528 results about "Fluorescence microscope" patented technology

A deep learning-based virtual HER2 IHC staining method uses a conditional generative adversarial network that is trained to rapidly transform autofluorescence microscopic images of unlabeled / label-free breast tissue sections into bright-field equivalent microscopic images, matching the standard HER2 IHC staining that is chemically performed on the same tissue sections. The efficacy of this staining framework was demonstrated by quantitative analysis of blindly graded HER2 scores of virtually stained and immunohistochemically stained HER2 whole slide images (WSIs). A second quantitative blinded study revealed that the virtually stained HER2 images exhibit a comparable staining quality in the level of nuclear detail, membrane clearness, and absence of staining artifacts with respect to their immunohistochemically stained counterparts. This virtual staining framework bypasses the costly, laborious, and time-consuming IHC staining procedures in laboratory, and can be extended to other types of biomarkers to accelerate the IHC tissue staining and biomedical workflow.

The invention discloses a rapid automatic focusing module and method suitable for a fluorescence microscope. The automatic focusing module comprises a laser, a diaphragm, a collimating lens group, a reflector, a dichroic mirror, an objective lens, a sample to be measured, a telescope lens group, a CCD2 detector, a control system and a displacement platform which are sequentially arranged along a focusing light path, and the sample to be measured, the objective lens, the dichroic mirror, a tube lens and the CCD2 detector which are sequentially arranged along an imaging light path; the collimated laser beam is emitted to the plane where the sample to be detected is located through the objective lens, and then the laser beam is reflected back to the telephoto lens group and converged to the CCD2 detector. According to the offset of the center of the reflected light beam, the relative distance between the objective lens and the to-be-tested sample is moved through a control system, and automatic focusing of a single view field of the to-be-tested sample is achieved; the to-be-measured sample is moved to the focal planes of different object view fields in the X direction and the Y direction and is quickly focused in real time, so that the large-size to-be-measured sample is always kept at the focal plane position, and the image quality and the working efficiency are improved.

A deep learning-based digital / virtual staining method and system enables the creation of digitally / virtually-stained microscopic images from label or stain-free samples. In one embodiment, the method of generates digitally / virtually-stained microscope images of label-free or unstained samples using fluorescence lifetime (FLIM) image(s) of the sample(s) using a fluorescence microscope. In another embodiment, a digital / virtual autofocusing method is provided that uses machine learning to generate a microscope image with improved focus using a trained, deep neural network. In another embodiment, a trained deep neural network generates digitally / virtually stained microscopic images of a label-free or unstained sample obtained with a microscope having multiple different stains. The multiple stains in the output image or sub-regions thereof are substantially equivalent to the corresponding microscopic images or image sub-regions of the same sample that has been histochemically stained.

The invention discloses a strong mineral interference and unconventional oily reservoir quantitative fluorescence experiment method which comprises the following steps: selecting a cleaned sample reservoir particle quantitative fluorescence analysis test, and taking a result as an original blank sample QGF index value; repeatedly grinding and thoroughly crushing the cleaned sample until the particle size is less than 30 microns; the standing time of spectroscopically pure dichloromethane is optimized to ensure that hydrocarbons released by the inclusion are fully extracted; a standard slice is manufactured, inclusion retention is observed under a fluorescence microscope, if the inclusion is observed under the microscope, further grinding and soaking are needed, if no inclusion exists, reservoir particle quantitative fluorescence analysis testing is conducted, and the testing result is the mineral QGF index value; and calculating the QGF index value of the inclusion, wherein the QGF index value is the difference value between the QGF index value of the original blank sample and the QGF index value of the mineral. According to the method, interference caused by the fact that mineral fluorescence and calculation errors are not considered in a traditional reservoir particle quantitative fluorescence experiment method is corrected.

The present invention relates to an image acquisition device and acquisition method for detecting a target material signal, which have improved sensitivity and uniformity compared to those of a conventional fluorescence microscope used for acquiring fluorescence images. The present invention provides the image acquisition device for detecting a target material signal, comprising: (a) a camera for detecting a fluorescence signal; (b) light sources that are disposed outside the camera at regular intervals in a circular shape; (c) a shielding guide that is positioned above the light sources and which transmit only vertical direction light of light emitted from the light sources; and (d) a prism that refracts the light passing through the shielding guide toward a sample.

