CN117730628A - Fluorescence imaging system and excitation area light source thereof - Google Patents
Fluorescence imaging system and excitation area light source thereof Download PDFInfo
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- CN117730628A CN117730628A CN202380012834.1A CN202380012834A CN117730628A CN 117730628 A CN117730628 A CN 117730628A CN 202380012834 A CN202380012834 A CN 202380012834A CN 117730628 A CN117730628 A CN 117730628A
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- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
According to the fluorescent imaging system and the excitation area light source thereof, the LED lamp bead array is adopted as the light emitting source of the area light source, the light spot area is increased, the switching delay is small under the drive of the LED drive circuit, the switching action of the excitation area light source can be matched with the exposure of the imaging device through the control of the control device, and the fluorescent material, particularly the time-resolved fluorescent material, is convenient to detect, so that the excitation area light source can be used in more use fields.
Description
Technical Field
The invention relates to the field of fluorescence analysis, in particular to a fluorescence imaging system and an excitation area light source thereof.
Background
Immunoblotting WesternBlot imaging systems usually use chemiluminescence or fluorescence methods, chemiluminescence can only be used for single signal acquisition, and fluorescence detection methods have the main advantages that multiple proteins can be detected simultaneously, and signals of the proteins are more consistent, so that quantitative research is facilitated compared with chemiluminescence detection. The excitation light source for fluorescence detection usually uses laser or a lamp tube, the focusing light spot of the laser is small, only spot imaging can be carried out at a time, the use scene is limited, the lamp tube can carry out surface imaging, the luminous uniformity is good, but the switching delay time is long, and the method is not suitable for measuring the fluorescence intensity of time-resolved fluorescent substances.
It can be seen that the existing excitation light source for exciting fluorescent substances to emit light has limited use scenes.
Disclosure of Invention
The invention mainly provides a fluorescence imaging system and an excitation surface light source thereof, which aim to enable an excitation light source for exciting fluorescent substances to emit light to be used in more use scenes.
One embodiment provides an excitation surface light source including:
an LED lamp bead array;
the LED driving circuit is used for driving the LED lamp bead array;
the light condensing cover covers the LED lamp bead array, an opening is formed in the top of the light condensing cover, and the opening is covered with an optical filter and a light homogenizing sheet; the optical filter is used for filtering the excitation light emitted by the LED lamp bead array, and the filtered excitation light is used for exciting fluorescent substances in the sample to emit emission light;
the control device is used for acquiring an exposure signal waveform, generating a light source switch waveform according to the exposure signal waveform, and driving the LED lamp bead array to be continuously turned on and off through the LED driving circuit according to the light source switch waveform so that the LED lamp bead array is continuously turned on and off to conform to the light source switch waveform; the exposure signal waveform is used for controlling the imaging device to continuously expose for a plurality of times to acquire the intensity of light emitted by fluorescent substances in the sample, and the light source switch waveform enables the LED lamp bead array to be turned on and off before the imaging device starts to expose.
One embodiment provides a fluorescence imaging system comprising:
the objective table is used for bearing a sample; the sample has a plurality of fluorescent substances for labeling target components in the sample;
an excitation surface light source as described above;
an imaging device for detecting the intensity of emitted light emitted from the fluorescent substance of the sample;
the control device is also used for:
controlling the excitation area light source to excite each fluorescent substance in the sample for multiple times to emit emission light, and controlling the imaging device to detect the emission light according to different exposure parameters when each fluorescent substance in the sample for multiple times emits the emission light so as to obtain actual total fluorescence intensity corresponding to the different exposure parameters; wherein the exposure parameters comprise exposure starting time and exposure ending time; and processing the actual total fluorescence intensity corresponding to the different exposure parameters to obtain the fluorescence intensity corresponding to the preset target exposure parameters of each fluorescent substance in the sample.
According to the fluorescent imaging system and the excitation area light source thereof, the LED lamp bead array is adopted as the light emitting source of the area light source, the light spot area is increased, the switch delay is small under the drive of the LED drive circuit, the switch action of the excitation area light source can be matched with the exposure of the imaging device through the control of the control device, the detection of fluorescent substances, particularly time-resolved fluorescent substances, is facilitated, and therefore the excitation area light source can be used in more use fields.
Drawings
Fig. 1 is a schematic structural diagram of an embodiment of an excitation surface light source according to the present invention;
FIG. 2 is an exploded view of an embodiment of an excitation area light source according to the present invention;
FIG. 3 is a block diagram illustrating an embodiment of an excitation surface light source according to the present invention;
FIG. 4 is a circuit diagram of an embodiment of an LED driving circuit in the excitation area light source according to the present invention;
FIG. 5 shows waveforms of exposure signals and waveforms of light source switches in the excitation area light source according to the present invention;
FIG. 6 is a block diagram illustrating an embodiment of a fluorescence imaging system according to the present invention;
FIG. 7 is a schematic diagram of a fluorescence imaging system according to an embodiment of the present invention;
FIG. 8 is a light path diagram of another embodiment of a fluorescence imaging system provided by the present invention;
FIG. 9 is a flow chart of an embodiment of a method of fluorescence imaging of a time-resolved fluorescent material;
FIG. 10 is a flow chart of one embodiment of a fluorescence imaging method in a conventional mode;
FIG. 11 is a flowchart of an embodiment of step 2 in FIG. 9;
FIG. 12 is a flowchart of an embodiment of step 21 of FIG. 11;
FIG. 13 is a flowchart of an embodiment of step 211 in FIG. 12;
FIG. 14 is a graph showing fluorescence intensity decay curves of two fluorescent substances;
FIG. 15 is a schematic diagram of the locations of four different exposure parameters in a fluorescence intensity decay curve;
FIG. 16 is a flowchart of another embodiment of step 211 in FIG. 12;
FIG. 17 is a flow chart of an embodiment of a fluorescence imaging method provided by the present invention;
FIG. 18 is a flow chart of an embodiment of a fluorescence imaging method in a hybrid mode.
Detailed Description
The invention will be described in further detail below with reference to the drawings by means of specific embodiments. Wherein like elements in different embodiments are numbered alike in association. In the following embodiments, numerous specific details are set forth in order to provide a better understanding of the present application. However, one skilled in the art will readily recognize that some of the features may be omitted, or replaced by other elements, materials, or methods in different situations. In some instances, some operations associated with the present application have not been shown or described in the specification to avoid obscuring the core portions of the present application, and may not be necessary for a person skilled in the art to describe in detail the relevant operations based on the description herein and the general knowledge of one skilled in the art.
Furthermore, the described features, operations, or characteristics of the description may be combined in any suitable manner in various embodiments. Also, various steps or acts in the method descriptions may be interchanged or modified in a manner apparent to those of ordinary skill in the art. Thus, the various orders in the description and drawings are for clarity of description of only certain embodiments, and are not meant to be required orders unless otherwise indicated.
The numbering of the components itself, e.g. "first", "second", etc., is used herein merely to distinguish between the described objects and does not have any sequential or technical meaning. The terms "coupled" and "connected," as used herein, are intended to encompass both direct and indirect coupling (coupling), unless otherwise indicated.
As shown in fig. 1 to 3, the excitation surface light source provided by the present invention includes: an array of LED light beads 15, an LED driver circuit 16, a light shield 14 and a control device 30.
The LED light bead array 15 includes a plurality of LED light beads, and each LED light bead is arranged at equal intervals, that is, each LED light bead is uniformly arranged on the substrate 17, so that the whole LED light bead array forms a surface light source, and the light emitted by the whole LED light bead array is also relatively uniform. The light emitted by the LED bead array is used to excite the fluorescent material in the sample to emit emission light, so the light emitted by the LED bead array may be referred to as excitation light. In an embodiment, the area of the LED bead array is greater than or equal to 10cm x 10cm, which may be a square or rectangular array. Each LED light bead in the LED light bead array is arranged in a plurality of rows and a plurality of columns, and the rows and columns are aligned, and fig. 3 shows 3 rows and 3 columns. The row spacing between the LED lamp beads is one of 2mm-20mm, the column spacing is also one of 2mm-20mm, and the row spacing and the column spacing can be equal. The arrangement mode and the size can better meet the requirements of a fluorescence imaging system on the light source luminous area, the intensity, the uniformity and the like. The LED lamp beads can adopt ultraviolet light LED lamp beads (UV LEDs), blue light LED lamp beads, and one part of the LED lamp beads can also adopt ultraviolet light LED lamp beads, and the other part of the LED lamp beads adopt blue light LED lamp beads.
