31 results about "Fluorescence-lifetime imaging microscopy" patented technology

A nucleic acid detection system and method are provided, in which excitation energy is transmitted from a pulsed excitation source to a reaction site including a fluorescence resonance energy transfer (FRET)-based dye system to generate a fluorescent signal at the reaction site, the fluorescent signal is detected by a detector from the reaction site, and detection of the fluorescent signal is respectively blocked and permitted at the detector by a detector gate this is timed based on an emission start time of the transmitted excitation energy.

A method and system for analysis of fluorescence emission spectroscopy data and fluorescence lifetime imaging microscopy data are described. A unique Laguerre expansion can be found for fluorescence intensity decays of arbitrary form with convergence to a correct solution faster than with conventional methods. The Laguerre expansion technique includes expansion coefficients highly correlated with intrinsic fluorescence lifetimes, allowing direct characterization of fluorescence dynamics. For complex systems, conventional analysis of fluorescence intensity decay in terms of discrete exponential components can not readily provide a true representation of underlying fluorescence dynamics. Utilizing the Laguerre expansion technique, an alternative non-parametric method for analysis of time-resolved fluorescence data from various systems is described, facilitating characterization and discrimination of a sample. An ultra-fast method for analysis of fluorescence lifetime imaging is also described, facilitating real-time analysis of compositional and functional changes in samples, at a microscopic or macroscopic level.

“A label-free imaging method to monitor stem cell metabolism discriminates different states of stem cell as they differentiate in a living tissues. We use intrinsic fluorescence biomarkers and the phasor approach to Fluorescence Lifetime Imaging Microscopy (FLIM). We identify and map intrinsic fluorophores such as collagen, retinol, retinoic acid, flavins, nicotinamide adenine dinucleotide (NADH) and porphyrin. We measure the phasor values of germ cells in C. Elegans germ line. Their metabolic fingerprint cluster according to their differentiation state, reflecting changes in FAD concentration and NADH binding during the differentiation pathway. The phasor approach to lifetime imaging provides a label-free, fit-free and sensitive method to identify different metabolic state of cells during differentiation, to sense small changes in the redox state of cells and may identify symmetric and asymmetric divisions and predict cell fate.”

The invention is applied to the fields of optics, biology, chemistry and the like and provides a super resolution fluorescence lifetime imaging method. The method comprises the following steps of: sparsely activating an optical switch dye molecule marked in a sample; exciting the activated optical switch dye molecule in the sample; collecting photons transmitted by the activated optical switch dye molecule and recording a fluorescent image of the optical switch dye molecule; carrying out the centroid positioning on the optical switch dye molecule in the fluorescent image; counting the photons received at the centroid positioning site and determining a fluorescence lifetime of the activated optical switch dye molecule; and constructing the super resolution fluorescence lifetime image by combining the centroid positioning result with the fluorescence lifetime of the obtained optical switch dye molecule. By combining the super resolution fluorescence microtechnique based on unimolecule positioning with the fluorescence lifetime imaging based on time relevant single photon counting, the invention realizes the super resolution fluorescence lifetime imaging, breaks through the traditional optical diffraction limit and has higher scientific significance and application value.

A method and system for analysis of fluorescence emission spectroscopy data and fluorescence lifetime imaging microscopy data are described. A unique Laguerre expansion can be found for fluorescence intensity decays of arbitrary form with convergence to a correct solution faster than with conventional methods. The Laguerre expansion technique includes expansion coefficients highly correlated with intrinsic fluorescence lifetimes, allowing direct characterization of fluorescence dynamics. For complex systems, conventional analysis of fluorescence intensity decay in terms of discrete exponential components can not readily provide a true representation of underlying fluorescence dynamics. Utilizing the Laguerre expansion technique, an alternative non-parametric method for analysis of time-resolved fluorescence data from various systems is described, facilitating characterization and discrimination of a sample. An ultra-fast method for analysis of fluorescence lifetime imaging is also described, facilitating real-time analysis of compositional and functional changes in samples, at a microscopic or macroscopic level.

Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique increasingly used in the life sciences during the past decades. An all-solid-state fluorescence-lifetime-imaging microscope (1) with a simple lock-in imager (4) for fluorescence lifetime detection is described. The lock-in imager (4), originally developed for 3D vision, embeds all the functionalities required for FLIM in a compact system. Its combination with a light-emitting diode (2) yields a cost-effective and user-friendly FLIM unit for wide-field microscopes. The system is suitable for nanosecond lifetime measurements and achieves video-rate imaging capabilities.

Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique increasingly used in the life sciences during the past decades. An all-solid-state fluorescence-lifetime-imaging microscope (1) with a simple lock-in imager (4) for fluorescence lifetime detection is described. The lock-in imager (4), originally developed for 3D vision, embeds all the functionalities required for FLIM in a compact system. Its combination with a light-emitting diode (2) yields a cost-effective and user-friendly FLIM unit for wide-field microscopes. The system is suitable for nanosecond lifetime measurements and achieves video-rate imaging capabilities.

The invention belongs to the technical field of medical equipment, in particular to an early cancer diagnosis device based on combination of auto-fluorescence lifetime imaging and fluorescence spectroscopy. The auto-fluorescence lifetime imaging detection and fluorescence spectroscopy detection are coupled with a laser scanning confocal microscope for observing cytologic morphology of a biological sample. The device comprises a laser scanning confocal microscope device and two fluorescence signal acquisition devices; different detectors receive fluorescence lifetime information and fluorescence spectroscopy information respectively, and a computer processing system processes the auto-fluorescence lifetime information and fluorescence spectroscopy information. The multiple dimensional information of fluorescence images, fluorescence lifetime and fluorescence spectroscopy of the biological sample can be detected directly, early detection and diagnosis of various cancers can be implemented, and the application prospect is promised in the fields of biomedicine and clinical diagnostics.

A demodulation pixel architecture allows for demodulating an incoming modulated electromagnetic wave, normally visible or infrared light. It is based on a charge coupled device (CCD) line connected to a drift field structure. The drift field is exposed to the incoming light. It collects the generated charge and forces it to move to the pick-up point. At this pick-up point, the CCD element samples the charge for a given time and then shifts the charge packets further on in the daisy chain. After a certain amount of shifts, the multiple charge packets are stored in so-called integration gates, in a preferred embodiment. The number of integration gates gives the number of simultaneously available taps. When the cycle is repeated several times, the charge is accumulated in the integration gates and thus the signal-to-noise ratio increases. The architecture is flexible in the number of taps. A dump node can be attached to the CCD line for dumping charge with the same speed as the samples are taken. Different implementations are described herein, which allow for smaller design or faster speed. The pixel structure can be exploited for e.g. 3D time-of-flight imaging. Both heterodyne and homodyne measurements are possible. Due to the highly-efficient charge transport enabled by static drift fields in the photo-sensitive region and small-sized gates in the CCD chain, high frequency bandwidth from just a few Hertz (Hz) up to greater GHz is supported. Thus, the pixel allows for highly-accurate optical distance measurements. Another possible application of this pixel architecture is fluorescence lifetime imaging microscopy (FLIM), where short laser pulses for triggering the fluorescence have to be suppressed.

A demodulation pixel architecture allows for demodulating an incoming modulated electromagnetic wave, normally visible or infrared light. It is based on a charge coupled device (CCD) line connected to a drift field structure. The drift field is exposed to the incoming light. It collects the generated charge and forces it to move to the pick-up point. At this pick-up point, the CCD element samples the charge for a given time and then shifts the charge packets further on in the daisy chain. After a certain amount of shifts, the multiple charge packets are stored in so-called integration gates, in a preferred embodiment. The number of integration gates gives the number of simultaneously available taps. When the cycle is repeated several times, the charge is accumulated in the integration gates and thus the signal-to-noise ratio increases. The architecture is flexible in the number of taps. A dump node can be attached to the CCD line for dumping charge with the same speed as the samples are taken. Different implementations are described herein, which allow for smaller design or faster speed. The pixel structure can be exploited for e.g. 3D time-of-flight imaging. Both heterodyne and homodyne measurements are possible. Due to the highly-efficient charge transport enabled by static drift fields in the photo-sensitive region and small-sized gates in the CCD chain, high frequency bandwidth from just a few Hertz (Hz) up to greater GHz is supported. Thus, the pixel allows for highly-accurate optical distance measurements. Another possible application of this pixel architecture is fluorescence lifetime imaging microscopy (FLIM), where short laser pulses for triggering the fluorescence have to be suppressed.

