Method, controller and apparatus for performing fluorescence measurements

Abstract

A method of performing a fluorescence measurement, the method comprising: exposing a sample to excitation light focused to an excitation focus at an illumination parameter, where the sample comprises a fluorescent label, and the illumination parameter of the excitation light is configured to cause a non-linear fluorescence response measured by a detector focused on a detection focus, wherein the non-linear fluorescence response is caused by one or more of bleaching, saturation and stimulated emission; measuring a first response of the fluorescent label to exposure of the excitation light; measuring a second response to exposure of excitation light; wherein the method further comprises one or more of: modeling fluorescence saturation, bleaching, and stimulated emission at the focus based on the measured first response and the measured second response; a transmittance of excitation light through the sample to the focus is calculated based on the modeled one or more non-linear effects.

Description

Technical Field

[0001] This invention relates to the field of fluorescence measurement. More specifically, this invention relates to a method for addressing one or both of light scattering and absorption by utilizing the nonlinearity of the fluorescence response. The invention also relates to a controller configured to enable a device to perform the method and a microscope configured to perform the method. Background Technology

[0002] Fluorescence measurement, especially in microscopy, is a powerful tool for observing specific molecules. For example, fluorescence microscopy, or a fluorometer, can be used to observe a target fluorescent sample or a sample stained with a fluorescent label. For instance, a fluorometer can be used to measure chlorophyll fluorescence in plant leaves. This is performed by illuminating the fluorescent target with excitation light and then observing the fluorescence re-emitted by the target.

[0003] However, a limitation of fluorescence measurements is that scattering and absorption of light between the focusing lens and the focal point within the sample make it impossible to quantitatively measure the observed fluorophore concentration. The same scattering and absorption between the focusing lens and the focal point makes it difficult to qualitatively interpret microscopic images by eye, as the images produced by microscopy are distorted due to scattering and absorption. Therefore, microscopic images often appear to lack the contrast they should exhibit, making them difficult to interpret. Summary of the Invention

[0004] According to a first aspect of this disclosure, a method for performing fluorescence measurement is provided, the method comprising: The sample is exposed to excitation light focused to the excitation focus under the following irradiation parameters, wherein the sample includes a fluorescent label, and the irradiation parameters of the excitation light are configured to induce a nonlinear fluorescence response measured by a detector focused on the detection focus, wherein the detection focus and the excitation focus are the same focus, and wherein the nonlinear fluorescence response is caused by one or more of the bleaching, saturation, and stimulated emission of the fluorescent label. Measure the first response of the fluorescent label to exposure to excitation light; Measure the second response of the fluorescent label to exposure to excitation light; The method may also include one or more of the following: Based on the measured first and second responses, fluorescence saturation at the focal point is modeled. Based on the measured first and second responses, the bleaching of the fluorescent marker at the focal point is modeled; and Based on the measured first and second responses, the stimulated emission of the fluorescent marker at the focal point is modeled; Based on one or more nonlinear effects modeled, the transmittance of the excitation light through the sample to the focal point is calculated.

[0005] In one or more embodiments, the method may further include: An excitation optics and a detection optics are arranged such that the path of the excitation light from the sample to the focal point is substantially the same as the path of the detection light from the focal point to the sample. The excitation optics define the path of the light from the sample to the focal point, and the detection optics define the light from the sample that the detector can detect. By utilizing the reciprocity of light, the transmittance of the probe light from the focal point through the sample is calculated based on the calculated transmittance of the excitation light through the sample toward the focal point.

[0006] In one or more embodiments, the method may further include estimating the concentration of a fluorescent marker in the sample based on the measured response and the calculated transmittance of the probe light from the focal point through the sample.

[0007] In one or more embodiments, fluorescence measurements can be performed using a confocal microscope.

[0008] In one or more embodiments, the second response may be measured at a time after the first response has been measured.

[0009] In one or more embodiments, the first response of the measured fluorescent marker to exposure to excitation light can be a measure of either the first harmonic or a higher harmonic of the modulated light source.

[0010] In one or more embodiments, the second response of the measured fluorescent marker to exposure to excitation light may be a harmonic different from the harmonic measured for the first response.

[0011] In one or more embodiments, the first and second responses can be measured simultaneously.

[0012] In one or more embodiments, exposing a sample to excitation light under illumination parameters may include exposing the sample to excitation light under a first set of illumination parameters and subsequently exposing the sample to excitation light under a second set of illumination parameters.

[0013] In one or more embodiments, the step of measuring the first response of the fluorescent label to exposure to excitation light may occur simultaneously with the start of excitation light exposure of the sample under a first set of irradiation parameters, but prior to exposure to excitation light under a second set of irradiation parameters; and the step of measuring the second response of the fluorescent label to exposure to excitation light may occur simultaneously with the start of excitation light exposure of the sample under a second set of irradiation parameters.

[0014] In one or more embodiments, the irradiation parameters of the first and second measurements can be selected to produce different degrees of one or more of the following: Fluorescence saturation at the focal point; Bleaching at the focal point; and Stimulated emission at the focal point.

[0015] In one or more embodiments, the excitation light and the probe light can be focused by the same focusing lens.

[0016] In one or more embodiments, a beam splitter can be used to combine excitation and probe light.

[0017] According to a second aspect of this disclosure, a controller for an apparatus is provided, the controller communicating with the apparatus and configured to cause the apparatus to perform the method according to the first aspect.