In one embodiment, the substrate 17 of the LED light bead array 15 is an aluminum substrate, and the aluminum substrate is used instead of a conventional PCB board, so that heat dissipation is easier and the service life of the LED light beads is prolonged. The aluminum substrate can further adopt a double-layer aluminum substrate, wires can be arranged on both sides of the double-layer aluminum substrate, and the LED driving circuit 16 is also arranged on the double-layer aluminum substrate, so that the layout of a circuit is planned, and each LED lamp bead is compacter in arrangement.
The LED driving circuit 16 is used for driving the LED lamp bead array 15.
The light-gathering cover 14 covers the LED lamp bead array 15, and is used for gathering the excitation light emitted by the LED lamp bead array 15 and then emitting the excitation light upwards. The top of the light-gathering cover 14 has an opening (also called a window) covered with the optical filter 12 and the light-homogenizing sheet 13. The filter 12 is used for filtering the excitation light emitted by the LED bead array 15, for example, filtering ultraviolet light and/or blue light. The filtered excitation light is used to excite fluorescent substances in the sample to emit emission light, and in this embodiment, the wavelength band of the filtered excitation light is an ultraviolet light wavelength band under the filtering action of the filter 12. The light homogenizing sheet 13 is also called a homogenizer, and is used for uniformly emitting light beams emitted by the LED lamp bead array 15, so that the energy of the excitation light is uniformly distributed in a larger range, and the light intensity consistency of the light spot section of the excitation surface light source is good. The area of the opening of the light-gathering cover 14 is greater than or equal to 10cm x 10cm. The area of the LED bead array 15 is not smaller than the area of the opening of the light-condensing cover 14. The projection of the opening of the light-gathering cover 14 towards the ground is positioned in the LED lamp bead array 15, so that the excitation light at the opening of the light-gathering cover 14 can be relatively uniform. The uniformity of the excitation light after passing through the light homogenizing sheet 13 is greater than 0.97, in other words, the variation coefficient of the brightness between any two points in the light spot which is greater than or equal to 10cm x 10cm and is emitted by the excitation surface light source is less than 3%.
The opening at the top of the light-gathering cover 14 is also covered with a transparent protection plate 11, such as transparent glass or transparent plastic. In this embodiment, the protection plate 11, the optical filter 12, and the light homogenizing sheet 13 are stacked from top to bottom.
The light-condensing cover 14 may include an enclosure 141 and a light-reflecting layer 142 disposed on an inner wall of the enclosure 141. The reflective layer 142 may be a reflective film or a reflective plate, and is used to reflect the light emitted from the LED bead array 15 to the opening of the light-gathering cover 14, so as to improve the light utilization rate.
It can be seen that the light emitted by the LED light bead array 15 can be efficiently and uniformly emitted upwards under the action of the light-condensing cover 14, the optical filter 12, the light-homogenizing sheet 13, and the like.
Referring to fig. 4, in one embodiment, the LED driving circuit 16 includes a dc power source 161 and a plurality of constant current LED driving ICs (chips) 162. Each constant current LED driving IC is used to drive a part of the LED beads in the LED bead array 15. That is, considering that the constant current LED driving ICs have limited pins, a plurality of ICs may be employed for driving. The dc power supply 161 is configured to provide dc power to at least one group of LED light beads, where a group of LED light beads is formed by connecting a plurality of LED light beads in series. The direct current provided by the direct current power source 161 is not too high, so that the number of the LED lamp beads in the group of LED lamp beads is not too large, and the group of LED lamp beads in fig. 3 and 4 is formed by connecting three LED lamp beads in series. As shown in fig. 4, the plurality of output pins (OUT 0-OUT 15) of the constant current LED driving IC162 are all connected to the cathodes of a group of LED beads, and the anodes of the LED beads of each group are connected to the output end of the dc power supply 161, so that when the constant current LED driving IC162 controls each output pin to be at a low level, each group of LED beads is turned on, the LED bead array 15 starts to emit light, and correspondingly, when the constant current LED driving IC162 controls each output pin to be at a high level, each group of LED beads is turned off, and the LED bead array 15 is turned off. Therefore, the control device 30 can control the on and off of the LED light bead arrays 15 driven by the respective constant current LED driving ICs 162 by only outputting the on signal and the off signal to the respective constant current LED driving ICs 162. In this embodiment, sixteen-bit constant current driving ICs of texas instruments are adopted as the constant current LED driving ICs 162, and the model is TLC59281. In the prior art, the switching of the LEDs or the switching of the lamp tube is controlled by a power supply, the control speed is low, and the constant-current LED driving IC162 is adopted for control, so that the sensitivity is high, the high-frequency switching of the LED lamp beads can be controlled to be performed for more than millions of times, the error of the delay time between the switching is less than or equal to 10 nanoseconds, and the method is very suitable for detecting the fluorescence of time-resolved fluorescent substances.
The excitation area light source may further comprise a base 18, and the substrate 17 and the light-condensing cover 14 are fixed on the base 18.
As shown in fig. 5, the control device 30 is configured to obtain an exposure signal waveform, generate a light source switching waveform according to the exposure signal waveform, and drive the LED lamp bead array 15 to be continuously turned on and off by the LED driving circuit 16 according to the light source switching waveform, so that the LED lamp bead array 15 is continuously turned on and off to conform to the light source switching waveform. Wherein the exposure signal waveform is used for controlling the imaging device to continuously perform exposure for a plurality of times to collect the intensity of the light emitted by the fluorescent substance in the sample, and the light source switch waveform enables the LED lamp bead array 15 to be turned on and off before the imaging device starts exposure. That is, the LED bead array 15 is turned on first to emit the excitation light to excite the time-resolved fluorescent material in the sample to emit the fluorescence, and then the LED bead array 15 is turned off to emit only the fluorescence of the time-resolved fluorescent material, so that the imaging device can start the exposure to collect the light intensity of the fluorescence. Therefore, the excitation area light source provided by the invention can be used for detecting time-resolved fluorescent substances.
Specifically, the exposure signal waveform may be a waveform with a fixed duty cycle and frequency, for example, the duty cycle of the exposure signal waveform is a value between 5% and 50%, and the frequency of the exposure signal waveform is a value between 100Hz and 1000 Hz. In one period of the exposure signal waveform, the imaging device is exposed once, and the duration between the exposure start time T2 and the exposure end time T3 of the period is the exposure duration. The light source switching waveform has the same frequency as the exposure signal waveform, and has a fixed phase difference, which allows the light source to be turned on and off once before the exposure start time T2 in each cycle to excite the time-resolved fluorescent substance to emit light. The duty cycles of the exposure signal waveform and the light source switching waveform may be the same or different.
The control device 30 may specifically acquire an exposure signal waveform, detect a rising edge (corresponding to an exposure start time T2) or a falling edge (corresponding to an exposure end time T3) in the exposure signal waveform, and if the rising edge is detected, obtain a time of the falling edge according to the duty ratio of the exposure signal waveform, so as to turn on and off the primary light source in a period from the time of the falling edge to the time of the next rising edge, thereby obtaining a corresponding light source switching waveform; if a falling edge is detected, the time of the rising edge is obtained according to the duty ratio of the exposure signal waveform, so that the primary light source is turned on and off in a period from the time of the falling edge to the time of the rising edge, and a corresponding light source switching waveform is obtained. The time difference between the rising or falling edge in the exposure signal waveform and the rising or falling edge in the light source switching waveform is the phase difference of the two waveforms. The control device 30 is further configured to phase-lock the light source switching waveform by using the exposure signal waveform, specifically, the control device 30 may obtain a corresponding phase difference according to the duty ratio of the exposure signal waveform after detecting a rising edge or a falling edge in the exposure signal waveform, or may use a preset phase difference, and further generate the light source switching waveform according to the phase difference and the frequency of the exposure signal waveform and perform phase locking.
When the excitation area light source is applied to detection of non-time-resolved fluorescent materials, the on/off control of the LED lamp bead array 15 is relatively simple, for example, the control device 30 controls the LED lamp bead array 15 to be turned on, the non-time-resolved fluorescent materials are excited to emit light, and after the imaging device detects the fluorescent light intensity, the control device 30 controls the LED lamp bead array 15 to be turned off.
Based on the excitation area light source described above, the present invention also provides a fluorescence imaging system, as shown in fig. 6 and 7, which includes the excitation area light source 10 described above, the imaging device 20, and the stage 40.