The invention relates to a fluorescence lifetime imaging system and method for synchronous measurement of photon arrival time and position. The system includes a scanning translation stage, a to-be-imaged sample, an excitation light source, a focusing lens, a polychromator, and an MCP position sensitive anode detector. The scanning translation stage is used for placing the to-be-imaged sample. Theexcitation light source is used for irradiating the to-be-imaged sample. The focusing lens, the polychromator, and the MCP position sensitive anode detector are arranged in the excited light propagation direction of the to-be-imaged sample in order. The outside of the MCP position sensitive anode detector is connected to a time signal extraction module. The position sensitive anode multiplexed output of the MCP position sensitive anode detector is connected to a charge sensitive preamplifier, a photon arrival time and position synchronous measurement circuit, and an image reconstruction module in order. The time signal extraction module is connected to the photon arrival time and position synchronous measuring circuit. And the scanning translation stage is connected to the image reconstruction module. The system and method provided by the invention can achieve finer spectral resolution and can acquire more dimensional information.

The invention belongs to the technical field of photoelectric functional organic materials and in particular relates to a preparation method and application of a metallic phosphorescence complex. Themetallic phosphorescence complex comprises a metal center and a cyclic metallic complex. The preparation method comprises the following step: performing a complexing reaction on N-N ligand and a C-N ligand with an amino chain, thereby obtaining the metallic phosphorescence complex. Through variations of service lives, the content of oxygen in organelle is relatively effectively judged, and different types of organelle can be well separated by using an FLIM (Fluorescence-Lifetime Imaging Microscopy) technique, so that the metallic phosphorescence complex has great significance for oxygen detection in cells, cytomembrane and mitochondria can be effectively separated by using the FILM technique, and the invention further discloses application of the metallic phosphorescence complex in fieldssuch as biological imaging, cell marking and oxygen detection.

A demodulation pixel architecture allows for demodulating an incoming modulated electromagnetic wave, normally visible or infrared light. It is based on a charge coupled device (CCD) line connected to a drift field structure. The drift field is exposed to the incoming light. It collects the generated charge and forces it to move to the pick-up point. At this pick-up point, the CCD element samples the charge for a given time and then shifts the charge packets further on in the daisy chain. After a certain amount of shifts, the multiple charge packets are stored in so-called integration gates, in a preferred embodiment. The number of integration gates gives the number of simultaneously available taps. When the cycle is repeated several times, the charge is accumulated in the integration gates and thus the signal-to-noise ratio increases. The architecture is flexible in the number of taps. A dump node can be attached to the CCD line for dumping charge with the same speed as the samples are taken. Different implementations are described herein, which allow for smaller design or faster speed. The pixel structure can be exploited for e.g. 3D time-of-flight imaging. Both heterodyne and homodyne measurements are possible. Due to the highly-efficient charge transport enabled by static drift fields in the photo-sensitive region and small-sized gates in the CCD chain, high frequency bandwidth from just a few Hertz (Hz) up to greater GHz is supported. Thus, the pixel allows for highly-accurate optical distance measurements. Another possible application of this pixel architecture is fluorescence lifetime imaging microscopy (FLIM), where short laser pulses for triggering the fluorescence have to be suppressed.

The invention discloses a method for representing a defect of a two-dimensional material and application thereof, and relates to the technical field of defect representation of a nanomaterial. The method for representing the defect comprises the step of: respectively and independently carrying out fluorescence lifetime imaging on a non-defect two-dimensional material substrate sample and a to-be-detected two-dimensional material substrate sample, and according to the change of a fluorescence lifetime, judging whether the to-be-detected two-dimensional material substrate sample has a defect, i.e., if the fluorescence lifetime of the to-be-detected two-dimensional material substrate sample is longer than that of the non-defect two-dimensional material substrate sample, determining that the to-be-detected two-dimensional material substrate sample is a defect sample, and if the fluorescence lifetime of the to-be-detected two-dimensional material substrate sample has no obvious change withrespect to the fluorescence lifetime of the non-defect two-dimensional material substrate sample, determining that the to-be-detected two-dimensional material substrate sample is a non-defect sample.The method disclosed by the invention adopts a fluorescence lifetime imaging method to represent the defect of the two-dimensional material; by the method, the change of the fluorescence lifetime canbe rapidly and visually observed so as to judge whether the material has the defect; and at the room temperature, representation can be carried out, a new defect cannot be introduced, and the method is a non-destructive detection method.