[0018] According to a third aspect of this disclosure, a microscope is provided that includes a controller as described in the second aspect. Attached Figure Description

[0019] One or more embodiments will now be described with reference to the accompanying drawings, which illustrate: Figure 1 An example embodiment of a confocal microscope with an indicated excitation beam according to one or more embodiments of the present disclosure is shown; Figure 2 An example embodiment of a confocal microscope according to one or more embodiments of the present disclosure is shown, wherein light reaches an indicated detector; Figure 3 The method according to this disclosure is shown; Figure 4 Uncorrected microscopic images are shown; Figure 5 Corrected microscopic images are shown, illustrating the results of the disclosed method; and Figure 6 An example system including a controller and a microscope according to one or more embodiments of the present disclosure is shown. Detailed Implementation

[0020] This disclosure provides a method for performing one or more fluorescence measurements that addresses scattering and absorption within a sample by utilizing the nonlinearity of the fluorescence response. This scattering and absorption is particularly problematic in confocal microscopy of 3D samples where the target focus is not at the sample surface. In this disclosure, the sample is typically a 3D sample, and the focus may be located at a location within the sample rather than at its surface. Scattering and absorption may not be unique to confocal microscopy; they are also problems in other types of fluorescence measurements.

[0021] For short, low-intensity exposures, the detector response is typically linear across the excitation light intensity. This means that exposure using the total light intensity of several exposures should produce a detector response equal to the sum of the detector responses obtained from individual exposures. However, this is not always the case due to fluorophore bleaching, fluorescence saturation, stimulated emission, and other nonlinear effects. In this disclosure, bleaching, saturation, and stimulated emission will be discussed as specific examples of nonlinear effects; however, it should be understood that other mechanisms of nonlinear fluorescence response can be induced, modeled, and used to calculate the transmittance of excitation light through the sample to the focal point.

[0022] By measuring nonlinearities, additional information inaccessible when measuring linear responses can be obtained. Saturation, bleaching, and other nonlinear interactions between the excitation light and the fluorescent label depend on the local intensity of light at the fluorescent label, such as a fluorophore. It has been found that the light intensity at the focal point can be modeled by measuring the intensity of these nonlinear effects and modeling the mechanism of the nonlinear response. The intensity of the nonlinear effects is obtained by acquiring at least two detector readings at different exposure times, for example, two different exposure intensities, after different total exposures and at different harmonics of the modulated excitation light. Based on the model of the light intensity at the focal point, the transmittance of the excitation light through the sample to the focal point can be obtained. This transmittance captures the portion of the excitation light that passes through the sample to the focal point, i.e., without loss due to scattering or absorption. It was also found that by arranging the detector to focus at the same focal point as the excitation source, and by arranging it such that the excitation and probe light pass through the sample at similar wavelengths using the same optical path (as in confocal microscopy), we can use the reciprocity of light (the principle of invariance stated under the exchange of light source and detector) to estimate the transmittance of emitted light from the focal point to the detector, given the transmittance from the excitation light to the focal point. Knowing both transmittances allows for the quantification of the fluorescent label concentration from the linear portion of the response, regardless of the absorption and scattering of the excitation and emitted light. For example, performing this operation on each pixel in a microscopic image allows for the correction of transmittance variations across the entire image, between different depths of the 3D sample, and between different 3D samples. By performing these steps and appropriate calibration of all components, transmittance can be obtained in molecules / μm. 3 The concentration of fluorescent markers is expressed in units, thus allowing for quantitative comparison of concentrations between different samples, objectives, and microscopes.

[0023] In this disclosure, a Cartesian coordinate system is used, where z is the depth of the sample entering the sample along the excitation beam path toward the focal point, and x and y are two directions orthogonal to each other and to z. It should be understood that these coordinate systems and assumptions are used for ease of reference and are in no way intended to limit this disclosure. The reader will understand that alternative coordinate systems, nomenclature, and assumptions may be employed without departing from the scope of this disclosure.

[0024] Figure 1 An example embodiment of a confocal microscope 100 with an indicated excitation ray is shown. The confocal microscope will be used as an exemplary form of the measuring apparatus in this disclosure; however, it should be understood that other measurements of fluorescence can utilize the same methods disclosed herein to obtain one or more of the beneficial effects described herein. For example, this method can be applied to light-sheet microscopy, multiphoton microscopy, or another form of microscopy, or to one or more of the following: fluorescence sensors (such as fluorometers, microfluorometers, fluorescence spectrophotometers, X-ray fluorescence spectrometers), or other devices that use confocal excitation and detection optics to measure fluorescence.

[0025] In microscopy or other fluorescence measurements, sample 101 can be received at a sample stage. The stage can be a typical sample stage in microscope 100. However, it should also be understood that the term "stage" is used herein to refer to the location on which the sample must be placed to perform the measurement. Thus, industrial-scale systems may use a conveyor belt that moves sample 101 to the correct position for measurement, or other means may be used to place sample 101 in the desired position. Handheld devices may require the device to be positioned such that the stage is in front of the leaves of a plant. In any case, for the purposes of this disclosure, the position of sample 101 relative to the apparatus 100 used for measurement is considered the stage. This apparatus may be, for example, a microscope, such as a confocal microscope.

[0026] In the case of a 3D sample, sample 101 can be, for example, a sphere composed of cells, organoids, or another 3D tissue sample. In other examples, sample 101 may not necessarily be a tissue sample, but rather a different type of sample, such as a part of a living organism, such as a leaf, a single cell, a mineral sample, an electronic device, or a colony of bacteria or fungi.