Stage 40 is used to carry sample F. Sample F has a plurality of fluorescent substances (fluorescent probes) for labeling target components in the sample. For example, different fluorescent substances are used to label different target components, so that the content of the labeled target components, etc. thereof can be determined by identifying the kind of fluorescent substance and its related parameters (such as the intensity of emitted light, etc.). The fluorescent substance may be a fluorescent dye, a fluorescent group, or the like. In this embodiment, the fluorescent substance may be specifically a time-resolved fluorescent substance. Preferably, the time-resolved fluorescence material comprises lanthanide rare earth ion chelate, and can realize high-sensitivity time-resolved fluorescence imaging by utilizing the characteristic of long luminous half-life and no background interference and matching with the subsequent steps.
The excitation surface light source 10 is used to excite each fluorescent substance in the sample to emit emitted light (e.g., fluorescence, phosphorescence). The excitation surface light source 10 may be located under the stage 40, and the excitation surface light source 10 emits excitation light to irradiate the sample F on the stage 40, thereby exciting each fluorescent substance in the sample F to emit emission light.
The imaging device 20 is configured to detect the intensity of the emitted light emitted by the fluorescent material of the sample, for example, when the light source excites each fluorescent material in the sample multiple times to emit the emitted light, the emitted light is detected (e.g., imaged) according to different exposure parameters, so as to obtain the actual total fluorescence intensity corresponding to the different exposure parameters. In the time resolution mode, the emitted light can be detected according to the exposure parameters, and the fluorescence intensity corresponding to the exposure parameters can be obtained by imaging the emitted light according to the exposure parameters.
The control device 30 is also used to control the fluorescence imaging system to implement the fluorescence imaging method provided by the present invention, as shown in fig. 9. Specifically, the control device is configured to control the excitation area light source 10 to excite each fluorescent material in the sample to emit emission light for multiple times, and control the imaging device 20 to detect the emission light according to different exposure parameters to obtain actual total fluorescence intensity corresponding to the different exposure parameters when each fluorescent material in the excitation area light source 10 excites each fluorescent material in the sample to emit emission light for multiple times, i.e., control the imaging device 20 to detect the emission light according to the corresponding exposure parameters to obtain the actual total fluorescence intensity corresponding to the exposure parameters when each fluorescent material in the excitation area light source emits the emission light for each time; wherein the exposure parameters comprise exposure starting time and exposure ending time; and processing the actual total fluorescence intensity corresponding to the different exposure parameters to obtain the fluorescence intensity corresponding to the preset target exposure parameters of each fluorescent substance in the sample. The detection (resolution) of various fluorescent substances is realized, the content of the target component is conveniently obtained by subsequent treatment, and the whole process does not adopt a light filtering mode, so that the detection sensitivity and the detection accuracy are improved.
In the second embodiment, the fluorescence imaging system may also be compatible with conventional filtering. As shown in fig. 8, the present embodiment includes a filter device 50 in addition to the various devices in fig. 6 and 7. Specifically, the fluorescence imaging system of the present embodiment includes the stage 40 as described above, the excitation surface light source 10 as described above, the imaging device 20 as described above, the filter device 50, and the control device 30.
The filter device 50 is used for filtering emitted light with different wavelengths. The filter device 50 may include a filter wheel on which a plurality of filters 510 or optical filters are disposed, each filter 510 or optical filter circumferential array, each filter 510 or optical filter filtering a different wavelength. The filter wheel is disposed between the imaging device 20 and the stage 40, specifically one of the filters 510 or the optical filter is located in the light path of the emitted light. The wavelength of the light emitted by one fluorescent material is the same as that of the light emitted by the corresponding fluorescent material, so that the light emitted by one fluorescent material passes through the corresponding filter 510 or the optical filter and then enters the imaging device 20 for detection (e.g. imaging), the light intensity detected by the imaging device 20 is the fluorescence intensity of the light emitted by the fluorescent material, the sample image obtained by imaging the light is only information of the fluorescent material, and then the light emitted by the other fluorescent material can be detected by rotating the filter wheel. The wavelength of the filtration of each filter 510 or filter may be between 400-1000nm, for example, the center wavelength of one filter 510 or filter is 450nm, the bandwidth is 50nm; the other filter 510 or the center wavelength of the filter is 543nm, and the bandwidth is 20nm; the center wavelength of the filter 510 or the filter is 620nm, and the bandwidth is 50nm.
The control device 30 has at least two modes of operation: time resolution mode and normal mode. The control device 30 is configured to determine a current operation mode, and execute the steps shown in fig. 9 when the current operation mode is a time resolution mode; when the current operation mode is the normal mode, the steps shown in fig. 10 are performed. The current mode of operation may be specifically determined according to a user instruction, for example, the fluoroscopic imaging system further comprises a man-machine interaction device 80. The man-machine interaction device 80 is used for performing man-machine interaction, and comprises a display and an input device. The input device is for receiving user input and may include one or more keys, a touch screen, etc. The control device 30 may determine the current operation mode by an operation mode command received by the man-machine interaction device 80. For example, the control device 30 displays display controls of various operation modes through a display, a user only needs to select a display control of a desired operation mode through an input device, and the control device 30 receives a selection instruction of the operation mode through the input device to determine the operation mode selected by the user, so as to activate (enter) the operation mode. Of course, an entity key corresponding to the operation mode may be set, and the user presses which entity key, so that the operation mode corresponding to the entity case is activated.
The following describes the specific procedure for the two modes of operation.
When the current operation mode is the time resolution mode, the control device 30 performs the steps shown in fig. 9, specifically including:
in step 1, the control device 30 controls the excitation area light source 10 (specifically, controls the LED bead array through the LED driving circuit 16) to excite each fluorescent material in the sample for multiple times to emit emission light, and when each fluorescent material in the multiple excitation sample emits emission light, the imaging device 20 is controlled to image the emission light according to different exposure parameters to obtain actual total fluorescence intensity and sample images corresponding to different exposure parameters. That is, the sample is excited once, imaging is correspondingly performed according to one exposure parameter, and the sample is excited once again, and imaging is performed according to different exposure parameters. The exposure parameters are different for different sample images. The actual total fluorescence intensity characterizes the total light intensity of the emitted light emitted by each fluorescent substance during the exposure period corresponding to the exposure parameter. The gray scale of the spot in the sample image is proportional to the actual total fluorescence intensity. The light spots in the sample image can enable a user to intuitively feel the actual total fluorescence intensity, and the sample image can be generated without generating. The excitation surface light source 10 is a controlled light source, and for example, an ultraviolet light source having a wavelength of 400nm or less, specifically, an ultraviolet light source having a center wavelength of 365nm, 395nm, or the like can be used. The control device 30 controls the excitation surface light source 10 to be turned on, so that the light source 10 emits excitation light to excite each fluorescent substance in the sample to emit emission light (laser), and the control device 30 controls the excitation surface light source 10 to be turned off, so that excitation is stopped. The excitation surface light source 10 is turned off after exciting the fluorescent substance, so that the camera can be ensured to have no interference of excitation light when imaging the emitted light, and the detection sensitivity is improved.
When the current operation mode is the time-resolved mode, the fluorescent substance in the sample is the time-resolved fluorescent substance, that is, when the target component is labeled with the time-resolved fluorescent substance, the time-resolved mode is required for detecting the sample. Imaging the emitted light is imaging the illuminated sample. The exposure parameters include an exposure start time and an exposure end time, and a time period from the exposure start time to the exposure end time is an exposure time period, so that the exposure parameters can be said to include an exposure time period. In the exposure signal waveform, the exposure start timing and the exposure end timing of each period are one exposure parameter. The exposure parameters of each period in the waveform can be different, or the exposure parameters of a plurality of adjacent periods can be the same, so that repeated exposure or accumulated exposure is realized. The exposure signal waveform may be generated by the control device 30 according to the kind of exposure parameters set by the user, the number of exposures of the same exposure parameter, the order of the exposure parameters, the frequency set by the user, and the like. The fluorescent substances in the sample are excited for multiple times to emit emitted light, and different exposure parameters are adopted to detect the fluorescence intensity (actual total fluorescence intensity) of the total emitted light during different excitation, so that the actual total fluorescence intensity corresponding to a plurality of different exposure parameters can be obtained, the fluorescence intensity decay curve of each fluorescent substance can be reversely deduced by utilizing the actual total fluorescence intensity, and the time-resolved fluorescent substances contained in the sample can be known by matching the fluorescence intensity decay curve with the known fluorescence intensity decay formula. The exposure parameters may be different, for example, at least one of exposure start time, exposure end time, and exposure time period.
As shown in fig. 7 and 8, the imaging device 20 may include a lens (e.g., an objective lens) 210 and a camera 220, and the emitted light from each time-resolved fluorescent material is collected into the camera 220 to be imaged after passing through the lens 210, i.e., the camera 220 is used to image the emitted light. In some embodiments, the focal length of the lens 210 may be adjustable, and the focal length may be in the range of 5mm-60 mm.
