# US 10,481,095 B2 · Methods and means for multispectral imaging > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,481,095 B2 - Document ID: US10481095B2 - Status: Granted - Kind: B2 - Application number: 15/316,455 - Filed: 2015-06-03 - Published: 2017-06-15 - Granted: 2019-11-19 - Earliest claimed: 2014-06-05 - Assignees: Universität Heidelberg - Applicants: Universität Heidelberg - Inventors: Nikolas Dimitriadis; Nikolaos Deliolanis - Web page: https://patentgrep.com/patent/US10481095B2 ## CPC - G01N 21/6456: {Spatial resolved fluorescence measurements; Imaging} - A61B 1/043: {for fluorescence imaging} - A61B 1/0638: {providing two or more wavelengths} - A61B 3/14: Arrangements specially adapted for eye photography - A61B 5/0071: {by measuring fluorescence emission} - A61B 5/14551: {for measuring blood gases} - G01J 3/10: Arrangements of light sources specially adapted for spectrometry or colorimetry - G01J 3/32: Investigating bands of a spectrum in sequence by a single detector - G01J 3/4406: {Fluorescence spectrometry} - G01N 21/6428: {Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53)} - G01N 21/80: Indicating pH value - G01N 21/91: using penetration of dyes, e.g. fluorescent ink - G01J 2003/102: {Plural sources} - G01J 2003/106: {the two sources being alternating or selectable, e.g. in two ranges or line:continuum} - G01N 2021/6417: {Spectrofluorimetric devices} - G01N 2021/6439: {with indicators, stains, dyes, tags, labels, marks} - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - A61B: DIAGNOSIS; SURGERY; IDENTIFICATION (usefulness limited to only animals A61D) - G01J: MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY (light sources F21, H01J, H01K, H05B; investigating properties of materials by optical means G01N) ## Abstract Multispectral imaging of samples, in particular of biological tissues. A method for acquisition of fluorescence and reflectance images of an object including alternatingly illuminating the object with at least a first light having several spectral regions of high intensity, wherein the first light has at least one region of low intensity that is of longer wavelength to a region of high intensity, and at least a second light having at least one spectral region of high intensity, recording a first image of the object during illumination of the object with the first light and a second image of the object during illumination of the object with the second light using a common sensor array, wherein the light recorded by the sensor array is attenuated in at least one of the spectral regions in which the first light has high intensities. ## Claims ### Claim 1 A method for acquisition of fluorescence and reflectance images of an object comprising the steps of alternatingly illuminating the object with at least a first light having at least two spectral regions of high intensity, wherein the first light has regions of low intensity that are of longer wavelengths to a region of high intensity, and at least a second light having at least one spectral region of high intensity, wherein the ratio of the light intensity between regions of high intensity at shorter wavelength and regions of low light intensity of longer wavelength for the first light is ≥1×102, recording a first image of the object during illumination of the object with the first light and a second image of the object during illumination of the object with the second light using a common sensor array, wherein the light recorded by the sensor array is attenuated in at least said two of the spectral regions in which the first light has high intensities, wherein the sensor array is a multichannel array where each channel has a distinct spectral sensitivity, wherein the light recorded by the sensor array is attenuated by a multiple-bandpass filter with transmission characteristics, which are complementary to the first light, so as to attenuate at least said two spectral regions in which the first light has high intensities, and wherein at least one of (a) the attenuation ratio between the intensity of the unattenuated to the attenuated spectral regions is ≥1×102, and (b) the amount of attenuation of the light recorded by the sensor array in at least one of the spectral regions in which the first light has high intensities is such that the intensity of the light recorded in unattenuated spectral regions is higher than the intensity of the light recorded in the sum of the attenuated spectral regions. ### Claim 2 (depends on 1) The method according to claim 1, wherein the multichannel array is a color sensor array. ### Claim 3 (depends on 1) The method according to claim 1, wherein the data, which are provided in the channel image space of the recorded images, are transformed into values of a component image space. ### Claim 4 (depends on 3) The method according to claim 3, wherein the components correspond to spatial distributions of fluorochromes, absorbers, derived values, or noise. ### Claim 5 (depends on 1) The method according to claim 1, wherein the ratio of the light intensity between at least one region of high intensity at shorter wavelength and at least one region of low light intensity of longer wavelength for at least one of the lights is >1×103. ### Claim 6 (depends on 1) The method according to claim 1, wherein the first light, the second light, or both the first light and the second light is/are generated by at least one of (a) by white light, from two broadband light sources, wherein said broadband lights are filtered by multiple bandpass filters in order to generate the first light and the second light, (b) multiple narrowband light emitting diodes (LED), where the light