[0027] The sample includes a fluorescent marker. In this specific embodiment, sample 101 may be referred to as being fluorescent itself; however, this is not necessary and is described in this manner only for ease of description. In other examples, sample 101 may be fluorescent itself or have a fluorescent component. In other examples, sample 101 may be stained with a fluorescent marker, or the fluorescent marker may be applied to the sample in some other way. The method according to this disclosure may include the step of staining sample 101 with a fluorescent marker before or after receiving sample 101 at a sample stage. The method may also include one or more additional sample preparation steps, such as clarifying the sample with a clarifying agent to make the sample transparent or substantially transparent.

[0028] Before exposing sample 101 to excitation source 102 in microscope 100, the methods of this disclosure may require setting one or more irradiation parameters to appropriate parameters for the excitation, bleaching, or stimulated emission of the selected fluorescent label. It should be understood that light refers not only to visible light but also generally to electromagnetic radiation. This disclosure can be applied to, for example, infrared, visible, ultraviolet, or X-ray radiation. The excitation source can be a laser, a light-emitting diode, an X-ray tube, another photon generator, or a combination of photon generators. It should also be understood that although the light to which the sample is exposed is referred to herein as excitation light, it may have purposes other than exciting the fluorescent label, such as inducing a nonlinear response by bleaching or stimulated emission. Specifically, as will be further described below, the irradiation parameters are configured to induce one or more of saturation, stimulated emission, and bleaching of the fluorescent label.

[0029] The method may also include setting one or more detection parameters for detector 103. For example, it may be necessary to set the gain of detector 103 or the interval of wavelengths used to perform detection. In one or more embodiments, it may be desirable to select a detection wavelength that is substantially similar to but still different from the excitation wavelength of excitation light 102. In one or more embodiments, the detection wavelength may be within 1%, 5%, 10%, or 20% of the wavelength of excitation light. In one or more embodiments, the detection wavelength may be within 10, 50, 100, or 150 nanometers from the excitation wavelength. Detector 103 may be a photomultiplier tube or any other suitable photon detector adapted to detect photons emitted by a fluorescent marker.

[0030] Figure 1 and Figure 2 A description of confocal microscopy is provided by way of example, and the following description introduces some concepts and assumptions according to this disclosure in the context of confocal microscopy. Following the following general introduction to confocal microscopy, full details of the method according to this disclosure will be provided.

[0031] like Figure 1 As shown, confocal microscopy begins by exciting a fluorescent marker on sample 101 using a set of illumination parameters of a light source 102. In one or more embodiments, the beam emitted by the excitation source 102 is directed to a beam splitter 104, which directs the incident beam to a focusing lens 105, which is configured to focus the light onto a target region 106.

[0032] Beam splitter 104 can be, for example, a dichroic mirror or a semi-transparent mirror, which can be configured to selectively reflect incident photons within a predetermined wavelength range and allow photons outside the predetermined wavelength range to pass through. In the case of confocal microscope 100, this allows excitation light to be reflected toward sample 101, while fluorescence emitted from sample 101 can pass through beam splitter 104 toward detector 103. It should be understood that beam splitter 104 is not a necessary component of all fluorescence measurement systems, and alternative arrangements without beam splitter 104 are also possible.

[0033] Focusing lens 105 is used to focus the excitation light toward focal point 106. When focusing lens 105 focuses the beam onto focal point 106, the focusing lens cannot focus the beam to a precise discrete point. Instead, it should be understood that focal point 106 can be defined as the volume in which the beam is focused in its narrowest region before the beam begins to diverge again. This can be alternatively referred to, for example, as the focal area or focal volume. This focal point can extend from an “idealized” or calculated focal position, for example, by one, two, five, or ten wavelengths. In practice, a finitely precise focal point cannot exist because the diffraction limit fundamentally requires that the range of the focal point be limited by the wavelength of the excitation light. When focusing lens 105 focuses the excitation light toward the intended focal point 106, the excitation light may be aberrated by the sample before reaching focal point 106, resulting in a larger volume of the beam reaching its narrowest region than if the beam were not affected by sample aberrations. As for focal point 106, we are referring to the volume of the narrowest region surrounding an aberration-free beam. The distribution of excitation light intensity in the focal point can be described by the excitation point spread function (PSF). The focusing lens 105 can be an excitation focusing lens and can form part of a larger set of focusing optics. The focal point 106 to which the excitation light is focused by the excitation focusing lens 105 can be referred to as the excitation focal point.

[0034] When the excitation light strikes sample 101, several possible interactions occur. Scattering of the incident photons by sample 101 is possible, resulting in scattered photons being sent in different directions at the same or substantially the same wavelength. Photons may also be lost due to absorption by the sample before reaching the focal point. Scattering and absorption reduce the number of excitation photons reaching the focal point.

[0035] Figure 2 The path taken by the probe photons received at detector 100 is shown. Probe photons are defined herein as those emitted in the direction from the probe optics toward the detector. These are a subset of all photons emitted by the fluorescent label, since the fluorescent label will emit photons equally in all directions of the isotropic material. Most of the fluorescence emission contributing to the detector response occurs at the focal point. Fluorescent photons are emitted in random directions; however, Figure 2The path taken by photons that can be detected by detector 103 is shown. These photons follow paths substantially opposite to those taken by excitation photons through the sample. In the case of confocal microscopy, the probe photons continue along the reverse path of the excitation photons until they reach beam splitter 104, where they pass through and enter detector 103. The detector has a response that focuses to the same focal point as the excitation light. That is, the system's probe optics are arranged such that photons originating from the focal point and striking the probe optics are directed toward the detector. The detector's focal point is similarly not a single point but a volume, and the detector's sensitivity to different points in space can be described by the probe PSF. As with excitation photons, probe photons can also be scattered and absorbed through the body of the 3D sample. Extinction of excitation and fluorescence photons is one of the main reasons why quantitative detection cannot be performed during microscopy and why images often end up distorted (especially within the body of a 3D sample). The probe optics may include a probe focusing lens that receives the probe light from the focal point and directs it toward the detector. In a reciprocal manner, the detector can also be described as being focused on a focal point within the sample by a detector focusing lens. The volume or region of the detector focus can be referred to as the detector focus. The detector focus coincides with the excitation focus, and specifically, the detector focus can be defined on a volume substantially the same as the excitation focus.