The imaging device 20 controls the exposure parameters and imaging can be accomplished in a number of ways, two of which are illustrated below.
In one manner, the imaging device 20 may further include a light chopping device, where the light chopping device is located on an emission light path between the lens 210 and the camera 220, and is configured to control on-off of the emission light path between the lens 210 and the camera 220, and the exposure parameter may be set by controlling on-off of the emission light path, for example, the exposure start time and/or the exposure end time are controlled by controlling on-off of the emission light path, the emission light path is turned on (unblocked) for a long time, and the corresponding exposure time is long, that is, the emission light path may be controlled on-off according to the exposure signal waveform, or the emission light path may be controlled on-off according to the exposure signal waveform to form the exposure signal waveform. That is, the light chopping device can control the on-off of the light path of the emitted light between the lens 210 and the camera 220 according to the corresponding exposure parameters when the light source 10 excites the time-resolved fluorescent material in the sample each time, and the camera 220 images the emitted light to obtain the actual total fluorescence intensity and the sample image corresponding to the current exposure parameters.
The light chopping device may include a chopper 230, and the chopper 230 may include a plurality of light shielding blades 231 for shielding the emitted light, and a transmission area between the two light shielding blades 231 is blank or transparent to facilitate the passage of the emitted light. In this way, the chopper 230 alternately blocks and passes the emitted light when rotated, and the duration of the transmission area scanning the path of the emitted light is the exposure duration of the image of the sample captured by the camera 220. That is, the chopper 230 adjusts the exposure parameter, that is, the exposure signal waveform, by adjusting the rotational speed or rotational frequency thereof.
As shown in fig. 5, the control device 30 controls the light source 10 to be turned on (emit excitation light) and turned off (stop emitting excitation light) for a plurality of times to excite each fluorescent substance in the sample to emit emission light, and the light path of the emission light between the sample and the camera is unblocked by controlling the light chopping device during each light source turn-off period, and as can be seen from fig. 5, the unblocked time period of the light path of the emission light is the exposure time period. The control device 30 can set the exposure parameters by controlling the switch of the light source 10 and/or controlling the switch of the light chopping device, and then the camera 220 images the emitted light to obtain the actual total fluorescence intensity corresponding to the exposure parameters and the sample image.
The control means 30 may set the exposure parameters by controlling the switching of the light source and/or controlling the switching of the light chopping means, for example, the control means 30 may control a first time difference between the time T1 when the light source starts to be turned off and the time T2 when the light path of the emitted light starts to be clear, the first time difference corresponding to the exposure start time being different, that is, the exposure start time being a time when the timing starts with the time when the fluorescent substance starts to emit the emitted light as a zero point. The control device 30 may make the frequency or the rotation speed of the chopper 230 unchanged, and adjust the first time difference by controlling the time T1 of each turn-off of the light source, so as to obtain a corresponding exposure start time. The control device 30 may also make the switching frequency of the light source unchanged, the duration of each turn on the same, and the duration of each turn off the same, and then adjust the frequency or the rotation speed of the chopper 230 to adjust the first time difference, so as to obtain the corresponding exposure starting time. The control device 30 may also control the frequency or the rotation speed of the chopper 230 and the time of turning off the light source each time to adjust the first time difference, thereby obtaining the corresponding exposure start time.
The control device 30 may also control a second time difference between the time T1 when the light source starts to be turned off and the time T3 when the light path of the emitted light starts to be turned off to set the corresponding exposure parameter, the second time difference being different in correspondence to the exposure termination time, that is, the time when the exposure termination time starts to be timed with the time when the fluorescent substance starts to emit the emitted light being the zero point, and the time when the light source is turned off may be the time when the fluorescent substance starts to emit the emitted light. The control device 30 may adjust the second time difference by controlling the time T1 of each turn-off of the light source so as to obtain the corresponding exposure termination time T3, while leaving the frequency or the rotation speed of the chopper 230 unchanged. The control device 30 may also make the switching frequency of the light source unchanged, the duration of each turn on the same, and the duration of each turn off the same, and then adjust the frequency or the rotation speed of the chopper 230 to adjust the second time difference, so as to obtain the corresponding exposure termination time T3. The control device 30 may also control the frequency or the rotation speed of the chopper 230 and the time T1 of each turn-off of the light source to adjust the second time difference, thereby obtaining a corresponding exposure termination time T3.
From the above, it is understood that the exposure parameters can be changed by the control device controlling the on and off timings of the light source 10 while keeping the switching frequency (rotation speed) of the chopper unchanged. Of course, the exposure parameter may also be changed by controlling the switching frequency (rotation speed) of the chopper, and the switching frequency of the light source 10 may be changed following the change of the switching frequency of the chopper. Of course, the switching frequency of the light source 10 may be unchanged, and the transmission area between the two light shielding blades of the chopper may be different, and one transmission area may correspond to one exposure parameter. In short, the exposure parameters can be changed.
Alternatively, the camera 220 itself can control the exposure start time and the exposure end time, such as controlling a shutter of the camera, to adjust the exposure parameters without the chopper 230. That is, each time a respective time-resolved fluorescent material in the sample is excited to emit emitted light, the control device 30 controls the camera 220 to image the emitted light according to the corresponding exposure parameter to obtain the actual total fluorescence intensity and the sample image corresponding to the exposure parameter.
Since the intensity of the emitted light is generally weak, the time-resolved fluorescent substance in the sample can be excited N times for the same exposure parameter, and then cumulative exposure imaging or repeated exposure imaging is performed. The following are respectively described:
the accumulated exposure imaging is to perform multiple exposure under the same exposure parameter, and the multiple exposure obtains accumulated actual total fluorescence intensity and finally generates a sample image. In this step, the control device 30 specifically controls the imaging device 20 to sequentially obtain the actual total fluorescence intensity and the sample image corresponding to the different exposure parameters. The obtaining of the actual total fluorescence intensity and the sample image corresponding to the current exposure parameter may be: the camera 220 is controlled to be continuously in an exposure state, as shown in fig. 5, and the light source 10 and the chopper 230 are controlled in a linkage manner so as to realize accumulated exposure imaging, when the light source is on, the chopper shields the camera, and when the light source is off, the chopper does not shield the camera. Specifically, the control device 30 controls the light source 10 to be turned on and off for a plurality of times, during the period when the light source is turned on, each time-resolved fluorescent substance in the sample is excited to emit emission light, and may also control the chopper 230 to block (close) the light path of the emission light during the period when the light source is turned on, so as to prevent the excitation light and the emission light from entering the camera, that is, the time when the chopper 230 blocks (closes) the light path of the emission light covers the period when the light source is turned on; the current exposure parameters may be set by controlling the timing at which the light source 10 is turned off, and/or by the chopper 230 during the time when the light source 10 is turned off. Specifically, during the period when the light source is turned off, if no chopper fan blade is blocking, the emitted light will continuously enter the camera 220, so that the moment when the chopper fan blade leaves the light path of the emitted light is the exposure start moment (the exposure start moment can be changed by changing the phase difference in fig. 5), the moment when the next fan blade is blocking the light path of the emitted light is the exposure end moment, and the time when the transmission area between the two fan blades passes through the light path of the emitted light is the exposure time; the corresponding exposure parameters may be set by controlling the times at which the light sources are turned on and off, and/or by controlling the times at which the choppers are turned on (do not block the light path of the emitted light) and off (block the light path of the emitted light), each time the choppers are turned on and off during the time that each light source 10 is turned off. When the number of cycles of turning on and off the light source 10 reaches the preset threshold N, the camera 220 is controlled to stop the exposure so as to obtain the actual total fluorescence intensity and the sample image corresponding to the current exposure parameter. The preset threshold N can be set according to the needs, and the preset threshold N is an integer greater than 1. Therefore, in the process of detecting (imaging) the sample by using the same exposure parameter, the light source and the chopper switch are turned on and off for N times, the camera is continuously exposed, the imaging of accumulated exposure of the emitted light corresponding to N times of excitation is accumulated into an actual total fluorescence intensity finally, and the actual total fluorescence intensity is accumulated into a sample image, so that the numerical value of the actual total fluorescence intensity is larger, and the light spots in the sample image are brighter, thus the accuracy of detecting the time-resolved fluorescent substance can be improved. Since the time of the emitted light is relatively short, the order of magnitude of N can be thousands, tens of thousands, etc., for example, N is a number greater than or equal to 1000, or N is a number greater than or equal to 10000, etc., that is, under the same exposure parameter, the emitted light is accumulated for thousands of times, so that even if there is disturbance in occasional several excitations, the disturbance can be ignored in thousands of excitations, and the data is stable, accurate, and reliable.
