CN115166960B

CN115166960B - LED excitation light source for quantitative FRET microscopic imaging and dimming method

Info

Publication number: CN115166960B
Application number: CN202210872007.7A
Authority: CN
Inventors: 陈同生; 左欢; 庄正飞; 胡敏
Original assignee: Normal University Rayleigh Optoelectronic Technology Qingyuan Co ltd; South China Normal University
Current assignee: Normal University Rayleigh Optoelectronic Technology Qingyuan Co ltd; South China Normal University
Priority date: 2022-07-19
Filing date: 2022-07-19
Publication date: 2023-08-04
Anticipated expiration: 2042-07-19
Also published as: CN115166960A

Abstract

The invention discloses an LED excitation light source for quantitative FRET microscopic imaging and a dimming method, wherein the light source comprises a shell, an optical waveguide adapter base, a dichroic mirror, a collimating mirror sleeve, LED lamp beads, a lamp bead base, a bottom plate, an external controller, a photoelectric sensor, a main control circuit and a power supply. The switch and the intensity of the LED with each wavelength are controllable in program, are not limited by a mechanical structure, can output a plurality of wavelengths at the same time, and are suitable for multicolor imaging; the wavelength can be switched rapidly without a rotating wheel, and the method is suitable for rapid measurement of living cells; the automatic light intensity control device can automatically adjust and maintain constant output light intensity inside and is suitable for quantitative analysis and calculation.

Description

LED excitation light source for quantitative FRET microscopic imaging and dimming method

Technical Field

The invention belongs to the technical field of fluorescent lighting imaging, and particularly relates to an LED excitation light source for quantitative FRET microscopic imaging and a dimming method.

Background

FRET microscopy based on fluorescent proteins has become an important tool for studying the dynamic processes of biochemical molecules in living cells. The occurrence of FRET requires that the emission spectrum of the donor and the excitation spectrum of the acceptor overlap greatly, and the excitation spectra of the two fluorescent dyes are partially overlapped, so that the fluorescence excitation of the acceptor is required to be reduced as much as possible while the donor can be sufficiently excited, the excitation crosstalk between the acceptors can be well reduced by configuring an excitation light source with proper wavelength, and the success rate of experiments and the reliability of results are improved. The traditional microscope fluorescence excitation light sources are mostly wide-spectrum light sources such as mercury lamps, metal halogen lamps and the like, and a narrow-band light source with a wanted wave band needs to be obtained by switching different optical filters through a rotating wheel/rotating disc, so that the defects of large heating value, short service life, long wavelength switching response time, low stability, inconvenient use and the like exist. The LED light source has high stability, long service life and convenient use, and is expected to replace the traditional light source. Most of the existing LED light sources for microscopic imaging are switched by rotating wheels, and the rotating wheels need to consume a certain mechanical rotation time, so that the existing LED light sources are not suitable for the requirement of rapid quantitative FRET imaging of living cells.

Disclosure of Invention

The invention aims to overcome the defects and shortcomings of the prior art and provides an LED excitation light source for quantitative FRET microscopic imaging and a dimming method.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

an LED excitation light source for quantitative FRET microscopic imaging comprises a shell, an optical waveguide adapter base, a dichroic mirror, a collimating mirror sleeve, LED lamp beads, a lamp bead base, a bottom plate, an external controller, a photoelectric sensor, a main control circuit and a power supply;

the bottom plate is arranged inside the shell;

the lamp bead base is fixed on the bottom plate through the straight slot; the lamp bead base is provided with a cylindrical bulge, the side surface of the cylinder is provided with external threads, and the upper surface of the cylinder is provided with two positioning screw holes and a through hole;

the LED lamp beads are welded on a round copper substrate which is consistent with the openings on the upper surface of the cylinder, the copper substrate is tightly attached to the cylinder through positioning screw holes, and heat conduction silicone grease is coated to fill the air gap;

the power line of the LED lamp bead is LED out through a through hole on the cylinder and is connected to the main control circuit; the main control circuit is powered by a power supply and is electrically connected with an external controller, and is used for controlling the switch and the intensity of each LED lamp bead;

the collimating lens is used for converging light, is fixed in a collimating lens sleeve with an inner side thread, is assembled on a cylindrical bulge of the lamp bead base through threads, and is fixed at any position within the length range of the cylindrical bulge through a clamping ring with an inner thread;

the dichroic mirror lens is clamped and fixed on the dichroic mirror base, and the dichroic mirror base can freely rotate and is used for adjusting the angle of the lens;

the photoelectric sensor module is positioned at the side of the nearest dichroic mirror at the position of the light outlet of the light path, and is symmetrically distributed with the light outlet by taking the lens as an axis;

the optical waveguide adapter comprises a female part deep part and an external thread part with a certain length, wherein the female part deep part is matched with the optical waveguide interface; the optical waveguide adapter base is provided with internal threads and internally provided with a focusing lens, and the optical waveguide adapter is assembled into the base through threads and locked through a set screw.