[0036] Figure 3 An example embodiment of method 300 according to this disclosure is shown. The following description will explain the method and provide examples in the context of the confocal microscopy method described above.

[0037] Method 300 may include receiving a sample 101 comprising a fluorescently labeled substance at the stage of a microscope, as described above. The method may also include setting one or more detection parameters of detector 103. For example, it may be necessary to set the gain of detector 103 or the interval of wavelengths performing the detection.

[0038] Before exposing sample 101 to excitation photons 102, the irradiation parameters of the excitation light can be set. The irradiation parameters are configured to induce at least one nonlinear response in the fluorescent label by selecting parameters suitable for the sample. This means that at least one of the responses is affected by fluorescence saturation, bleaching, or stimulated emission. The parameters will be any parameters that will induce one or more of bleaching, saturation, or stimulated emission in the fluorescent label, such as a first laser intensity, wavelength, or exposure time. The selected parameters can be sample-specific and selected by the user or by the measuring device itself (in some examples, a confocal microscope) based on automatic detection of the type of fluorescent label or based on received input indicating the type of fluorescent label. In the case of automatic selection, the device may include a sample type detector configured to detect the type of sample located at the sample stage. In one or more embodiments, multiple different parameters can be tested before obtaining a desired set of parameters that induce the desired nonlinear response. It should be understood that in one or more embodiments, the step of setting the irradiation parameters may not be necessary, as these parameters may be pre-programmed or set as default parameters in the microscope or the controller controlling the microscope. The irradiation parameters of the excitation light 102 before the first measurement can be the first set of irradiation parameters.

[0039] Method 300 includes exposing sample 101 to excitation light 102 under illumination parameters. At least one set of illumination parameters is configured to induce a nonlinear response due to one or more of bleaching, saturation, and stimulated emission of the fluorescent label, as described above. The set of illumination parameters inducing the nonlinear response may be a first set of illumination parameters or a second set of illumination parameters.

[0040] The method may include the following steps: arranging an excitation optics and a probe optics such that the path of the excitation light from the sample to the focal point is substantially the same as the path of the probe light from the focal point through the sample, wherein the excitation optics are configured to define the path of the light from the sample to the focal point, and the probe optics are configured to define the light from the sample that the detector can detect.

[0041] Method 300 also includes measuring the first response of the fluorescent label 302 to exposure to excitation light 102.

[0042] Method 300 also includes measuring a second response of the fluorescent label to exposure to excitation light 102.

[0043] In one or more embodiments, the second response can be measured at a time point following the first response. For example, sample 101 is exposed to excitation light 102, and the first response can be measured substantially simultaneously with or after a delay from the exposure of the sample to excitation light 102. The second response can be measured substantially simultaneously with the exposure of the sample to excitation light 102, or after a second delay from the measurement of the first response. The first delay and the second delay can be the same or different.

[0044] In one or more embodiments, method 300 may include the step of exposing the sample to excitation light under a second set of irradiation parameters after measuring a first response of the fluorescent label to exposure to excitation light 102 but before or simultaneously measuring a second response of the fluorescent label to exposure to excitation light 102.

[0045] The first and second sets of illumination parameters can differ, for example, in the intensity of the excitation light 102 (if a second set exists). In other examples, the exposure time can differ, as can the repetition rate, wavelength, or any other parameter of the excitation light. In one or more examples, the first and second sets of illumination parameters can be identical. Using similar parameters can be particularly effective in distinguishing between the saturation effect and the bleaching effect of the sample, since saturation can be expected to be the same in both the first and second measurements, while bleaching caused by the first, second, or any additional exposure of the sample 101 to the excitation light continues to affect the second measurement, where the second exposure of the sample to the excitation light 102 combines the effect of bleaching. Using different excitation wavelengths to selectively excite and induce stimulated emission allows for the measurement of the efficiency of the stimulation.

[0046] In one or more alternative embodiments, the first and second responses can be measured simultaneously under a second set of illumination parameters without exposing the sample 101 to the excitation light 102. For example, when the sample is exposed to a harmonic modulation light source, different harmonic components of the response can be measured simultaneously. For example, the fundamental frequency (first harmonic) can be measured simultaneously with the second or third harmonic. One way to achieve this is by using a lock-in amplifier; however, alternative specific implementations can be used to simultaneously measure the first and second responses of the fluorescent label on the sample to the excitation light.