The repeated exposure imaging is to perform multiple exposure under the same exposure parameter, so as to obtain multiple actual total fluorescence intensities and generate multiple sample images. In this step, the control device 30 specifically controls the imaging device 20 to sequentially obtain the actual total fluorescence intensity and the sample image corresponding to the different exposure parameters. The obtaining of the actual total fluorescence intensity and the sample image corresponding to the current exposure parameter may be: and carrying out exposure imaging for N times under the current exposure parameters to obtain N actual total fluorescence intensities and N sample images, accumulating the N actual total fluorescence intensities to obtain the actual total fluorescence intensities corresponding to the current exposure parameters, superposing the N sample images into one sample image, and taking the superposed sample image as the sample image corresponding to the current exposure parameters. N is an integer greater than 1. This also improves the accuracy of detection of the fluorescent substance.
After the actual total fluorescence intensity and the sample image corresponding to the current exposure parameter are obtained according to the method, the actual total fluorescence intensity and the sample image corresponding to the preset next exposure parameter can be obtained according to the same method, and the cycle is performed until the actual total fluorescence intensity and the sample image corresponding to all the exposure parameters are obtained.
The number of different exposure parameters is at least two more than the number of time-resolved fluorescent species. The kind of time-resolved fluorescent substance contained in the sample is known, and thus the number of different exposure parameters is also known or can be set in advance, as well as different exposure parameters. The exposure parameters may be different, for example, in one embodiment, exposure start time is different between different exposure parameters (the control device sets the method of different exposure start time through the light source and/or the chopper, see the foregoing description), exposure end time exceeds the longest theoretical lifetime parameter in various time-resolved fluorescent materials, and exposure end time may be the same. Various theoretical lifetime parameters of time-resolved phosphors are known. The exposure start time is different with respect to the time at which the emitted light is excited. In each imaging, the time when the emitted light is just excited can be used as a time zero point to start timing, and different exposure starting time corresponds to different time periods between the exposure starting time and the time zero point. The exposure termination time exceeding the longest theoretical lifetime parameter among the various time-resolved phosphors means that the emitted light of the various time-resolved phosphors is all collected by the camera after the exposure is started. The theoretical lifetime parameter is used to characterize the lifetime of the time-resolved fluorescent material in terms of emitted light, and may be the lifetime of the fluorescent light, for example, the time required for the fluorescence intensity (the intensity of emitted light) of the time-resolved fluorescent material to drop to 1/e of the maximum intensity of fluorescence (phosphorescence) upon excitation, when the excitation light is removed, referred to as the fluorescence lifetime. e is a natural constant. The theoretical lifetime parameter may also be the half-life, which is the time required for the fluorescence intensity of the time-resolved fluorescent substance to drop to 1/2 of the maximum fluorescence (phosphorescence) intensity upon excitation, after removal of the excitation light.
For another example, in another embodiment, the exposure start time is the same as the exposure start time, the exposure end time is different and the longest theoretical lifetime parameter of the various time-resolved fluorescent materials is not exceeded, and the control device sets the method of the different exposure end times through the light source and/or the chopper. Preferably, each exposure termination time does not exceed the shortest theoretical lifetime parameter of the various time-resolved phosphors, i.e. the exposure is terminated before the lifetime of any time-resolved phosphor is terminated. The exposure start time may be the time when the emitted light has just been excited.
For example, in yet another embodiment, the exposure start time and the exposure end time are different between different exposure parameters, but the exposure time period is the same. Taking t as the exposure starting time and x as the exposure time, the exposure parameters are (t, t+x), and x is fixed and known, and different exposure parameters can be obtained by changing t. The same exposure time length means that the structure and control of the chopper are relatively simple and convenient to operate.
The fluorescence intensity of the emitted light from all the fluorescent substances in the sample is obtained by exposure imaging, but the fluorescence intensity of the emitted light from each fluorescent substance is unknown, and the light intensity of the emitted light from each fluorescent substance needs to be extracted from the actual total fluorescence intensity. This is achieved by the following step 2.
The light source 10 is located below the stage, for example at the bottom of the sample. When the light source is turned on, the chopper fan blades shield the camera, no light can be transmitted to the camera at the moment, when the light source is turned off, the transmitted light is transmitted to the camera through the transmission area of the chopper, and after the light source is turned off, the transmitted light intensity of the sample can be exponentially reduced, so that the rotation frequency of the chopper is improved as much as possible, the transmitted light of the sample can be captured by the camera as soon as possible after the light source is turned off, the image size of the transmitted light beam of the chopper can be reduced under the condition that the rotation frequency of the chopper is fixed, namely the width of the transmitted light beam of the chopper is reduced, and at the moment, the chopper with smaller fan blade gaps (smaller transmission area) can be used for high-frequency accumulated exposure imaging, so that the signal intensity can be improved, and the imaging efficiency can be improved. Thus, in some embodiments, as shown in FIG. 7, the fluorescence imaging system further includes a light cone 60 and a beam expanding lens group 70. The cone of light 60, chopper 230, beam expanding lens group 70, and camera 220 are disposed in that order on the light path of the emitted light. The cone 60 serves to aggregate the individual emitted light, thereby converging the emitted light, i.e. reducing the width of the emitted light beam. The beam expanding lens group 70 is used to diverge the respective emitted light to facilitate camera imaging. The beam expander lens group 70 may have only one beam expander lens, or may have a plurality of beam expander lenses. The light cone 60 has the advantages of high resolution, small imaging distortion and the like, the emitted light is imaged on the large end face of the light cone 60 through the lens 210, the small end face of the light cone 60 is reduced after emergent, and the emitted light is amplified by the beam expanding lens group 70 after passing through the chopper and finally captured by the camera, so that imaging is realized.
Step 2, the control device 30 processes the actual total fluorescence intensities corresponding to the different exposure parameters to obtain fluorescence intensities corresponding to the preset target exposure parameters of each fluorescent substance in the sample. Specifically, as shown in fig. 11, the steps may be as follows:
step 21, the control device 30 determines the fluorescence intensity corresponding to the preset target exposure parameter of each fluorescent material in the sample according to the fluorescence intensity attenuation formula (curve) of the fluorescent material in the sample, different exposure parameters and the actual total fluorescence intensity corresponding to the different exposure parameters.
Immunoblotting WesternBlot analysis is generally based on fluorescence imaging, and fluorescence dye excitation and detection wavelengths are all in the visible spectrum, and in the wavelength range, chemical high polymer substances, films, glue, microporous plastic plates and the like can emit fluorescence, so that high background fluorescence interference is easy to generate, and even if a filter with a high OD value (cut-off depth) is used, a small part of background fluorescence penetrates through the filter, so that the sensitivity of detecting protein or nucleic acid on the film is greatly limited. Although this state is improved by using infrared laser fluorescence, the requirement of extremely high sensitivity for some specific proteins is not satisfied. The fluorescence of multicolor fluorescent materials covers a relatively wide wavelength range, and the wavelength range of fluorescence of one fluorescent material is usually overlapped with the wavelength range of fluorescence of another fluorescent material, and the fluorescence intensity of the overlapped area is usually weak, but the fluorescence intensities still affect each other, so that the detection sensitivity is also affected. The above-mentioned contents show that the time resolution mode of the invention matches time resolution fluorescent material by the corresponding method, and has the advantages of no need of filtering, no background interference and high detection sensitivity.
As shown in fig. 12, the present step 21 may include:
step 211, the control device 30 analyzes the lifetime parameter value and the maximum fluorescence intensity value of each time-resolved fluorescence substance according to the fluorescence intensity decay formula of the time-resolved fluorescence substance, different exposure parameters, and the actual total fluorescence intensities corresponding to the different exposure parameters. Wherein, the fluorescence intensity attenuation formulas of different time-resolved fluorescent materials are different, and the theoretical life parameters are also different. The analyzed life parameter value can be regarded as actual life, can be matched with each known theoretical life parameter, and can determine the type of the time-resolved fluorescent substance through matching. The kind of time-resolved fluorescent material contained in the sample is known, and therefore, a fluorescence intensity decay formula, a theoretical lifetime parameter, and the like of each time-resolved fluorescent material can be acquired in advance, which is also known.
