissue region. The edge is at nm that is higher than the excitation wavelength 59 of the dye ICG, so that the fluorescence is excited with the light beam 81 both which is observable for the pictures taken by the camera 55 images then, as well as the region of tissue is illuminated with visible light so that it can be viewed as a normal light image by either insight into the eyepieces 16, 17 or by observing the image taken by the cameras 32, 43 images.

However, the edge is lower than the emission wavelength of the fluorescent dye from nm to nm, so that the object is not illuminated with fluorescent light and the detected fluorescent light by the camera 55 is thus caused solely by fluorescent fluorescent dyes.

A transmission characteristic of the filter 85 is configured such that the filter 85 visible light and to an edge at about nm well transmitted right and light with wavelengths above the edge at about nm does not transmit substantially. The filter 85 serves as a heat-absorbing filter and eliminates long-wave radiation from the illumination light beam 81 to protect the examined tissue 9 from unnecessary thermal stress.

The edge at about nm is below the excitation wavelength of nm of the fluorescence dye ICG, so that the fluorescence of the dye is not substantially excited, when the filter is arranged in the beam 74 85th.

Figure 2 illustrates functions of the camera 55 and the controller