[0047] In one or more embodiments, the method may include measuring more than two responses of the fluorescent label to exposure to excitation light. For example, the method may include measuring at least 3, 5, 10, or 20 responses of the fluorescent label to exposure to excitation light. Additional measurements can provide additional data points that can be used to subsequently obtain a more accurate model of saturation, bleaching, or stimulated emission, and to account for error sources such as noise and background signals. Performing multiple measurements of the fluorescent label's response to exposure to excitation light can utilize corresponding numbers of light exposures, as described above regarding the second exposure of the sample to excitation light. In other embodiments, more than two different harmonics may be measured simultaneously. In one or more embodiments, fluorescence at more than one wavelength may be measured simultaneously to distinguish between stimulated emission and spontaneous emission. In one or more embodiments, combinations of different harmonic measurements and time interval measurements may be performed.

[0048] The method further includes one or more of the following: modeling saturation of the fluorescent marker at the focal point of the excitation light 102 based on the measured first response and the measured second response 304a; modeling bleaching of the fluorescent marker at the focal point based on the measured first response and the measured second response 304b; and modeling stimulated emission of the fluorescent marker at the focal point based on the measured first response and the measured second response 304c. It will be understood here that the focal point of the excitation light will generally be considered as the focal point described above.

[0049] There are various methods available for modeling saturation at the focal point of the excitation light. For example, saturation can be modeled using a measured or estimated excitation PSF along with a model of the saturation of each fluorescently labeled molecule; it can be modeled using empirically motivated parameterization of the focal emission rate as a function of the excitation intensity at the focal point, obtained by calibrating the measurements; or a machine learning model can be trained on known reference samples to output transmittance given a measured first and second response.

[0050] Similarly, various methods exist for modeling bleaching at the focal point of the excitation light. For example, when imaging a single z-slice, such as in confocal microscopy with well-distinguished focal points, or when measuring the concentration of a single fluorescent label at a single focal point without imaging, the amount of bleaching can be modeled as varying across different portions of the focal point. The amount of bleaching at the focal point and its effect on measurements determined using the measured or approximate probing PSF can then be modeled using the measured or approximate probing PSF. In another approach, numerous z-slices and overlapping pixels can be repeatedly imaged, such that exposure can be assumed to be uniform except for variations in transmittance, and any spatial variation in the amount of bleaching is due to differences in transmittance.

[0051] Similarly, various methods exist for modeling stimulated emission at the focal point of the excitation light. For example, the amount of stimulated emission can be modeled as varying across different portions of the focal point. Then, the amount of stimulated emission at the focal point and its effect on measurements determined using the measured or approximate detector PSF can be modeled using a measured or approximate detector PSF. By repeatedly exposing the sample to the same amount of excitation light but different amounts of stimulation light, the amount of stimulation light reaching the focal point can be estimated based on the varying amounts of autofluorescence and stimulated fluorescence reaching the detector. The estimate of the stimulation light reaching the focal point can be used to determine the transmittance through the sample.

[0052] In implementations of saturation, bleaching, and stimulated emission that affect the response, some of these effects may need to be modeled simultaneously.

[0053] When modeling saturated, bleached, or stimulated emission, it should be recognized that many details of the model may become irrelevant once the model is used to fit the detector response. For example, the shape of the PSF may not be important for calculating the expected measurement results, and a function summarizing the intensity distribution of the PSF can be used instead, as done in the example implementation below.

[0054] For illustrative purposes, a full example of one method for modeling saturation and bleaching to estimate transmittance will be provided later in this disclosure.

[0055] Based on modeling one or more of saturation, bleaching, and stimulated emission, method 300 also includes calculating 305 the transmittance from the microscope objective (the focusing lens of the apparatus) to the focal point. That is, one or more of the saturation model, bleaching model, and stimulated emission model can be used to estimate how many photons travel from the focusing lens of the apparatus to the focal point and how many photons are lost due to one or both of scattering and absorption. It should be recognized that some parts of the modeling may contain uncertain constants that make the transmittance undeterminable over a range of overall constant multiples.

[0056] Method 300 then includes calculating the transmittance of fluorescence from the objective lens through the sample based on one or more modeled nonlinear effects. This may include using the reciprocity of light to calculate the transmittance of fluorescence from the objective lens to the objective lens based on the calculated transmittance from the objective lens to the focal point. The reciprocity of light means that light with a specific wavelength and polarization state behaves identically when the source and detector are exchanged. Excitation light and fluorescence have similar but not identical wavelengths, making the reciprocity approximation. This method may include adjusting for this by estimating errors based on the wavelength dependence of light absorption and scattering in the sample. The method may also include using a filter on one or both of the excitation source and detector to bring the excitation wavelength and detector wavelength closer to each other, thereby making the approximation of reciprocity more accurate. The filter may include a portion of one or both of the excitation optics and the detector optics. Additionally, the method may include arranging the excitation and detector optics such that the excitation beam and detector beam are opposite in direction but similar in the portion of the optical path through the sample. This is to enable the estimation of the detector transmittance based on the transmittance of the excitation beam using the reciprocity of light. Typically, excitation and detection optics can be configured and arranged to ensure one or more of the following: defining the correct optical path for the excitation and detection light; selecting a predetermined wavelength for the excitation and detection light; and selecting a predetermined polarization for the excitation and detection light. Depending on the requirements of the measurement in question, the light can be unpolarized or polarized.

[0057] This disclosure will later provide a complete example of how to calculate the transmittance from the focal point to the detector based on the transmittance from the excitation light to the focal point.

[0058] Method 300 may ultimately include estimating the concentration of the fluorescent marker at the focal point 307 using an estimated transmittance from the focal point (focal region) to the objective lens. In other embodiments, the method may alternatively include estimating and correcting the intensity of the fluorescence image based on the estimated transmittance from the focal point to the objective lens to compensate for the effects of scattering and absorption of excitation and probe light passing through the sample.