There are a variety of specific implementations of this step 211, and two examples are provided for illustration. The first way is to solve the equation set, as shown in fig. 13, which may specifically include:
in step 2111, control device 30 integrates the time-resolved fluorescence intensity decay formula during the exposure period corresponding to the different exposure parameters, respectively, to obtain a plurality of theoretical fluorescence intensity expressions of the time-resolved fluorescence intensity during the exposure period corresponding to the different exposure parameters. The theoretical fluorescence intensity expression comprises two parameters, namely a life parameter tau and a maximum fluorescence intensity k, and the two parameters need to be solved later. Although the two parameters of the life parameter tau and the maximum fluorescence intensity k of the time-resolved fluorescent substance can be measured before the test is performed, the two measured values are not accurate enough, so that the two parameters are solved in the invention when the sample is detected, and the method is more accurate and reliable.
The exposure period is a period of time between an exposure start time and an exposure end time of an exposure parameter.
The fluorescence intensity decay formula of the time-resolved fluorescence substance is as follows:
y＝k×e -t/τ equation (1),
wherein y is fluorescence intensity, k is maximum fluorescence intensity, i.e., fluorescence intensity when k is t=0; τ is the lifetime parameter of the fluorescent substance, and t is time. Different phosphors, k and τ are typically different. e is a natural constant.
In this embodiment, (t, t+x) is used to represent the exposure parameter, where t is the exposure start time, t+x is the exposure end time, and x is the exposure duration. Integrating the fluorescence intensity attenuation formula of the time-resolved fluorescence substance to obtain a theoretical fluorescence intensity expression corresponding to the exposure parameters of the time-resolved fluorescence substance:
y＝k×τ×e -t/τ ×(1-e -x/τ ) Formula (2);
for convenience of explanation and understanding, this embodiment will be described by taking two time-resolved fluorescent materials X and Y contained in a sample as an example. Assuming that the actual lifetime parameter of X is Tx, the actual lifetime parameter of Y is Ty, the fluorescence intensity decay formula (curve) of the time-resolved fluorescence substance X is Y1, and the fluorescence intensity decay curve of the time-resolved fluorescence substance Y is Y2, as shown in fig. 14. In this embodiment, the exposure start time is different for each exposure, and in the four different exposure parameters of this embodiment, as shown in fig. 15, the four different exposure start times are t1, t2, t3 and t4, respectively.
The sample of this example has two time-resolved phosphors, and the theoretical fluorescence intensity expression of time-resolved phosphor x for an exposure parameter of time t1 at the start of exposure is k1×τ1×e -t1/τ1 ×(1-e -x1/τ1 ) That is, the superposition (integration) of the theoretical fluorescence intensities from t1 to t1+x1 (x 1 is known, e.g., a fixed value may be used, or Tx may be used, etc.) in the y1 curve; k1 is the maximum fluorescence intensity of the time-resolved fluorescence substance X, τ1 is the lifetime parameter of the time-resolved fluorescence substance X. The theoretical fluorescence intensity expression of the time-resolved fluorescence substance Y is k2×τ2×e -t1/τ2 ×(1-e -x1/τ2 ) That is, the superposition (integration) of the theoretical fluorescence intensities in the y2 curve for a period of time from t1 to t1+x1; k2 is the maximum fluorescence intensity of the fluorescent substance Y, and τ2 is the lifetime parameter of the fluorescent substance Y. For an exposure parameter of which the exposure start time is t2, the theoretical fluorescence intensity expression of the fluorescent substance X is k1×τ1×e -t2/τ1 ×(1-e -x2/τ1 ) That is, the superposition (integration) of the theoretical fluorescence intensities in the y1 curve for a period of time from t2 to t2+x2; the theoretical fluorescence intensity expression of the fluorescent substance Y is k2×τ2×e -t2/τ2 ×(1-e -x2/τ2 ) I.e. the superposition (integration) of the theoretical fluorescence intensities in the y2 curve for a period of time from t2 to t2+x2. For an exposure start time t 3, the theoretical fluorescence intensity expression of the fluorescent substance X is k1×τ1×e -t3/τ1 ×(1-e -x3/τ1 ) The theoretical fluorescence intensity expression of the fluorescent substance Y is k2×τ2×e -t3/τ2 ×(1-e -x3/τ2 ). For an exposure parameter at an exposure start time of t4, the theoretical fluorescence intensity expression of the fluorescent substance X is k1×τ1×e -t4/τ1 ×(1-e -x4/τ1 ) The theoretical fluorescence intensity expression of the fluorescent substance Y is k2×τ2×e -t4/τ2 ×(1-e -x4/τ2 ). In order to simplify the operation, the exposure start time can be different in different exposure parameters, and the exposure time is the same, that is, x1, x2, x3 and x4 are all the same. Of course, in some embodiments, the exposure start time is different from one exposure parameter to another, and the exposure end time exceeds the longest theoretical lifetime parameter of the various phosphors, which can simplify the operation. In still other embodiments, the exposure start time is the same between different exposure parameters, the exposure end time is different and does not exceed the longest theoretical lifetime parameter of various fluorescent materials, which can also simplify the operation. The present embodiment will be described taking the first example (exposure start times are different and exposure time periods are the same).
In step 2112, the fluorescence intensity is the intensity of the emitted light, and the control device 30 sums the theoretical fluorescence intensity expressions of the same exposure parameters of each time-resolved fluorescent material to obtain a theoretical total fluorescence intensity expression of the sample, i.e. one exposure parameter corresponds to one theoretical total fluorescence intensity expression of the sample.
The theoretical total fluorescence intensity expression of the sample is obtained by adding the theoretical fluorescence intensity expressions of the respective fluorescent substances. Taking the sample containing two fluorescent substances as an example, the theoretical total fluorescence intensity expression of the sample is:
equation (3).
Considering that there is background noise interference and also random interference noise when the camera collects fluorescence intensity, various interferences can be classified as an unknown constant c. The noise figure at the time of camera imaging can be considered as c. That is, the theoretical total fluorescence intensity expression of the sample may further include c, specifically:
equation (4).
Step 2113, the control device 30 equalizes the theoretical total fluorescence intensity expression of the same exposure parameter with the actual total fluorescence intensity, thereby establishing an equation containing the lifetime parameter and the maximum fluorescence intensity of each time-resolved fluorescent material under the exposure parameter; wherein, one exposure parameter corresponds to one equation, and the equations corresponding to a plurality of exposure parameters form an equation set. That is, different exposure parameters are substituted into the formula (3) or (4), respectively, and are made equal to the actual total fluorescence intensity corresponding to the same exposure parameter, thereby creating a system of equations.
In this embodiment, taking substitution formula (3) as an example, for an exposure parameter with an exposure start time t1, the theoretical total fluorescence intensity expression is: k1×τ1×e -t1/τ1 ×(1-e -x1/τ1 )+k2×τ2×e -t1/τ2 ×(1-e -x1/τ2 ). And the actual total fluorescence intensity corresponding to the exposure parameter with the exposure starting time of t1 is G1, the equation (1) corresponding to the exposure parameter (t 1, t1+x1) can be obtained: g1 =k1×τ1×e -t1/τ1 ×(1-e -x1/τ1 )+k2×τ2×e -t1/τ2 ×(1-e -x1/τ2 )。
Similarly, for an exposure parameter with an exposure start time of t2, the theoretical total fluorescence intensity expression is: k1×τ1×e -t2/τ1 ×(1-e -x2/τ1 )+k2×τ2×e -t2/τ2 ×(1-e -x2/τ2 ). And the actual total fluorescence intensity corresponding to the exposure parameter with the exposure starting time of t2 is G2, the equation (2) corresponding to the exposure parameter (t 2, t2+x2) can be obtained: g2 =k1×τ1×e -t2/τ1 ×(1-e -x2/τ1 )+k2×τ2×e -t2/τ2 ×(1-e -x2/τ2 )。
Similarly, for an exposure parameter with an exposure start time of t3, the theoretical total fluorescence intensity expression is: k1×τ1×e -t3/τ1 ×(1-e -x3/τ1 )+k2×τ2×e -t3/τ2 ×(1-e -x3/τ2 ). And the actual total fluorescence intensity corresponding to the exposure parameter with the exposure starting time of t3 is G3, the equation (3) corresponding to the exposure parameter (t 3, t3+x3) can be obtained: g3 =k1×τ1×e -t3/τ1 ×(1-e -x3/τ1 )+k2×τ2×e -t3/τ2 ×(1-e -x3/τ2 )。
Similarly, for an exposure parameter with an exposure start time of t4, the theoretical total fluorescence intensity expression is: k1×τ1×e -t4/τ1 ×(1-e -x4/τ1 )+k2×τ2×e -t4/τ2 ×(1-e -x4/τ2 ). And the actual total fluorescence intensity corresponding to the exposure parameter with the exposure starting time of t4 is G4, the equation (4) corresponding to the exposure parameter (t 4, t4+x4) can be obtained: g3 =k1×τ1×e -t4/τ1 ×(1-e -x4/τ1 )+k2×τ2×e -t4/τ2 ×(1-e -x4/τ2 )。
The number of different exposure parameters is not less than the minimum number required by solving the equation set, so that the unknown number in the equation set can be solved, such as the life parameter, the maximum fluorescence intensity, the noise coefficient c during camera imaging and the like. For example, if the noise factor c is ignored, then only the lifetime parameters and the maximum fluorescence intensities of the fluorescent substances X and Y are in the equation set for the specific embodiment, and a total of four unknowns, at least 4 different exposure parameters are required. If the noise c is considered, the lifetime parameters and the maximum fluorescence intensities of the fluorescent substances X and Y and the noise c are five unknowns in the equation set of the embodiment, and at least 5 different exposure parameters are required.