The invention provides novel non-invasive in vitro methods for assessing the metabolic condition of oocytes and / or embryos with fluorescence lifetime imaging microscope, that can be used, for example, in assessment of oocytes and embryos in assisted reproductive technologies.

A label-free imaging method to monitor stem cell metabolism discriminates different states of stem cell as they differentiate in a living tissues. We use intrinsic fluorescence biomarkers and the phasor approach to Fluorescence Lifetime Imaging Microscopy (FLIM). We identify and map intrinsic fluorophores such as collagen, retinol, retinoic acid, flavins, nicotinamide adenine dinucleotide (NADH) and porphyrin. We measure the phasor values of germ cells in C. Elegans germ line. Their metabolic fingerprint cluster according to their differentiation state, reflecting changes in FAD concentration and NADH binding during the differentiation pathway. The phasor approach to lifetime imaging provides a label-free, fit-free and sensitive method to identify different metabolic state of cells during differentiation, to sense small changes in the redox state of cells and may identify symmetric and asymmetric divisions and predict cell fate.

The present invention discloses a DMD (digital micromirror device) computing holographic scanning-based fully automated TCSPC-FLIM (time-correlated single photon counting-fluorescence lifetime imagingmicroscopy) system and a time detection method. According to the DMD (digital micromirror device) computing holographic scanning-based fully automated TCSPC-FLIM system and the time detection method,a continuous laser excites a sample to generate fluorescence under a wide field condition; an SCMOS camera transmits obtained sample fluorescence information to a computer; the computer performs calculation and processing so as to obtain the position and intensity information of a fluorescence image, selectively generates DMD two-dimensional hologram distribution corresponding to a region of interest, and at the same time controls a DMD lens group to achieve selective optical excitation; under the control of the computer, a DMD can transmit laser light from a first laser to the sample througha light microscope group, a chromatic dispersion elimination system, the DMD and the like, so that the laser light can excite the sample to realize FLIM. According to the system of the invention, theDMD is applied to a fully-automated TCSPC-FLIM system; the DMD is adopted as a scanner, and is added to an automatic selection optical path system; fully automated fluorescence service life microscopic imaging can be realized according to the definition and selection of a user. With the optical path system of the present invention adopted, an imaging scanning speed can be greatly increased, and selective optical excitation is realized, and the fluorescence imaging of a weak fluorescent region can be effectively improved.

The invention relates to the field of fluorescent lifetime imaging, and the lifetime estimation accuracy is improved for expanding the fluorescent lifetime estimation range under the same laser frequency condition. An RLD (Rapid Lifetime Determination) pre-estimation method is improved, so that the iterative operation is avoided. According to the adopted technical scheme, a correction method of a center of mass method used for calculating fluorescent lifetime comprises the following steps that to-be-recovered decay lifetime is tau, the laser frequency adopted by a time-correlated single-photon counting (TCSPC) system is F, the recorded total photon number is NC, and the photon arrival time summation obtained by quantification is NT; a threshold is defined and determination is carried out; when the lifetime is smaller than the threshold, a lifetime deviation is compensated by adopting nonlinear one-iteration; or otherwise, the fluorescent lifetime is estimated through the RLD method; and correction is carried out through nonlinear compensation to obtain final fluorescent lifetime. The method is mainly applied to the occasion of designing and manufacturing fluorescent lifetime imaging products.

Disclosed are methods of detecting enzymatic activity on a fluorophore-labeled substrate using by monitoring the fluorescence lifetime of the fluorophore.