Furthermore, an aspheric lens is arranged in front of each LED lamp bead and used for converging divergent light to achieve an approximate collimation effect, and loss in the light transmission process is reduced.

Further, the distance between the collimating lens and the LED lamp beads can be adjusted in a certain range through threaded fit of the cylinder and the collimating lens sleeve.

Further, the wavelengths of the LED lamp beads are 385, 435, 488, 515, 561 and 635nm respectively;

emergent light of LED lamp beads with different wavelengths is uniformly combined on an optical waveguide adapter base on a bottom plate in a mode of transmission and reflection of a dichroic mirror.

Further, the optical waveguide adapter is positioned at the focal plane position of the focusing lens and is fixedly connected with the liquid optical waveguide, so as to provide a certain adjustable stroke, thereby more accurately finding the focal plane;

the focusing mirror gathers the received LED light and focuses the LED light into the optical waveguide, and the LED light is output through the liquid optical waveguide.

Furthermore, the main control circuit adopts a constant current source design, and each constant current source independently controls the corresponding LED lamp beads, and drives the switch and the intensity of the LED lamp beads in a mode of controlling the output constant current.

Further, the temperature sensor and the cooling fan are further arranged, the temperature sensor monitors the temperature inside the shell of the light source in real time and feeds back the temperature to the main control circuit, when the temperature is normal, the control circuit outputs a PWM signal with a low duty ratio to the cooling fan, the cooling fan operates at a low speed, when the ambient temperature is higher than a preset value, the control circuit outputs a PWM signal with a high duty ratio, and the cooling fan operates at a high speed, so that the cooling effect is improved.

Further, be equipped with the wind channel mouth that looses on the shell, wind channel mouth department sets up black sponge, prevents that inside light from leaking when guaranteeing to ventilate.

Furthermore, the external controller can also adopt upper computer software;

the external controller or the upper computer software or both of the external controller and the upper computer software can be selected to be used during manual control, and the external controller and the soft upper computer software can be operated in real time and synchronized during simultaneous use.

The invention also comprises a dimming method based on the LED excitation light source, wherein the current output intensity of the light source is monitored by adopting a photoelectric sensor, and the output voltage of the photoelectric sensor is changed along with the change of the detected light intensity and is electrically connected with a main control circuit;

when the crosstalk coefficient is calculated, the light source is configured into a standard value mode, and a software program is used for recording the return values of the photoelectric sensor when the donor excitation light and the acceptor excitation light are respectively output at the moment, and recording the return values as standard values and storing the standard values;

if the current return value of the photoelectric sensor is lower than the stored standard value, the fact that the output intensity of the light source is attenuated is indicated, the control circuit adjusts the output signal to the main control circuit constant current source according to the difference between the return value and the standard value, and then adjusts the driving current of the LED lamp beads until the return value of the photoelectric detector is restored to the standard value level, and the fact that the output light intensity is not reduced is ensured.

Compared with the prior art, the invention has the following advantages and beneficial effects:

1. the six wave bands selected by the LED can efficiently excite the C-V/C-Y/G-R/GM system used for quantitative FRET measurement and the system is similar to the spectrum thereof.

2. The switch and the intensity of the LED with each wavelength are controllable in program, are not limited by a mechanical structure, can output a plurality of wavelengths at the same time, and are suitable for multicolor imaging; the wavelength can be switched rapidly without a rotating wheel, and the method is suitable for rapid measurement of living cells.

3. The LED self-provided output intensity correction device has the output intensity correction function, and keeps the output intensity not to be reduced due to the performance reduction of the LED.

4. The upper computer software and the external controller are independently controlled, so that the method is applicable to various experimental loop mirrors; the structure is simple, the device is not locked, and any part is damaged, so that the new accessory can be replaced conveniently.

Drawings

FIG. 1 is a diagram of the internal structure of an LED excitation light source of the present invention;

FIG. 2 is a schematic view of the light path of the LED excitation light source of the present invention;

FIG. 3 is a diagram of the LED excitation light source housing of the present invention;

FIG. 4 is a schematic diagram of an external controller for an LED excitation light source according to the present invention;

FIG. 5 is a flow chart of the calibration and compensation of the LED excitation light source of the present invention;

FIG. 6 is a software control flow chart of the LED excitation light source of the present invention;

FIG. 7 is a graph of output light power versus input current for several different LEDs;

reference numerals illustrate: 1-an optical waveguide adapter; 2-an optical waveguide adapter base; a 3A-dichroic mirror mount; 3B-dichroic mirror; 4-a collimator sleeve; 5-a lamp bead base; 6-a bottom plate; 7-a master control circuit; 8-a housing; 9-an external controller; 10-photosensor.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.