In step 2114, the control device 30 solves the equation set to solve a plurality of sets of lifetime parameter values and maximum fluorescence intensity values, and if the equation is established by using the formula (4), the noise coefficient c can also be solved. In this embodiment, k1 and k2, τ1 and τ2 are solved according to equations (1) - (4). Where k1 and τ1 are one time-resolved phosphor and k2 and τ2 are another time-resolved phosphor, but whether k1 and τ1 belong to time-resolved phosphor X or Y is not yet determined, as determined by step 2115.
Step 2115, control device 30 determines a time-resolved phosphor to which each set of lifetime parameter values and maximum fluorescence intensity value belong, based on each set of lifetime parameter values and/or maximum fluorescence intensity value. The lifetime parameters and the maximum fluorescence intensity of time-resolved phosphors are generally known and are usually in a range, which is not very accurate. The calculated lifetime parameter values can be matched with the lifetime parameters (intervals) of known fluorescent substances one by one, so that each group of lifetime parameter values and the maximum fluorescence intensity value belong to which fluorescent substance can be matched; and/or, the solved maximum fluorescence intensity value is matched with the known maximum fluorescence intensity (interval) of the fluorescent substance one by one, so that each group of life parameter values and the fluorescent substance to which the maximum fluorescence intensity value belongs can be matched. For example, τ1 has a value closer to the fluorescence lifetime of fluorescent substance X, so τ1 and k1 belong to fluorescent substance X.
If the lifetime parameter value and/or the maximum fluorescence intensity value is 0, it is indicated that there is no corresponding fluorescent substance.
The second way is to use a nonlinear fit, as shown in fig. 16, step 211 may specifically include the following steps:
in step 2111', the control device 30 obtains a time-dependent variation curve of the actual total fluorescence intensity according to the different exposure parameters and the corresponding actual total fluorescence intensity. The exposure start time and the exposure end time of the exposure parameters are known, and their corresponding fluorescence intensities (actual total fluorescence intensities) of the detected samples are also known. In this way, in order to facilitate the subsequent calculation process, different exposure parameters are: the exposure start time is different, the exposure end time is different, but the exposure duration is the same. The time t can be taken as an abscissa and the actual total fluorescence intensity as an ordinate, so as to obtain a time-dependent change curve of the actual total fluorescence intensity.
In step 2112', the control device 30 obtains a time-dependent change curve of the theoretical total fluorescence intensity of the sample according to the fluorescence intensity decay formula of the time-resolved fluorescence substance and different exposure parameters. For this example, equation (4) is obtained.
In step 2113', the control device 30 performs nonlinear fitting on the time-dependent change curve of the actual total fluorescence intensity according to the time-dependent change curve of the theoretical total fluorescence intensity of the sample, so as to obtain a plurality of sets of lifetime parameter values and maximum fluorescence intensity values. The theoretical total fluorescence intensity of the sample obtained in the last step is a curve with time, wherein the life parameter tau, the maximum fluorescence intensity k and the noise coefficient c of each fluorescent substance are unknown, the actual total fluorescence intensity is obtained by measuring a plurality of exposure times, and the two curves are essentially a curve, so that each unknown number can be obtained by carrying out nonlinear fitting on the actual curve through a theoretical curve formula. In the method, the analyzed c, the life parameter value and the maximum fluorescence intensity value of each fluorescent substance are very accurate through nonlinear fitting.
Step 214', control device 30 determines the fluorescent substance to which each set of lifetime parameter values and maximum fluorescence intensity value belongs, based on each set of lifetime parameter values and/or maximum fluorescence intensity value. This step is the same as step 2115, and will not be described here.
In step 212, the control device 30 calculates the fluorescence intensity corresponding to the target exposure parameter of the fluorescent material according to the lifetime parameter value, the maximum fluorescence intensity value, the fluorescence intensity decay formula and the target exposure parameter of the time-resolved fluorescent material. By the same method, the fluorescence intensity corresponding to the target exposure parameter of each fluorescent substance can be obtained.
The control device 30 may substitute the resolved maximum fluorescence intensity value, lifetime parameter value and target exposure parameter of the time-resolved fluorescence substance into its theoretical fluorescence intensity expression to obtain fluorescence intensity corresponding to the target exposure parameter of the time-resolved fluorescence substance in the sample, and may further consider the noise coefficient c to add the fluorescence intensity corresponding to the target exposure parameter to the resolved noise coefficient c to obtain fluorescence intensity corresponding to the target exposure parameter finally. This is equivalent to separating the fluorescence intensity of each fluorescent substance from the actual total fluorescence intensity, and the whole process does not need to filter by a filter. The target exposure parameters can be set as required, and in general, the smaller the initial exposure time is, the stronger the corresponding fluorescence intensity is, so t=0 and the like can be taken. After various fluorescent substances in the sample are excited by the light source, the fluorescent intensity under the target exposure parameter is calculated, and a single fluorescent image can be generated according to the fluorescent intensity of the fluorescent substances under the target exposure parameter, so that the single fluorescent image of each time-resolved fluorescent substance is separated from one total sample image. In this embodiment, two single fluorescent images are separated from one single fluorescent image, wherein one single fluorescent image is generated by imaging only the emitted light of fluorescent substance X and the other single fluorescent image is generated by imaging only the emitted light of fluorescent substance Y.
The curve of the fluorescence intensity and the concentration of each time-resolved fluorescence substance can be obtained in advance by detecting, for example, for the time-resolved fluorescence substance X, time-resolved fluorescence substances of different concentrations are configured, and the fluorescence intensity corresponding to the different concentrations under the target exposure parameters is obtained by exciting the time-resolved fluorescence substance to emit light and detecting the fluorescence intensity under the target exposure parameters, so that the curve of the fluorescence intensity and the concentration of the time-resolved fluorescence substance X is obtained. Whereas the concentration of the time-resolved fluorescent material is proportional to the concentration of its labeled target component, e.g. 1:1, the control device 30 substitutes the fluorescence intensity corresponding to the preset target exposure parameter of the time-resolved fluorescence substance into a preset (known) curve of fluorescence intensity and concentration, and the concentration of the target component of the time-resolved fluorescence substance label is obtained, thereby completing the detection of the content of the target component in the sample.
In summary, the method and the system provided by the invention can accurately distinguish various time-resolved fluorescent substances, and can separate the fluorescence intensity of the emitted light emitted by each time-resolved fluorescent substance from one total fluorescence intensity, so that the content of the target component marked by the fluorescent substance can be calculated according to the fluorescence intensity of the emitted light emitted by the time-resolved fluorescent substance, and the whole process does not need to filter light, so that the method and the system have no background interference and high sensitivity and are very suitable for immunoblotting WesternBlot analysis and detection of other molecules.