The invention relates to a fluorescence-lifetime imaging microscopy method having time-correlated single-photon counting, wherein a sample (36) is periodically excited with excitation light pulses toemit fluorescence photons by means of a pulsed light source (12), wherein a measurement interval is defined between each pair of consecutive excitation light pulses, the fluorescence photons are detected by means of a detector (42) and an analog detector signal representing the detected fluorescence photons is produced, detection times at which the fluorescence photons are detected by the detector(42) within the measurement intervals are determined on the basis of the detector signal, at least one value characterizing the fluorescence decay behavior is determined on the basis of the detectiontimes of the detected fluorescence photons, and imaging is performed on the basis of the characterizing value. The analog detector signal is sampled within each measurement interval in a plurality ofsampling intervals and is converted into a series of discrete signal values associated with the individual sampling intervals. On the basis of the series of discrete signal values that belongs to theassociated measurement interval, it is determined whether more than a predefined number of fluorescence photons has been detected within the measurement interval. This measurement interval is discarded for the determination of the characterizing value if more than said predefined number of fluorescence photons has been detected.

The invention relates to a novel fluorescent lifetime microimaging device and method on the basis of optical addition heterodyne modulation. The device comprises a fluorescent exciting light path, a reference light path and a fluorescent imaging light path, wherein a cosinusoidal modulated high-frequency exciting light excites a fluorescent sample to generate the florescent light with the same frequency, and the florescent light images on a light-sensitive surface of an image intensifier; moreover, a reference light which has the modulation frequency which is similar to the exciting light frequency is also irradiated to the light-sensitive surface of the image intensifier; a fluorescent image and the reference light are superposed on the light-sensitive surface of the image intensifier to form a heterodyne image; and finally, a fluorescent lifetime image is synthesized through phase-locking amplifying and polar coordinate phase diagram analyzing. The fluorescent lifetime microimaging device and method have the advantages of high fluorescent lifetime measurement resolution, high imaging efficiency, strong antijamming capability and the like, and moreover, the technical requirements on a core part-the image intensifier are low.

The invention belongs to the technical field of medical equipment, and in particular relates to a device for early diagnosis of cancer in combination with autofluorescence lifetime imaging and fluorescence spectrum. The invention couples fluorescence lifetime imaging measurement and fluorescence spectrum measurement with a laser scanning confocal microscope to observe the cytological morphology of biological samples, and combines autofluorescence lifetime and spectral information to realize a novel device for early diagnosis of cancer. The device includes a laser scanning confocal microscope device and a two-way fluorescence signal acquisition device. Different detectors respectively receive fluorescence lifetime information and fluorescence spectrum information, and use a computer processing system to process autofluorescence lifetime information and fluorescence spectrum information. The device can directly detect multi-dimensional information such as fluorescence images, fluorescence lifetimes, and fluorescence spectra of biological samples to be tested, and realize early detection and diagnosis of various cancers. It has broad application prospects in the fields of biomedicine and clinical diagnosis.

The invention relates to an apparatus (200) for incubation of a viable biological material (2); said apparatus comprises: a housing (4) having an extension in a longitudinal direction X, in a transversal direction Y, and in a direction Z perpendicular to the longitudinal direction and the transversal direction; said housing comprising: two or more culture dish compartments (6), each being adapted to accommodate, one or more culture dishes (8) comprising a biological material (2); wherein said apparatus comprises an image capturing device (10); wherein said apparatus comprises a control unit (12) for controlling the operation thereof; wherein at least part of said image capturing device is being configured to be movable in relation to the two or more culture dish compartments (6), thereby allowing capture of images of one or more of said biological materials (2) accommodated in said one or more culture dishes (8); and wherein said apparatus comprises a FLIM unit (11) (fluorescent lifetime imaging microscope); wherein at least part of said FLIM unit (11) is being configured to be movable in relation to the two or more culture dish compartments (6), thereby allowing capture of FLIM spectra of one or more of said biological materials (2) accommodated in said one or more culture dishes (8).