The invention belongs to the field of biological pharmacy, and relates to a temperature-sensitive hydrogel, in particular to a temperature-sensitive hydrogel SV (at) HP based on hyaluronic acid as well as a preparation method and application of the temperature-sensitive hydrogel SV (at) HP. The temperature-sensitive hydrogel SV (at) HP entraps CS-siRNA nanoparticles and VEGF (at) PLGA microspheres, and is used for realizing microenvironment responsive drug delivery to treat intrauterine adhesion. Chlorogenic acid and TGF beta1 siRNA are self-assembled through disulfide bonds to form CS-siRNA nano-particles, the CS-siRNA nano-particles are used for treating intrauterine adhesion through gene and chemical therapy, and cell and animal experiments prove that the CS-siRNA nano-particles have a good effect of improving intrauterine adhesion. Through evaluation of a real-time fluorescence quantification method, a western blot method, a cell scratch experiment, laser confocal microscope observation, inverted fluorescence microscope observation, an in-vivo experiment and the like, the SV-coated HP gel has the effects of resisting inflammation, improving endometrial fibrosis, promoting IUA rat endometrial angiogenesis and repair, inhibiting intrauterine adhesion and improving the intrauterine environment.

The invention discloses an automatic focusing method, device and equipment for a fluorescence microscope. The method comprises the following steps: S10, acquiring an image acquired by the microscope and extracting a target cell contour in the image acquired by the microscope; s20, the contour of the target cell is identified, an internal area and an external area are obtained, the internal area is a cell main body part, and the external area is a part except the cell main body part; s30, the definition value of the internal area and the definition value of the external area are calculated respectively, weighting processing is carried out respectively, and the weight corresponding to the definition value of the internal area is larger than the weight corresponding to the definition value of the external area; s40, calculating a global definition score according to a preset formula; and S50, automatically adjusting the focal length of the microscope based on the global definition score. The definition values of the internal region and the external region are respectively calculated and weighted, and the detail definition of the cell main body part is preferentially considered, so that the interference of the halo effect on the focusing result is effectively reduced, and the focusing precision and the imaging quality are improved.

The invention relates to the technical field of medical animal models, in particular to a zebrafish intestine toxicity modeling and detection method based on podophyllotoxin and application thereof, and the method comprises the following steps: S1, randomly selecting 3-day (3dpf) wild-type AB-line zebrafish after fertilization in a 6-pore plate, and treating 30 zebrafish in each pore (experimental group); s2, dissolving the podophyllotoxin in water, performing modeling, setting a normal control group, setting the volume of each pore to be 3mL, and treating at 28 DEG C for 2 days; and S3, performing intestinal histopathology analysis and intestinal apoptosis analysis on the zebrafish obtained by modeling in the step S2. The method comprises the following steps: Hamp; according to the method, the change of the tissue structure can be clearly observed, and the damage degree of the podophyllotoxin to the intestinal tract and brain tissue of the zebra fish can be intuitively evaluated. AO staining is combined with a fluorescence microscope, so that the apoptosis condition can be quantitatively analyzed; the damage mechanism of the podophyllotoxin to the brain cell structure and function is defined, and the blank of the podophyllotoxin in the aspect of brain toxicity research is filled.

The invention discloses a construction method and application of a caenorhabditis elegans inflammation model, belongs to the technical field of model biology, and provides a construction method of a caenorhabditis elegans inflammation model, which comprises the following steps: putting L1-stage caenorhabditis elegans into a lipopolysaccharide solution, and carrying out induced culture to obtain induced cultured caenorhabditis elegans; placing the caenorhabditis elegans in a chromosome system, performing staining culture and centrifugation to obtain dyed caenorhabditis elegans, and placing the dyed caenorhabditis elegans on a culture medium for recovery to obtain the caenorhabditis The surface of the caenorhabditis elegans is provided with a transparent cuticle anatomical structure, the intestinal barrier damage condition is easy to observe after the intestinal tract is dyed by a dye, and the cell proliferation, tissue development and tissue integrity of the caenorhabditis elegans are easy to carry out fluorescence microscope and morphological analysis. A caenorhabditis elegans inflammation model is constructed through lipopolysaccharide induction, and a new experimental animal model is provided for screening anti-inflammatory drugs.