When the current operation mode is the normal mode, the control device 30 performs the steps shown in fig. 10, specifically including:
step 3, the control device 30 controls the light source 10 to excite each fluorescent substance in the sample to emit emitted light, controls the light filtering device 50 to filter the emitted light, controls the imaging device 20 to detect (e.g. image) the filtered emitted light to obtain the fluorescence intensity corresponding to a single fluorescent substance, and can also obtain a filtered image. When the fluorescent substance is a non-time-resolved fluorescent substance, the normal mode may be selected, and of course, the time-resolved fluorescent substance may be selected. The conventional mode requires filtering because the light source continues to emit light, and the sensitivity is lower than that of the time resolution mode. The non-time-resolved fluorescent material emits emission light when excitation light is present (light source is on), but does not emit emission light when excitation light is not present (light source is off), so that the light source needs to be continuously turned on when imaging a sample in the conventional mode, and thus optical filtering is needed when imaging. The first specific process of the step can be as follows: the control device 30 controls the light source 10 to continuously excite each fluorescent material in the sample to emit emission light, controls the light filtering device 50 to filter the emission light, for example, controls the rotation of the light filtering wheel, and controls one filter 510 or the light filtering sheet of the light filtering wheel to stop when the light filtering sheet rotates to the light path of the excitation light, so as to control the imaging device 20 to image the emission light filtered by the current filter 510 or the light filtering sheet to obtain the fluorescence intensity and the filtered image of the fluorescent material corresponding to the current filter 510 or the light filtering sheet; and then controlling the rotation of the filter wheel to enable the next filter 510 or the filter to rotate to the light path of the excitation light, and controlling the imaging device 20 to image so as to obtain the fluorescence intensity and the filtering image of the fluorescent substances corresponding to the next filter 510 or the filter, so that the circulation is performed, and the fluorescence intensity and the filtering image of each fluorescent substance in the sample are obtained. The types of fluorescent substances in the sample in the normal mode are known, and the center wavelength thereof is also known, so that the matched filter means 50 may be provided in advance, for example, a matched filter wheel on which each filter 510 or the wavelength filtered by the filter corresponds to (coincides with) the center wavelength of each fluorescent substance substantially one by one, is selected according to the known center wavelength of each fluorescent substance.
The subsequent control device 30 may process the fluorescence intensity of each fluorescent substance in the sample. For example, the fluorescence intensity of the fluorescent substance is substituted into a preset curve of fluorescence intensity and concentration to obtain the concentration of the target component of the fluorescent substance label.
The fluorescence imaging system may further include a limiting device for limiting the chopper 230 when the current operation mode is the normal mode, so that the emitted light is not blocked by the chopper 230 and enters the camera 220, in other words, the chopper 230 is not required to be used in the normal mode, and the limiting device fixes the transmission area of the chopper 230 on the light path of the emitted light.
Based on the fluorescence imaging system provided in the second embodiment, the present invention further provides a corresponding fluorescence imaging method, as shown in fig. 17, including the following steps:
step 0, determining a current working mode, wherein the working mode at least comprises two types: time resolution mode and normal mode. The specific process is described in the above embodiments, and will not be described herein. When the current operation mode is the time resolution mode, the method flow shown in fig. 9 is performed. When the current operation mode is the normal mode, the method flow shown in fig. 10 is performed. Since the specific processes of fig. 9 and 10 are described in detail in the above embodiments, they are not described in detail herein.
In the third embodiment, the operation mode of the control device 30 further includes a mixed mode, and the mixed mode may be selected when the sample contains both the time-resolved fluorescent substance and the non-time-resolved fluorescent substance. As shown in fig. 18, the control device 30 in the hybrid mode performs the steps shown in fig. 18, and specifically includes:
step 3, the control device 30 controls the light source to continuously excite each fluorescent substance in the sample to emit emission light, the light filtering device filters the emission light, and the imaging device is controlled to detect (e.g. image) the filtered emission light to obtain the fluorescence intensity corresponding to each non-time-resolved fluorescent substance in the sample. The wavelength of the light emitted by the time-resolved fluorescent material and the wavelength of the light emitted by the non-time-resolved fluorescent material in the sample are different, so that even if the light is excited by the light source, the light can be sequentially filtered through the filtering device, and any filtering image obtained by imaging only contains the light intensity information (the gray scale of the light spot is reflected on the image) of the light emitted by the non-time-resolved fluorescent material. This step may be performed before step 1 or after step 2. The specific process of this step is the same as that of step 3 in the second embodiment, and will not be described here.
Step 1, the control device 30 controls the light source to excite each time-resolved fluorescent material in the sample for multiple times to emit emission light, and when each time-resolved fluorescent material in the sample is excited for multiple times to emit emission light, the imaging device is controlled to detect (e.g. image) the emission light according to different exposure parameters, so as to obtain the actual total fluorescence intensity corresponding to the different exposure parameters. Although the sample contains non-time-resolved fluorescent material, the sample is detected and imaged after the light source is turned off, and the non-time-resolved fluorescent material does not emit emitted light after the light source is turned off, so that the detection of the time-resolved fluorescent material is not interfered. The specific process of this step is the same as that of step 1 in the above embodiment, and will not be described here again.
Step 2, the control device 30 processes the actual total fluorescence intensities corresponding to the different exposure parameters to obtain fluorescence intensities corresponding to the preset target exposure parameters of each time-resolved fluorescent material in the sample. The specific process of this step is the same as that of step 2 in the above embodiment, and will not be described here again.
Reference is made to various exemplary embodiments herein. However, those skilled in the art will recognize that changes and modifications may be made to the exemplary embodiments without departing from the scope herein. For example, the various operational steps and components used to perform the operational steps may be implemented in different ways (e.g., one or more steps may be deleted, modified, or combined into other steps) depending on the particular application or taking into account any number of cost functions associated with the operation of the system.
Claims (10)
1. An excitation surface light source, comprising:
an LED lamp bead array;
the LED driving circuit is used for driving the LED lamp bead array;
the light condensing cover covers the LED lamp bead array, an opening is formed in the top of the light condensing cover, and the opening is covered with an optical filter and a light homogenizing sheet; the optical filter is used for filtering the excitation light emitted by the LED lamp bead array, and the filtered excitation light is used for exciting fluorescent substances in the sample to emit emission light;
the control device is used for acquiring an exposure signal waveform, generating a light source switch waveform according to the exposure signal waveform, and driving the LED lamp bead array to be continuously turned on and off through the LED driving circuit according to the light source switch waveform so that the LED lamp bead array is continuously turned on and off to conform to the light source switch waveform; the exposure signal waveform is used for controlling the imaging device to continuously expose for a plurality of times to acquire the intensity of light emitted by fluorescent substances in the sample, and the light source switch waveform enables the LED lamp bead array to be turned on and off before the imaging device starts to expose.
2. The excitation surface light source according to claim 1, wherein the control device is further configured to phase lock the light source switching waveform with an exposure signal waveform.
3. The excitation surface light source according to claim 1, wherein the duty ratio of the exposure signal waveform is a value between 5% and 50%, and the frequency of the exposure signal waveform is a value between 100Hz and 1000 Hz.
4. The excitation surface light source according to claim 1, wherein the opening has an area of 10cm x 10cm or more and the uniformity of the excitation light after passing through the light homogenizing sheet is greater than 0.97.
5. The excitation area light source according to claim 1, wherein the substrate of the LED bead array is an aluminum substrate.
6. The excitation surface light source according to claim 1, wherein the LED driving circuit includes a plurality of constant current LED driving ICs, each for driving a part of the LED beads in the LED bead array.
7. The excitation area light source according to claim 6, wherein the LED beads are ultraviolet LED beads; the row spacing and the column spacing between the LED lamp beads in the LED lamp bead array are respectively one of 2mm-20 mm; the wave band of the filtered excitation light is ultraviolet light wave band.
8. A fluorescence imaging system, comprising:
the objective table is used for bearing a sample; the sample has a plurality of fluorescent substances for labeling target components in the sample;
The excitation surface light source according to any one of claims 1 to 7;
an imaging device for detecting the intensity of emitted light emitted from the fluorescent substance of the sample;
the control device is also used for:
controlling the excitation area light source to excite each fluorescent substance in the sample for multiple times to emit emission light, and controlling the imaging device to detect the emission light according to different exposure parameters when each fluorescent substance in the sample for multiple times emits the emission light so as to obtain actual total fluorescence intensity corresponding to the different exposure parameters; wherein the exposure parameters comprise exposure starting time and exposure ending time; and processing the actual total fluorescence intensity corresponding to the different exposure parameters to obtain the fluorescence intensity corresponding to the preset target exposure parameters of each fluorescent substance in the sample.
9. The system of claim 8, wherein the control means processes the actual total fluorescence intensity corresponding to the different exposure parameters to obtain fluorescence intensities corresponding to preset target exposure parameters for each fluorescent material in the sample, comprising:
and determining the fluorescence intensity corresponding to the preset target exposure parameter of each fluorescent substance in the sample according to a fluorescence intensity attenuation formula of the fluorescent substance, different exposure parameters and the actual total fluorescence intensity corresponding to the different exposure parameters.
10. The system of claim 9, wherein the control device is further configured to substitute a fluorescence intensity corresponding to a preset target exposure parameter of the fluorescent substance into a preset fluorescence intensity versus concentration curve to obtain a concentration of the target component of the fluorescent substance label.
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