emanating from the narrowband light source or light emitting diodes is filtered by a multiband filter in the light path, and (c) a combination of broadband light sources according to (a) and narrowband light sources according to (b). ### Claim 7 (depends on 1) The method according to claim 1, wherein the image recorded during illumination of the object with the second light is processed in order to correct the image for the attenuated spectral regions. ### Claim 8 (depends on 1) The method according to claim 1, wherein the object is illuminated with second light for generation of first reflected light reflected by the object and with two or more spectrally different first lights for excitation of two or more, spectrally different, fluorescence lights emitted by the object, alternating between the second light and each of the two or more first lights, and recording first images of the object during illumination of the object with each of the first lights and a second image of the object during illumination of the object with the second light using a common sensor array, wherein the light recorded by the sensor array is permanently attenuated in one, several or all the spectral regions of all first lights, in which the first lights have high intensity to an amount that the intensity of the light recorded in unattenuated spectral regions is higher than the intensity of the light recorded in the attenuated spectral regions. ### Claim 9 (depends on 1) The method according to claim 1, wherein alternatingly illuminating the object with at least the first light, having a first illumination period, and the second light, having a second illumination period, and illuminating the object with pulses of a further light, wherein the pulse duration of the further light is short compared to the first illumination period and short compared to the second illumination period, and holding the recording of the first image and the second image during said illumination with further light, or alternatingly illuminating the object with at least the first light, having a first illumination period, the second light, having a second illumination period, and a third light having a third illumination period. ### Claim 10 An imaging apparatus for acquisition of fluorescence and reflectance images of an object comprising at least a first light source providing a first light having at least two spectral regions of high intensity and at least a second light source providing a second light having at least one spectral region of high intensity, wherein the first light has at least regions of low intensity that are of longer wavelengths to a region of high intensity, wherein the ratio of the light intensity between regions of high intensity at shorter wavelength and regions of low light intensity of longer wavelength for the first light is ≥1×102, wherein the at least one first light source and the at least one second light source are configured to alternatingly illuminate the object with the first light and the second light, a sensor array arranged for recording a first image of the object during illumination of the object with the first light and a second image of the object during illumination of the object with the second light, wherein the sensor array is a multichannel array where each channel has a distinct spectral sensitivity, and an attenuator, wherein the attenuator comprises a multiple bandpass filter positioned between the object and the sensor array and having transmission characteristics, which are complementary to the first light, so as to attenuate the light recorded by the sensor array in at least said two spectral regions in which the first light has high intensities, and wherein at least one of (a) the attenuation ratio between the intensity of the unattenuated to the attenuated spectral regions is ≥1×102, and (b) the amount of attenuation of the light recorded by the sensor array in at least one of the spectral regions in which the first light has high intensities is such that the intensity of the light recorded in unattenuated spectral regions is higher than the intensity of the light recorded in the sum of the attenuated spectral regions. ### Claim 11 (depends on 10) An apparatus according to claim 10, wherein the sensor array is a color sensor array. ### Claim 12 (depends on 10) The apparatus according to claim 10 configured to transform the data, which are provided in the channel image space of the recorded images, into values of a component image space. ### Claim 13 (depends on 12) The apparatus according to claim 12, wherein the components correspond to spatial distributions of fluorochromes, absorbers, derived values, or noise. ### Claim 14 (depends on 10) The apparatus according to claim 10, wherein the ratio of the light intensity between at least one region of high intensity at shorter wavelength and at least one region of low light intensity of longer wavelength for at least one of the lights is >1×103. ### Claim 15 (depends on 10) The apparatus according to claim 10, wherein the image recorded during illumination of the object with second light is processed in order to correct the image for the attenuated spectral regions. ### Claim 16 (depends on 10) An endoscope or a surgical microscope comprising an imaging apparatus according to claim 10. ### Claim 17 (depends on 10) A use of a method, apparatus, endoscope or surgical microscope according to claim 10 for recording reflection images or fluorescence images for at least one of (a) internal inspection in automotive applications, (b) in medical diagnostics, (c) in medical treatment, (d) in chemical analysis and (e) in physical analysis.