The invention discloses a biological experiment automatic touch-free adding system based on optical control and an operation method thereof, and relates to the technical field of biological experiment equipment, the system comprises the following components: an optical marking module, an optical imaging and detection module, an optical control module and an automatic control module; high automation of biological experiments is realized by integrating a plurality of modules including optical marking, optical imaging and detection, optical control and automatic control, efficient and stable marking of biological samples is ensured by utilizing specific marking of a quantum dot solution and combining accurate control of a microfluidic channel in the optical marking stage, and then, the accuracy of the biological samples is ensured. Through advanced equipment such as a high-resolution fluorescence microscope and an optical spectrum analyzer, the system can accurately obtain optical information of a marked biological sample, a solid foundation is provided for subsequent control and recognition, in the optical control stage, the optical tweezers system achieves accurate control over the biological sample in a touch-free mode, and the accuracy of the biological sample is improved. Pollution and damage possibly caused by traditional physical contact are avoided.

The invention discloses a method and a device for detecting errors of a rotary table and a rotary shaft of an overall measurement light path connected upper rotary shaft type laser tracker, and belongs to the technical field of laser tracking measurement. The device comprises a microscope, a fluorescent microsphere, a substrate and a six-dimensional precision calibration displacement table, the microscope is located above the fluorescent microsphere, and the six-dimensional precision calibration displacement table is located above the substrate. The fluorescent microspheres are fixed on the substrate, and the substrate is fixed on the six-dimensional precise calibration displacement table. By combining the fluorescence microscope with the astigmatism imaging function, the precise displacement table, space coordinate calculation and other operations, the determination of the axis space linear equation of the upper and lower rotary tables of the whole measurement light path connected upper rotary shaft type laser tracker and the detection of the shaft radial runout in a complete rotation period are realized under one coordinate system; a necessary technical foundation is laid for realizing error compensation of the rotary table and the rotary shaft of the rotary shaft type laser tracker connected with an overall measurement light path and improving the tracking precision and the stability of the laser tracker.

The invention discloses an oral risk assessment method and system based on image recognition and a medium, and relates to the technical field of image recognition, and the method comprises the steps: carrying out the block segmentation of a user oral image, activating a lesion recognizer to recognize the features of an image block, carrying out the matching of an oral disease, and obtaining a corresponding flora type set; imaging the target flora by using a fluorescence microscope, and extracting fluorescence characteristics; and generating an oral risk decision of the target user in combination with the identified disease information and the fluorescence characteristics. Therefore, the technical effects of multi-dimensional image comprehensive analysis and evaluation accuracy and efficiency improvement are achieved.

A fluorescence microscopy inspection system includes light sources able to emit light that causes a specimen to fluoresce and light that does not cause a specimen to fluoresce. The emitted light is directed through one or more filters and objective channels towards a specimen. A ring of lights projects light at the specimen at an oblique angle through a darkfield channel. One of the filters may modify the light to match a predetermined bandgap energy associated with the specimen and another filter may filter wavelengths of light reflected from the specimen and to a camera. The camera may produce an image from the received light and specimen classification and feature analysis may be performed on the image.

The present application discloses methods for characterising vesicles. The method involves (1) a sample preparation step, comprising providing a test specimen with vesicles attached to a substrate, wherein the vesicles are labelled with one or more fluorescent probes; (2) an image acquisition step, comprising imaging said one or more fluorescent probes on the vesicles to generate image data; (3) an image processing step which identifies individual vesicles and constructs a feature vector containing characterising parameters for individual vesicles characterising parameters (including a morphological parameter) (4) a data transformation step to calculate modified feature vectors of lower dimensionality for individual vesicles; and (5) a characterisation step, which characterises the vesicles based on the modified feature vectors. The application also discloses methods for immobilising vesicles on a substrate, as well as substrates functionalised to capture vesicles.

The invention discloses an ANA detection image-text report intelligent generation method and system, and relates to the technical field of immunodetection, the system uses different fluorescent markers to mark ANA, signal sensitivity is enhanced, and detection accuracy is ensured. Then, collecting fluorescence signals of different wavelengths by adopting a fluorescence microscope and a multi-wavelength excitation light source, and obtaining dynamic change data of reaction intensity by combining a time-resolved fluorescence imaging technology to generate a multi-channel imaging data set; thirdly, noise removal, signal enhancement and image alignment are carried out through an image preprocessing technology, and data consistency is ensured; based on a multi-channel data set, a convolutional neural network is used for training and testing an initial model, an intelligent detection model is constructed, a fluorescence reaction mode is extracted, finally, a detailed ANA detection report is automatically generated by calculating a comprehensive scoring coefficient and comparing the comprehensive scoring coefficient with a preset threshold value, and an accurate immunodiagnosis basis is provided for doctors.