[0059] This method may include the step of repeating the method described above multiple times at multiple different locations (x, y, z) through the sample. Repeating the method multiple times allows an image to be constructed from the individual measurements. If each pixel is measured in the (x, y) plane using the same depth z, the image can be provided as a 2D image. In other examples, different depths z can be used to determine a 3D image. In one or more alternative embodiments, method 300 may not include the step of estimating the fluorescent marker concentration. In such an embodiment, method 300 may alternatively include the step of estimating the corrected intensity of image pixels associated with a location in the sample. The method can then be repeated for multiple points throughout the sample to construct a corrected image within the sample. In other embodiments, both fluorescent marker concentration estimation and estimation of the corrected intensity of image pixels can be performed.

[0060] Figure 4 Example uncorrected image 400 is shown. It can be seen that the interior of the sample appears dark due to the increased number of scattering and absorption events occurring on the light reaching the sample body.

[0061] Figure 5 An example corrected image 500 is shown, which has been corrected using the methods of this disclosure. It can be seen that the interior of the sample is not affected by the same artificial darkening caused by scattering and absorption, but instead provides a more accurate representation of the sample.

[0062] Figure 6 An example controller 601 communicating with microscope 602 is shown. Controller 601 is configured to cause microscope 602 to perform each step of method 300. In one or more embodiments, controller 601 may be remote from microscope 602, and in one or more alternative embodiments, controller 601 may be part of microscope 602. For example, controller 601 may be part of or a computer configured to control a connection to microscope 602. In other examples, microscope 602 may not require external control and may be configured to operate independently, and therefore may include controller 601.

[0063] Example of an implementation plan Not wishing to be bound by theory, the following sections provide details relating to a more academically rigorous description of one or more embodiments of this disclosure. It should be understood that, as noted above, one or more steps derived below may not be necessary in all embodiments, but are provided by way of example in this specification. The scope of protection of this invention is defined by the appended claims.

[0064] We consider the case of confocal microscopy, where the sample is in the same... z The slice was used to excite the laser at several different intensities. Repeated imaging, in which k Exposures were labeled according to the acquisition order, k=1,2,3,… Each detector reading (image) was represented as the count of photons detected at each pixel. , where i and j are the indices of the labeled pixels. We show how the obtained image can be used to compute a new image corrected for the scattering and absorption of excitation light and fluorescence.

[0065] We assume the sample is stained with a simple fluorophore (or another fluorescent marker), which can be in one of two states: a relaxed state or an excited state. When in the relaxed state, it is stained with a fluorophore having an extinction coefficient. The incident photon excitation, i.e., the excitation rate is... ,in It is the local intensity of the excitation light. When in the excited state, it emits light at a rate of... Relaxation, and each relaxation is done with probability. (i.e., quantum yield) emits photons. Additionally, we believe the fluorophore emits photons at a rate... Bleaching, regardless of its excited state. This is an approximation, but it's good enough for low levels of saturation. Initially, we don't consider bleaching, but we will eventually consider its effect. In this example, there is no stimulated emission.

[0066] When local intensity During irradiation, we obtain the probability that the fluorophore is in an excited state. The evolutionary pattern is as follows:

[0067] And the expected emission rate of each fluorophore It is given by the following formula:

[0068] In the setup considered here, the exposure time of a single pixel to excitation light is on the order of 1 µs, and the lifetime of the fluorophore is... The order of magnitude is 1 ns. Therefore, a good approximation is that the system has reached equilibrium and the emission rate is given by the steady-state value:

[0069] in: .

[0070] Now consider as follows Figure 1 The confocal microscope setup. We have defined plane 107A behind the dichroic mirror but in front of the sample, and plane 107B in front of the region from which the fluorescence response originates.

[0071] Excite the laser to intensity The emitted light travels through the microscope optics, through plane 107A and into the sample, through plane 107B and into the focal point. The excitation point spread function (PSF) describes the intensity distribution of the excitation light at the focal point. Similarly, there exists a probe PSF that indicates the detector sensitivity to fluorescence emission at different points in space. The precise 3D shape of the PSF depends on the microscope optics (e.g., excitation and detector pinhole sizes), the objectives, and the transmittance of each possible beam path from the objectives to the focal point. However, we have found that, for the purposes described here, a reasonable approximation is that the shape of the excitation and probe PSFs is independent of the transmittance of the individual rays between the objectives and the focal point, and changes only by a multiplicative factor as the average transmittance changes over all rays. We denote this average transmittance as… And the local intensity around the focal point is written as:

[0072] in It is an excitation PSF independent of transmittance. Is in The coordinates of the focal point when the pixel is imaged. It is the depth of the focal plane, and It depends on the microscope optics but is related to And a constant independent of the sample's transmittance. We have labeled the transmittance with pixel indices because the transmittance varies between focal points corresponding to different pixels. It is used to stimulate The intensity of the excitation laser.

[0073] Similarly, for those from space Pixels of a single point The density of light entering the detector is given by the following formula:

[0074] in It is a detection PSF that is independent of transmittance, and It is the average transmittance from plane 107B to 107A, and It is a constant that depends on the microscope optics but is independent of pixel index and sample transmittance. Local relaxation rate. It is given by the product of the concentration of fluorophores and their respective emission rates:

[0075] in It is the concentration of fluorophores, and It is the local excitation light intensity. Reaching the pixel. The total amount of light from the detector It is obtained by integrating the contributions from the entire sample volume:

[0076] Here we have already performed formal integration over all 3D spaces; however, we only need to know the shape of the PSF near the focal point, as the region near the focal point is the main contributor to this integration.