Examples

As shown in fig. 1, 3 and 4, the LED excitation light source for quantitative FRET microscopic imaging of the present invention includes a housing 8, an optical waveguide adapter 1, an optical waveguide adapter base 2, a dichroic mirror base 3A, a dichroic mirror 3B, a collimator mirror sleeve 4, LED lamp beads, a lamp bead base 5, a bottom plate 6, an external controller 9, a photosensor 10, a main control circuit 7, and a power supply;

the bottom plate is arranged inside the shell;

the LED lamp beads are welded on a round copper substrate consistent with the openings on the upper surface of the cylinder, and the copper substrate is tightly attached to the cylinder through positioning screw holes, so that the LED lamp beads are ensured to be positioned at the center of the surface of the cylinder; the copper substrate is also coated with heat-conducting silicone grease to fill the air gap so as to ensure heat dissipation;

the dichroic mirror lens is clamped and fixed on the dichroic mirror base, and the dichroic mirror base can freely rotate and is used for adjusting the angle of the lens so as to change the propagation direction of the light beam; the dichroic mirror is capable of transmitting light having a wavelength greater than the nominal wavelength of the mirror and reflecting light having a transmission wavelength less than the nominal wavelength of the mirror, and typically at an angle of 45 degrees, allows the two directions of light to be combined.

The photoelectric sensor module is positioned at the side of the nearest dichroic mirror at the position of the light outlet of the light path, and is symmetrically distributed with the light outlet by taking a lens as an axis, so that the photoelectric sensor module can detect light beams at all different positions under the condition of not shielding the light path output;

In this embodiment, an aspheric lens is disposed in front of each LED lamp bead, and is used for converging divergent light to achieve an effect of approximate collimation, so as to reduce losses in the light transmission process.

In this embodiment, LED beads with wavelengths 385, 435, 488, 515, 561, 635nm are selected to be configured and combined into a six-band light source.

In this embodiment, the distance between the collimator and the LED lamp beads can be adjusted within a certain range by threads.

In this embodiment, the outgoing light of the LED lamp beads with different wavelengths is uniformly combined to the optical waveguide adapter base on the bottom plate in a mode of transmission and reflection of the dichroic mirror.

In the embodiment, the optical waveguide adapter is positioned at the focal plane position of the focusing lens and is fixedly connected with the liquid optical waveguide, so as to provide a certain adjustable stroke, thereby more accurately finding the focal plane manually in the later period;

the focusing lens gathers the received LED light and focuses the LED light into the optical waveguide, the LED light is output through the liquid optical waveguide, and the liquid optical waveguide is compatible with each large mainstream microscope.

As shown in fig. 2, the light path coupling adopts a dichroic mirror combination mode, uses a dichroic mirror a to reflect 385, 435 and 488nm wave band light, and transmits 515, 561 and 635nm; dichroic mirror b reflects 385nm transmission 435 and 488nm; dichroic mirror c reflects 435nm transmission 488nm; dichroic mirror d reflects 515nm transmission 561nm; dichroic mirror e reflects 515 and 561nm transmits 635nm. The combination mode can complete the rapid control and switching of the output wavelength without mechanical rotation.

In this embodiment, the main control circuit adopts a constant current source design, and each constant current source independently controls the corresponding LED lamp bead, and drives the switch and intensity of the LED lamp bead by controlling the mode of outputting constant current.

In this embodiment, a temperature sensor and a cooling fan are further provided, the temperature sensor monitors the internal temperature of the light source in real time and feeds back the internal temperature to the main control circuit, when the temperature is normal, the main control circuit outputs a PWM signal with a low duty ratio to the cooling fan, the cooling fan operates at a low speed, when the ambient temperature is higher than a preset value, the control circuit outputs a PWM signal with a high duty ratio, and the cooling fan operates at a high speed, thereby increasing the cooling effect.

In this embodiment, the heat dissipation air duct port is further provided with a black sponge, so that internal light is prevented from leaking while ventilation is ensured.

In this embodiment, the external controller may also be replaced by upper computer software;

the external controller or the upper computer software or both of the external controller and the upper computer software can be selected to be used during manual control, and the external controller and the soft upper computer software can be operated in real time and synchronized during simultaneous use. As shown in fig. 6, a software control flow chart is shown.