The invention discloses a microscopic image integer pixel alignment method based on sub-pixel edge offset detection, and relates to the field of microscopic image processing. The problems that an existing sub-pixel registration technology depends on iterative optimization or a complex deformation model, and real-time processing is difficult; sensor sampling errors and mechanical translation errors need to be compensated through a high-resolution camera or a precise displacement table, and the system cost is high. According to the invention, through sub-pixel-level offset compensation, sub-pixel alignment can be realized without depending on a high-precision displacement table, and the hardware cost is remarkably reduced. The method comprises the following steps: extracting image edge feature points by using an edge detection algorithm, fitting a gray centroid to calculate a sub-pixel offset, and counting an average offset; generating an affine transformation matrix according to the average offset, resampling the image, and aligning pixel values to an integer coordinate grid; and finally, cutting the image edge according to the interpolation algorithm type to eliminate the ghost. The method is also suitable for the application fields of fluorescence microscope multi-view splicing, super-resolution reconstruction and the like.

A deep learning-based digital / virtual staining method and system enables the creation of digitally / virtually-stained microscopic images from label or stain-free samples. In one embodiment, the method of generates digitally / virtually-stained microscope images of label-free or unstained samples using fluorescence lifetime (FLIM) image(s) of the sample(s) using a fluorescence microscope. In another embodiment, a digital / virtual autofocusing method is provided that uses machine learning to generate a microscope image with improved focus using a trained, deep neural network. In another embodiment, a trained deep neural network generates digitally / virtually stained microscopic images of a label-free or unstained sample obtained with a microscope having multiple different stains. The multiple stains in the output image or sub-regions thereof are substantially equivalent to the corresponding microscopic images or image sub-regions of the same sample that has been histochemically stained.

The present invention discloses a method and system for fusing visible light images and fluorescence images, and relates to the field of medical image processing technology. The method comprises: performing image registration on a visible light camera and a near-infrared camera to obtain registration parameters; synchronously acquiring visible light images and fluorescence images, preprocessing the received fluorescence images, and aligning the pixels of the visible light images and the preprocessed fluorescence images according to the registration parameters; performing pixel fusion on the aligned visible light images and fluorescence images to generate a multispectral image; and sequentially merging the fused multispectral images to generate a corresponding video sequence. The present invention adopts offline camera registration, and utilizes the registration parameters to efficiently and accurately align visible light images and fluorescence images. By enhancing the preprocessing, a high-quality fluorescence image can be generated. The fusion weight is calculated based on the edge intensity value and the fluorescence intensity value, so that the weighted fused multispectral image can simultaneously maintain edge and fluorescence intensity information, and is suitable for medical equipment such as fluorescence microscopes / endoscopes.

The invention discloses a single molecule microscopic imaging industrial camera imaging system and imaging method. The system comprises an inverted fluorescence microscope, a laser light source, a coupler, a light source adjusting device, an optical filter, an objective lens, a CMOS camera, a microcomputer processing system and an image enhancement device. The inverted fluorescence microscope is used for laser scanning confocal microscopic imaging or single molecule PALM microscopic imaging; the laser light source is used for providing incident light for the camera system; the coupler is used for obtaining high-power excitation and activation laser to enter the microscope system; the light source adjusting device is used for switching light sources; the optical filter is used for collecting the maximum fluorescence signal and blocking the exciting light at the same time; the objective lens is used for total internal reflection of light beams on the sample surface; the CMOS camera is used for photographing to obtain an image; the image intensifier is used for intensifying the image. And the microcomputer processing system carries out picture synthesis and result output through a preset correction algorithm. According to the invention, the living cell single-molecule trajectory detection efficiency can be improved.

A method for automatically ascertaining illumination brightnesses to be adjusted of at least two light sources for exciting at least one respective fluorophore in a sample to be imaged in a fluorescence microscope includes separately controlling, in terms of illumination brightness, each of the at least two light sources, detecting an image intensity of a microscopically imaged sample with at least two detectors, and automatically ascertaining the illumination brightnesses to be adjusted of the at least two light sources in such a way that a predefined setpoint of a signal-to-noise ratio is reached for each fluorophore. In order to ascertain the illumination brightnesses of the at least two light sources, cross-talk of a detector for different emission spectra of the fluorophores and / or cross-excitation of a fluorophore for different illumination spectra of the light sources are / is taken into account.

