Radiant energy – Luminophor irradiation
Reexamination Certificate
2001-06-14
2004-06-08
Porta, David (Department: 2878)
Radiant energy
Luminophor irradiation
C250S459100
Reexamination Certificate
active
06747280
ABSTRACT:
The invention relates to a method for the individual adaptation of excitation intensities in a multiband fluorescence microscope and a multiband fluorescence microscope to execute the method corresponding to the features of the introductory clause of the independent claims.
With the multiband fluorescence microscope, the user frequently confronts the problem that the different fluorescence bands in the microscopic image have varying intensities and are not uniformly visible. The cause lies frequently in the difference in excitation intensities in the illumination beam path or even in the varying blockage of the fluorescence intensities by a barrier filter in the imaging beam path. Also, different concentrations of the fluorescence dye for the different excitation bands, even when staining the objects to be considered, or the progressive bleaching of the dye, the so-called fading, lead to differing intensities of the fluorescence bands in the microscopic image. The different intensities of the fluorescence bands prove to be especially problematic then if the microscopic image is to be photographically recorded. Then the portion of the fluorescent light of weak intensity on the photo is too weakly reproduced or is not visible at all. Only with intensities of the fluorescence bands that are as uniform as possible can there be defect-free photos of the microscopic image.
U.S. Pat. No. 5,371,624 specifies a fluorescence microscope having only two excitation bands in which the intensities of the two excitation bands can be intermittently affected. It includes an illumination beam path having a light source and an excitation filter that produces several excitation bands of varying light wavelengths from the light of the light source. Furthermore, it has a splitter mirror, an output filter (also designated as a barrier filter or emission filter) for the fluorescent light and a filter element for affecting the intensities of the excitation bands.
The filter element can, by tilting continuously with respect to the optical axis, be switched between two limit positions having two fixed, predetermined values of the transmission factors of one or the other excitation band. In the one limit position, only the first excitation band is attenuated, in the other limit position only the second excitation band is attenuated and between the two limit positions, neither of the excitation bands is attenuated. A drop in the transmission factor of the particular excitation band is only possible up to the fixed, predetermined value. However, variation between a maximum transmission and a zero value, i.e., up to full suppression of one of the two excitation bands, is not possible. Moreover, only two excitation bands can be affected.
It is object of the present invention to specify a method for individual adaptation of excitation intensities in a multiband fluorescence microscope and a multiband fluorescence microscope to execute the method, in which one or more of the excitation bands can be partially or completely filtered out. For this purpose the transmission factor for each excitation band is to be continuously adjustable using simple means.
This objective is met by the invention via the characterizing features of the independent claims. Advantageous embodiments arise from the features of the dependent claims.
The method of the invention starts from a known multiband fluorescence microscope in which several excitation bands of varying wavelengths are produced using an excitation filter in an illumination beam path from the light of a light source. The excitation bands illuminate a fluorescence object prepared using fluorescence dyes and are converted by it into frequency-shifted fluorescence bands.
According to the invention, the fluorescence intensities of the individual fluorescence bands are first determined in the microscopic image and compared to previously set intensity setpoint values. The fluorescence intensities can be determined, for example, either visually or by using an intensity meter. This can consist for example of a video or CCD camera having an image analysis system connected in an outgoing circuit.
In this context a different intensity setpoint value can be set for each fluorescence band. However, in practice, the setpoint values are oriented toward concrete problems posed by the microscope user. If, for example, the microscopic image is to be documented either photographically or by video camera, and thus each fluorescence band in the photo or in the video image is to be reproduced with equal brightness, then the level of the setpoint values depends on the spectral sensitivity of the film or the camera. Therefore, their spectral sensitivity must be taken into consideration in the determination of setpoint values for the various excitation bands.
Therefore, the desired setpoint values must all be equal—and in particular equal to the lowest fluorescence intensity—provided that the film or the camera reproduces all spectral colors with equal intensity. However, if the spectral sensitivity of the video camera or of the film is not constant, then correspondingly varying setpoint values must be set for the different fluorescence bands in order to be able to reproduce the fluorescence bands with equal brightness.
On the other hand, if specific fluorescence bands do not appear on the photo or the video image, and thus are masked out, then the setpoint values for said bands must be equal to zero. In this context, it proves to be beneficial if the setpoint values are equal to the lowest of their intensities also for the fluorescence bands that are not masked out. Then these fluorescence bands all appear equally bright.
For each excitation band that is assigned to a fluorescence intensity deviating from the setpoint values, a selective filter according to the present invention tuned to the pertinent excitation band is brought into the illumination beam path. Its spectral transmission curve is configured so that exclusively the intensity of the pertinent excitation band is reduced, but the remaining spectral regions pass through unhindered.
In a multiband fluorescence microscope according to the invention for executing the method according to the invention, a filter draw assembly made of a multiplicity of individually movable filter draws is perpendicularly inserted in the illumination beam path tightly next to the aperture diaphragm plane.
The structure of the filter draw is a function of the number of the different excitation bands of the multiband fluorescence microscope. For a number of n excitation bands, each filter draw has n selective filters harmonized to the excitation bands and having surface regions with high and low transmission factors.
The different transmission factors are achieved by virtue of the fact that only certain area portions of the beam cross-section are occupied using separate filter-area elements. In this context as even a surface coverage of the beam cross-section as possible is sought so that no unilateral shading, and thus also no unilateral illumination of the pupils, is made. As a result a crooked illumination, and thus a lateral migration of the picture elements, is prevented during focusing.
It must be possible to insert the filters in the illumination beam path independently of each other individually or in combination using the desired surface region or the desired transmission. In this context, it must be possible with n excitation bands to combine a maximum of n−1 filters with each other, i.e. simultaneously insert them in the illumination beam path. This is sufficient since all excitation bands must never be attenuated simultaneously, because as a rule an excitation band supplies the intensity setpoint value and remains unchanged. Likewise, all excitation bands must not be triggered simultaneously, since this is equivalent to turning off the lighting.
In order to achieve the required combinations from n−1 filters, n−1 individually movable filter draws that are situated tightly parallel next to the aperture diaphragm plane with little
Leica Microsystems Wetzlar GmbH
Porta David
Simpson & Simpson PLLC
Sung Christine
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