[0077] We will now make another simplifying assumption; we assume that the fluorophore concentration within each focal point is uniform. This is generally a poor approximation, but for our purpose of measuring transmittance, it proves to be good enough. The reason is that although the concentration varies within the focal point, the transmittance variation due to scattering and absorption within a single focal point is negligible. The concentration variation remains a problem for the expression here, but once we average it over many pixels (e.g., by reducing the image for transmittance calculation), this error averages to 0 because the average concentration distribution across several focal points is uniform. We therefore make an approximation. Note that we still have a dependency on pixel indices, because the density may differ at the focal point corresponding to different pixels. We will now... Written as

[0078] in:

[0079] This can be considered as a function Extend this to the entire focal point. Given the normalized incident light intensity at the focal point. This parameter characterizes the amount of excitation light emitted that can be detected by the detector. (Function) It does not capture the transmittance of light arriving between the objective and the focal point; it only captures the nonlinearity of the response of light that has already reached the focal point. As we will see later, it will be important to optimize the excitation and probe wavelengths to be close to each other and to optimize the beam shapes used for excitation and probe to be similar. Because of this, we can approximate the excitation and probe PSFs to be the same. We noticed that, The integrand in the definition depends on position only with respect to the value of PSF. Therefore, we can change the variable to integrate with respect to the value of PSF instead. We will change this variable... Marked as:

[0080] in:

[0081] and It was Dirac function. Regardless of the shape of the PSF, it is a measure of the volume of each value of the PSF, thus we can obtain a fairly coarse approximation of the PSF. Instead of measuring the PSF, we use an idealized Gaussian PSF (here in cylindrical coordinates). ):

[0082] here Set the width of the PSF's waist (limited by the wavelength) and It is the beam divergence, proportional to the numerical aperture of the objective lens. This PSF is normalized so that... The integral on the plane is 1. The total factor can be simply incorporated into the constant. and We can use this PSF in Integral operations are performed within the definition to obtain: .

[0083] Therefore, we calculate

[0084] , Among them, the dimensionless saturation function Defined as:

[0085] Finally, we get .

[0086] Now, we assume no bleaching and a constant fluorophore concentration. However, the fluorophore concentration decays as the fluorophore is bleached by excitation light. When the fluorophore is in the excited state, bleaching is usually caused by fluorophore oxidation, meaning the bleaching rate also saturates with increasing intensity. However, for simplicity, we neglect this effect (which only relates to bleaching in the focal plane at high excitation intensities) and assume a linear bleaching rate:

[0087] in This is a parameter indicating how quickly the fluorophore bleachs. We added the time dependence of concentration and intensity, which was implicit until now. We can write the solution to this differential equation as follows:

[0088] We further assume that the excitation laser scans uniformly across the entire focal plane, and therefore the bleaching is uniform. This is a good approximation outside the focal plane, but at the focal plane, depending on the pixel pitch, bleaching exhibits slight inhomogeneity compared to the PSF beam waist size. It should be noted that both of our assumptions about bleaching become very good approximations outside the focal plane, and therefore if bleaching is primarily due to scanning multiple... Stacking-induced effects work well. We also note that since the light rays are straight, the angular distribution of the light rays does not change within the sample. This means that, using the same laser intensity, when scanning the entire image, the amount of light reaching a particular fluorophore is always the same (except for some edge effects), regardless of the focal plane. This also means that, except for edge effects, bleaching at a particular fluorophore is similar to integrating the laser intensity multiplied by the local transmittance. It is proportional, and independent of which focal plane we are scanning.

[0089] Now consider that we have equal time. But different laser intensity settings Several exposures. The number of photons detected in the detector for a specific pixel. It follows a Poisson distribution (we assume the signal originates from many individual fluorophores), where the average value is given by the integrated intensity over the exposure time. Consider from arrive Exposure:

[0090] In the The average concentration at a specific pixel at the start of the second exposure is given by the following formula:

[0091] We are on the first Integrating the concentration from each exposure yields:

[0092] We noticed It may be unknown. These are similarly possibly unknown fluorophore parameters. However, we don't need all of these factors because they only appear in specific combinations. We define...

[0093] And this simplifies to:

[0094] in .

[0095] It is known and It can be measured or fitted individually. For each pixel, we have two unknowns:

[0096] By acquiring two or more images We can use the results to solve for these two unknowns. Since these unknowns are random due to shot noise, we can obtain better estimates of them by acquiring further images.

[0097] Due to normalized concentration and focal-to-objective transmittance Here, they always appear as a product, so we cannot solve them individually. To obtain the concentration, we arrange the excitation and probe beam paths as similar as possible. This allows us to use the reciprocity of light to ensure that the transmittance from the objective to the focal point and from the focal point to the objective is the same, regardless of the sample placed in the microscope. This is similar to the existing construction principle of confocal microscopes. The geometry of the excitation and probe rays is identical. We label each ray with the point intersecting plane 107A and write the total transmittance as a weighted sum of the transmittances of the individual rays that make them up: .

[0098] here and Marked on wavelength From point 107A in plane To the focal point and at wavelength The transmittance of each ray of light propagating in the opposite direction. and This depends on the contribution of the multiplication factor of the PSF for each ray, the intensity distribution of the light source across plane A, and the weights of the detector sensitivity for different rays across plane 107A. Note that... and Both can be designed separately by using apodization filters on the excitation light or the detector, so we can make them identical: .