The invention discloses a denoising method based on R2U-Net and an adaptive noise addition model, and the method comprises the steps: obtaining an original noise image from a fluorescence microscope, carrying out the preprocessing, and reducing the noise level of the original noise image through median filtering; building a noise adding module to add noise to the image, wherein the noise adding comprises regional noise adding and global noise adding; the method comprises the following steps: establishing an R2U-Net network, and introducing a residual learning and recursion unit on the basis of U-Net; the R2U-Net network is trained, a new noise image is generated through self-supervised learning and serves as input of the R2U-Net network, the R2U-Net network is used for predicting a denoising result and optimizing the network, and the optimized R2U-Net network is used for denoising an image to be processed. According to the method, the noise in the fluorescent microscopic image can be dynamically and accurately removed without a large amount of annotation data, the detail information of the image is reserved, and the image quality is improved.

Disclosed is a method for gene expression by transient transformation of a wild rice seed using Agrobacterium. The method includes the following steps: subjecting a wild rice seed to induction culture and subculture in sequence, and selecting a resulting callus with a dense structure to allow pre-culture to obtain a pre-cultured callus; transforming a 1305Ubi-Ubi-GFP-H plasmid carrying a green fluorescent protein (GFP) gene into an Agrobacterium strain EHA105 or LBA4404 to obtain a positive Agrobacterium strain; subjecting the positive Agrobacterium strain to culture to obtain a bacterial suspension with an optical density (OD) value of 0.02 to 0.5 at a wavelength of 600 nm to obtain an infection bacterial solution; and infecting the pre-cultured callus with the infection bacterial solution to allow co-culture and recovery culture in sequence; observing an infection status of a recovered callus under a stereo fluorescence microscope, and calculating a transient expression rate.

This disclosure provides microscope-based systems and methods that significantly improve image contrast and have better vertical / horizontal resolution. This disclosure uses confocal microscopy in replacement of conventional fluorescent microscopy, for example, adding a spinning disk or a confocal microscopy scanning unit on the prior invention. Using a spinning disk vastly improves the speed of image acquisition (allowing for imaging of fast dynamic processes and live specimens), and considerably reduces photo damage. Disclosed herein are some embodiments to demonstrate how to set up the spinning disk microscopy system for image-guided illumination and photolabeling.

The invention discloses a fixing reagent and method for double-color fluorescence detection of an anti-NMDAR antibody and application, the fixing reagent comprises paraformaldehyde and a mixture of methanol and acetone, and the mixture comprises 10-25% by volume of methanol, 75-90% by volume of acetone, 10-25% by volume of paraformaldehyde, 10-25% by volume of paraformaldehyde and 10-25% by volume of paraformaldehyde. The fixing effect is better by adopting paraformaldehyde with the mass-volume ratio concentration of 4% and a mixture of methanol with the volume fraction of 15% and acetone with the volume fraction of 85% in two steps. By adopting the fixing liquid and the fixing method, the antigenicity of NMDAR cannot be damaged, obvious green fluorescence cells can be seen in a positive sample, red fluorescence quenching is avoided, and co-localization of red fluorescence and green fluorescence can be observed by using a fluorescence microscope.

The invention belongs to the field of molecular sieve material characterization, and particularly relates to a method for analyzing aluminum atom pairs in a ZSM-5 molecular sieve based on super-resolution fluorescence microscopic imaging, which comprises the following steps: activating a ZSM-5 molecular sieve sample; contacting the obtained sample with probe molecules; washing the ZSM-5 molecular sieve sample, and removing unbound or nonspecifically bound probe molecules; imaging the marked sample by using a super-resolution fluorescence microscope to obtain an image sequence containing a single fluorescent molecule emission event; processing the image sequence, determining a space coordinate of each fluorescence event, and reconstructing a super-resolution fluorescence image exceeding an optical diffraction limit; and based on the space coordinates of the fluorescent dots in the super-resolution fluorescence image, carrying out statistics to obtain the position, surface density or distribution information of the aluminum atom pairs in the ZSM-5 molecular sieve. According to the method disclosed by the invention, nanoscale spatial resolution, direct imaging and quantitative analysis of Al-pair in ZSM-5 (Zeolite Socony Mobil-5) can be realized.

