Method for investigating a sample
AssigneeCarl Zeiss Jena GmbH
InventorsRalf Wolleschensky, Bernhard Zimmermann, Sesbastian Tille
Life of this patent
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Abstract
A method for investigating specimens, wherein a spectral splitting of the radiation coming from the specimen is carried out for specimen points or point distributions, for the operation of a laser scanning microscope or a fluorescence screening arrangement or a flow cylinder comprising the steps of generating a λ-stack so that the spectral distribution is measured by individual detection channels and storing the signals so as to be correlated to the detection signals with at least one of the spatial coordinates x, y and z and/or so as to be correlated to the measurement time t.
Claims29
Method for investigating specimens, wherein a spectral splitting of the radiation coming from the specimen is carried out for specimen points or point distributions, preferably for the operation of a laser scanning microscope or a fluorescence screening arrangement or a flow cytometer, wherein a λ-stack is generated in that the spectral distribution is measured by individual detection channels and the signals are stored so as to be correlated to the detection signals with at least one of the spatial coordinates x, y or z and/or so as to be correlated to the measurement time t.
Method according to at least one of the preceding claims, wherein ROIs are marked as relevant specimen areas by means of an input device or automatically.
Method according to at least one of the preceding claims, wherein the ROIs are used to form reference spectra.
Method according to at least one of the preceding claims, wherein a color-coded image is generated by superimposing a plurality of image channels when a plurality of dyes are contained in a specimen area.
Method according to at least one of the preceding claims, wherein ROIs are marked as relevant specimen areas by means of an input device, reference spectra are formed from the ROIs and a color-coded image is generated from the reference spectra by quantitative analysis.
Method according to at least one of the preceding claims, wherein ROIs are marked as relevant specimen areas by means of an input device and a quantitative analysis is carried out by means of reference spectra from a stored database.
Method according to at least one of the preceding claims, wherein a qualitative analysis is carried out and relevant specimen areas are formed as ROIs which are used for forming and storing reference spectra.
Method according to at least one of the preceding claims, wherein an input device is provided for selecting dyes which are quantitatively analyzed by means of a dye database and are displayed.
Method according to at least one of the preceding claims, wherein the ratio of the spectral components is formed from a color-coded image formed by superposition of a plurality of image channels for determining the ion concentrations.
Method according to at least one of the preceding claims, wherein image channels or detection channels corresponding to unwanted signals such as autofluorescence or reflection or nonspecific fluorescence are eliminated.
Method according to at least one of the preceding claims, wherein k-stacks corresponding to the measured spectral distribution with individual detection channels are calculated from a stored color-coded image and a reference spectrum.
Method according to claim, wherein additional images are formed and/or ROIs are marked and evaluated from these λ-stacks.
Method according to at least one of the preceding claims, wherein the spectral width of the detection within which the spectral distribution is detected collectively and to which an image channel is allocated is variable.
Method according to at least one of the preceding claims, wherein the spectra are evaluated in ROIs.
Method according to at least one of the preceding claims, wherein adjusting elements are provided for adjusting the spectral width.
Method according to at least one of the preceding claims, wherein the adjusted spectral width is generated by digital signal combination or electronic combination of detection channels.
Method according to at least one of the preceding claims, wherein adjusting elements and the spectral width are displayed on a monitor in such a way that the adjusting areas of the adjusting elements are spatially correlated to the spectral width.
Method according to at least one of the preceding claims, wherein sliders are provided, whose position on the monitor corresponds to the position of the associated spectral region.
Method according to at least one of the preceding claims, wherein the adjusted wavelength is indicated by adjusting the pointer of a computer mouse on the adjusting element, and the adjusted wavelength area is indicated on the monitor by adjusting between two adjusting elements.
Method according to at least one of the preceding claims, wherein a wavelength-dependent display of the temporal behavior of specimen areas or of the entire specimen is carried out on a monitor.
Method according to at least one of the preceding claims, wherein a gray scale image is generated from the lambda stack in that the maximum intensity value is determined and displayed for every x,y pixel position over the wavelength regions.
Method according to at least one of the preceding claims, wherein the individual images of the lambda stack are displayed on a screen at least partially in series so as to overlap adjacent to one another or one behind the other.
Method according to at least one of the preceding claims, wherein time-dependent and/or z-dependent views are also generated and displayed in addition to an x,y lambda display.
Method according to at least one of the preceding claims, wherein the lambda stack is sectioned and the resulting section images are displayed.
Method according to one of the preceding claims, wherein the quantitative analysis involves an unmixing process.
Method according to one of the preceding claims, wherein the qualitative analysis involves a PCA process.
Dependencies are inferred from the claim text.