[0099] We can further bring the excitation and detection wavelengths close to each other by, for example, selecting fluorescent markers and detector filters. If they are close enough, we can use the Stokes-Helmholtz reciprocity principle. This principle states that from point U, with polarization... Emitting, with polarization The proportion of light reaching point V is related to switching the positions of the light source and detector, i.e., from point V with polarization. Emitting, with polarization The proportions of the light rays reaching point U are the same. In our scheme, where U is a point in plane 107A and V is the corresponding point in plane 107B, this means: , And we obtain: , It is independent of the transmittance of each ray passing through the sample between planes 107A and 107B. Using this setup, we can now... Seek And ultimately from the value obtained Calculate .

[0100] absolute concentration Through constants and The relevant parameters can be determined using the above formula and measurements of the quantities therein, or simply by calibrating the measurements. Otherwise, It can be used directly to create images that are corrected for changes in transmittance, but displayed in arbitrary units.

[0101] Another nonlinear fluorescence effect that can be used for transmittance calculations is stimulated emission. As in stimulated emission depletion microscopy (STED), a fluorescent sample can be exposed to both excitation and emission excitation light, and the response comprising only spontaneously emitted (non-stimulated emission) photons can be measured. Selective measurement of spontaneous emission can be accomplished, for example, by filtering wavelength differences or gating the response based on the timing of excitation and excitation measurements. The ratio of spontaneous to stimulated emission can be obtained by measuring a first response comprising both spontaneous and stimulated emission and a second response comprising only spontaneous emission. The ratio of stimulated to spontaneous emission depends on the intensity of the excitation light reaching the focal point. As is the case in the depletion region of STED, high-intensity excitation light causes most emitted photons to be generated in a stimulated manner. When no excitation light reaches the focal point, all emission will be spontaneous. Therefore, we can use this ratio as a measure of the intensity of the excitation light at the focal point and use this intensity to calculate the transmittance from the objective to the focal point.

Claims

1. A method for performing fluorescence measurement, the method comprising: A sample is exposed to an excitation light focused to an excitation focal point under irradiation parameters, wherein the sample includes a fluorescent label, and the irradiation parameters of the excitation light are configured to induce a nonlinear fluorescence response measured by a detector focused on a detection focal point, wherein the detection focal point and the excitation focal point are the same focal point, and wherein the nonlinear fluorescence response is caused by one or more of bleaching, saturation, and stimulated emission of the fluorescent label. Measure the first response of the fluorescent label to exposure to the excitation light; Measure the second response of the fluorescent label to exposure to the excitation light; The method further includes one or more of the following: Based on the measured first and second responses, fluorescence saturation at the focal point is modeled. Based on the measured first response and the measured second response, the bleaching of the fluorescent marker at the focal point is modeled; as well as Based on the measured first response and the measured second response, the stimulated emission of the fluorescent marker at the focal point is modeled; Based on one or more nonlinear effects modeled, the transmittance of the excitation light through the sample to the focal point is calculated.

2. The method according to claim 1, wherein the method further comprises: An excitation optics and a detection optics are arranged such that the path of the excitation light through the sample to the focal point is substantially the same as the path of the detection light from the focal point through the sample, wherein the excitation optics define the path of light through the sample to the focal point, and the detection optics define the light from the sample that the detector can detect; Utilizing the reciprocity of light, the transmittance of the probe light from the focal point through the sample is calculated based on the calculated transmittance of the excitation light through the sample toward the focal point.

3. The method according to claim 2, wherein the method further comprises: The concentration of the fluorescent marker in the sample is estimated based on the measured response and the calculated transmittance of the probe light from the focal point through the sample.

4. The method according to any preceding claim, wherein the fluorescence measurement is performed in a confocal microscope.

5. The method according to any preceding claim, wherein the second response is measured at a time following the measurement of the first response.

6. The method according to any preceding claim, wherein the measured first response of the fluorescent marker to exposure to the excitation light is a measure of one of the first harmonic or higher harmonics of the modulated light source.

7. The method of claim 4, wherein the second response of the measured fluorescent marker to exposure to the excitation light is a harmonic different from the harmonic measured for the first response.

8. The method of claim 5, except when subordinate to claim 3, wherein the measured first response and the second response are measured simultaneously.

9. The method according to any preceding claim, wherein exposing the sample to the excitation light under the irradiation parameters comprises: The sample was exposed to the excitation light under a first set of irradiation parameters, and then the sample was exposed to the excitation light under a second set of irradiation parameters.

10. The method according to claim 9, wherein: The step of measuring the first response of the fluorescent label to the exposure of the excitation light occurs simultaneously with the start of exposing the sample to the excitation light under the first set of irradiation parameters, but occurs before the exposure to the excitation light under the second set of irradiation parameters; and The step of measuring the second response of the fluorescent label to the excitation light occurs simultaneously with the start of exposing the sample to the excitation light under the second set of irradiation parameters.

11. The method according to any preceding claim, wherein: The irradiation parameters of the first and second measurements are selected such that one or more of the following exhibit different degrees: Fluorescence saturation at the focal point; The bleaching at the focal point; and Stimulated emission at the focal point.

12. The method according to any preceding claim, wherein the excitation light and the probe light are focused by the same focusing lens.

13. The method according to any preceding claim, wherein a beam splitter is used to combine the excitation light and the probe light.

14. A controller for a device, the controller communicating with the device, the controller being configured to cause the device to perform the method according to any one of claims 1 to 13.

15. A microscope comprising the controller according to claim 14.

Images