2.1. To increase the resolution of quasi-confocal line scanning microscopes with a main beam splitter for coupling the illumination and detection beam paths, the "re-scan" method involves descanning the sample light in the detection beam path by the illuminating beam deflection unit. After descanning, the sample light is scanned again in the detection beam path by moving it across the sensor behind the confocal aperture by a second beam deflection unit in a direction transverse to the linear focus. To increase resolution in the longitudinal direction, the illumination line can be structured. This optical arrangement has the disadvantage of significant light losses due to the large number of optical interfaces that must be passed through. 2.2. A microscope in which the illuminating beam deflection unit is optically positioned between the light source and the main beam splitter, allowing the sample light to reach the sensor away from the illuminating beam deflection unit (non-descanned), is more light-sensitive and flexible. A control unit, in one operating mode, moves the sample light beam across the sensor using only the second beam deflection unit. Depending on the operating mode, synchronization with the first beam deflection unit may be provided. 2.3. Due to its high light sensitivity, the microscope is particularly suitable for fluorescence microscopy.

The invention relates to the technical field of equipment calibration, in particular to a fluorescence microscope metering calibration method and system.The detection end is connected with a host, the detection end is provided with a standard light source and a micropore array plate matched with the magnification times of an objective lens, the standard light source serves as the standard light source, and the host controls the luminous intensity of the host to be adjustable through an electric signal; by adopting the micropore array plate, the light source is in uniform light transmission, equal-scale and gradient dot matrix arrangement, and objective lens magnification, imaging uniformity, imaging linearity and optical linearity calibration are carried out in sequence, so that the system not only has traceability, reusability and controllability, but also is low in use cost and high in reliability; meanwhile, the magnification, the light uniformity, the imaging linearity and the optical linearity of the eyepiece of the measured fluorescence microscope are gradually calibrated, so that the accuracy and the reliability of measured data are ensured.

The invention discloses an image recognition analysis method and system for heart macrophage inflammation regulation and control, and relates to the technical field of macrophage inflammation regulation and control recognition, and the method comprises the following steps: observing and obtaining a cell image through a fluorescence microscope, and preprocessing the cell image to obtain a preprocessed image; constructing a cell recognition deep learning model, and training the cell recognition deep learning model by extracting fluorescence features of macrophages and gray features of inflammatory cells; carrying out statistics on macrophages and inflammatory cells in the inflammation area, and outputting a regulation and control inspection result; the method is used for solving the problem that the judgment of doctors on the condition of cardiac inflammation is easily affected due to insufficient recognition accuracy of macrophages and inflammatory cells in the existing macrophage inflammation regulation and control recognition technology.

The invention discloses a method for characterizing the degree of fusion of new and old asphalt based on fluorescence microscopy, and the process is as follows: using a confocal microscope to observe the surface of a wood simulated aggregate coated with homogenous SBS modified asphalt, obtaining fluorescence images at different depths, calculating an average fluorescence intensity curve, and fitting a fluorescence intensity attenuation correction function; observing the mixed asphalt on the surface of the wood simulated aggregate under the confocal microscope; scanning from the outermost layer to the innermost layer of the wood simulated aggregate, and outputting a plurality of fluorescence images of the new and old asphalt in the asphalt film thickness layer; performing noise reduction processing on the fluorescence image, obtaining a concentration change curve of the new asphalt and correcting it using an attenuation correction function; calculating a concentration attenuation change curve of the new asphalt, judging the influence of noise and the asphalt fusion layer, retaining the layer position with a fitting coefficient less than or equal to 95%, and calculating the concentration attenuation change curve after removing the influence of noise and fluorescence signal attenuation; and defining the degree of fusion of the mixed asphalt by an area method.

A method of analyzing a mixed fluorescence response of a plurality of fluorophores in a microscopic sample includes reconstructing individual fluorescence responses from a mixed fluorescence response using spectral un-mixing based on reference emission spectra for fluorophores to be reconstructed, and a procedure for determining and validating reference emission spectra including providing a plurality of image acquisition settings for a sequence of images of the sample equal to, or greater than, the plurality of fluorophores and including an illumination setting for each image, acquiring the sequence of images using the plurality of image acquisition settings and storing each image together with the corresponding illumination setting, determining candidate reference emission spectra for the fluorophores to be reconstructed from the sequence of images of the sample using one or more reference emission spectra determination algorithms, and conditionally using the candidate reference emission spectra as the reference emission spectra in the spectral un-mixing.