Nano-structured anodic aluminum oxide ceramic films and membranes doped with chelated metals for fluorescence enhancement. Controlled doping during production steps results in the inclusion of traces of ions in the finished materials while maintaining high control over the film structure. This approach yields products suitable for optical applications, including fluorescence enhancement. The nano-structured anodic aluminum oxide ceramic films and membranes are particularly useful for in vitro diagnostics, drug discovery, DNA sequencing, proteomics, immunofluorescence, immunohistochemistry, biosensing, and bio-assay fluorescence technologies such as time resolved Forster resonance energy transfer (TR-FRET), Fluorescence in situ hybridization (FISH), Fluorescence-lifetime imaging microscopy (FLIM), Fluorescence polarization immunoassay (FPIA), Fluorescence anisotropy or fluorescence polarization, Fluorescence recovery after photobleaching (FRAP), Fluorescence Loss in Photobleaching (FLIP), Fluorescence correlation spectroscopy (FCS), and Falck-Hillarp fluorescence (F-H).

The invention is applied to the fields of optics, biology, chemistry and the like and provides a super resolution fluorescence lifetime imaging method. The method comprises the following steps of: sparsely activating an optical switch dye molecule marked in a sample; exciting the activated optical switch dye molecule in the sample; collecting photons transmitted by the activated optical switch dye molecule and recording a fluorescent image of the optical switch dye molecule; carrying out the centroid positioning on the optical switch dye molecule in the fluorescent image; counting the photonsreceived at the centroid positioning site and determining a fluorescence lifetime of the activated optical switch dye molecule; and constructing the super resolution fluorescence lifetime image by combining the centroid positioning result with the fluorescence lifetime of the obtained optical switch dye molecule. By combining the super resolution fluorescence microtechnique based on unimolecule positioning with the fluorescence lifetime imaging based on time relevant single photon counting, the invention realizes the super resolution fluorescence lifetime imaging, breaks through the traditional optical diffraction limit and has higher scientific significance and application value.

The invention relates to the field of fluorescent lifetime imaging, and the lifetime estimation accuracy is improved for expanding the fluorescent lifetime estimation range under the same laser frequency condition. An RLD (Rapid Lifetime Determination) pre-estimation method is improved, so that the iterative operation is avoided. According to the adopted technical scheme, a correction method of a center of mass method used for calculating fluorescent lifetime comprises the following steps that to-be-recovered decay lifetime is tau, the laser frequency adopted by a time-correlated single-photon counting (TCSPC) system is F, the recorded total photon number is NC, and the photon arrival time summation obtained by quantification is NT; a threshold is defined and determination is carried out; when the lifetime is smaller than the threshold, a lifetime deviation is compensated by adopting nonlinear one-iteration; or otherwise, the fluorescent lifetime is estimated through the RLD method; and correction is carried out through nonlinear compensation to obtain final fluorescent lifetime. The method is mainly applied to the occasion of designing and manufacturing fluorescent lifetime imaging products.

A fluorescence lifetime imaging microscopy method using time-correlated single-photon counting, by means of a pulsed light source (12) to periodically excite a sample (36) with excitation light pulses to emit fluorescent photons, in every two consecutive excitations A measurement interval is defined between the light pulses; the fluorescent photons are detected by means of a detector (42) and an analog detector signal representing the detected fluorescent photons is generated; the detection time is determined based on the detector signal, and the fluorescent photons are within the corresponding measurement interval on the detection time Detected by a detector (42); based on the detection time of the detected fluorescent photons, at least one parameter characterizing the decay characteristic of the fluorescence is determined, and imaging is performed by means of the characterizing parameter. The analog detector signal is sampled in a plurality of sampling intervals within the corresponding measurement interval and converted into a sequence of discrete signal values ​​assigned to the individual sampling intervals. With the aid of the sequence of discrete signal values ​​belonging to the respective measurement interval, it is determined whether more than a predetermined number of fluorescent photons are detected within the measurement interval, and if so, the measurement interval is discarded for determining the characteristic variable.

Methods and devices for single cell analysis using fluorescence lifetime imaging microscopy (FLIM) are disclosed. The methods utilize microfluidic devices which use traps to immobilize cells for FLIM analysis. The analysed cells may be sorted before or after imaging and may be plant, animal, or bacterial cells. Analysis of the FLIM data may use a phasor plot and may be used to identify a metabolic pattern of the single cells.