When computer software is not used, the external controller can be manually touched to firstly configure the intensity percentage and then click the switch button of the corresponding LED to carry out the intensity of output light and the switch control, so that the visual field of a sample can be conveniently found in advance; when the software is used for operation, the main control circuit is communicated with the serial port through the USB port and is in millisecond level or CAN communication with higher speed is carried out through the double buses, when the software is used for one-key operation, the corresponding SDK instruction set is preset, the light source CAN be operated rapidly, meanwhile, in order to consider different experimental environments, the software CAN control not only single wavelength but also multiple wavelength LEDs at the same time, each channel has the option of being in a selected state or not, when the system is actually used, only the selected wavelength is required to be set in the selected state, the LED light with the multiple wavelengths CAN be turned on or off at the same time, and other unselected LEDs are not affected in a controlled manner and are kept in the state before operation.

As shown in fig. 5, the flow chart of the output light correction part is that the dichroic mirror has a passing/reflecting rate of about 90% and inevitably has the phenomena of reflection and light leakage, so that the photoelectric sensor with the spatial position shown in fig. 2 can detect the emergent light of all LEDs, and the output voltage value of the photoelectric sensor changes linearly along with the change of the photon number received by the photosensitive surface of the detector and is electrically connected with the main control circuit. The main control circuit samples and converts the voltage value output by the sensor into a digital signal through AD conversion, and the attenuation condition of the output light intensity can be reflected by analyzing the change of the digital signal value.

In quantitative FRET calculations, in addition to the prepared donor-acceptor pair samples, each experiment requires the additional preparation of separately expressed donor and acceptor samples to calculate the crosstalk coefficients, which vary widely from illumination intensity to illumination intensity. In actual experiment operation, the types of the samples are relatively fixed by a commonly used donor and acceptor in a laboratory, and in order to reduce unnecessary repeated calculation, after a group of crosstalk coefficients are measured under a certain fixed light intensity in advance, the calculated values are only required to be called under the condition that the output intensity of the light source is kept unchanged in later experiments, so that the complexity of an experiment flow is greatly reduced.

Therefore, when the crosstalk coefficient is measured through photographing, after the crosstalk coefficient is manually operated and adjusted to the excitation intensity gear of the donor and acceptor which are most suitable for FRET photographing, the light source can be configured to enter a FRET parameter correction mode through the upper computer software, and the return value of the photoelectric sensor is recorded as a standard value and stored in the internal memory of the hardware circuit. In the subsequent experiments, the light source can be controlled to be started through the software of the upper computer and enter a FRET photographing mode, at the moment, the excitation light of the donor and the excitation light of the receptor are automatically switched to the corresponding intensity gear, and if the return value of the photoelectric sensor and the internal storage value are different when the excitation light of the donor/receptor is started, the change of the output light intensity is indicated. According to the characteristics of the relation between the output light power of the LEDs and the current and the P-I curves of the LEDs actually measured, as shown in FIG. 7, the P-I curves of the LEDs are approximately in a linear relation, and as the adjustment precision of the driving current of the adopted constant current source module is 0.1%, when the difference between the current output acquisition value and the standard value is more than 0.1%, the main control circuit changes the duty ratio coefficient of the PWM signal output by the corresponding light path according to the ratio of the current output acquisition value and the standard value, thereby adjusting the input current of the constant current module to the LEDs and comparing the difference again until the difference between the acquisition values is less than 0.1%, and the effect that the output light used in the current FRET experiment is consistent with the output light intensity used in the measurement of the crosstalk coefficient is achieved.

In another embodiment, a dimming method based on the LED excitation light source of the embodiment is also provided, the current output intensity of the light source is monitored by adopting a photoelectric sensor, and the output voltage of the photoelectric sensor is changed along with the detected light intensity and is electrically connected with a main control circuit;

if the current return value of the photoelectric sensor is lower than the stored standard value, the fact that the output intensity of the light source is attenuated is indicated, the main control circuit adjusts the output signal to the constant current source according to the difference between the return value and the standard value, and then adjusts the driving current of the LED until the return value of the photoelectric detector is restored to the standard value level, and the fact that the output light intensity is not reduced is ensured.

It should also be noted that in this specification, terms such as "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The LED excitation light source for quantitative FRET microscopic imaging is characterized by comprising a shell, an optical waveguide adapter base, a dichroic mirror, a collimating mirror sleeve, LED lamp beads, a lamp bead base, a bottom plate, an external controller, a photoelectric sensor, a main control circuit and a power supply;

the bottom plate is arranged inside the shell;

the optical waveguide adapter comprises a female part deep part and an external thread part with a certain length, wherein the female part deep part is matched with the optical waveguide interface; the optical waveguide adapter base is provided with internal threads and internally provided with a focusing lens, and the optical waveguide adapter is assembled into the base through threads and locked through a set screw;

the distance between the collimating mirror and the LED lamp beads can be adjusted in a certain range through the threaded fit of the cylinder and the collimating mirror sleeve;

the wavelengths of the LED lamp beads are 385nm, 435nm, 488nm, 515nm, 561nm and 635nm respectively;

2. The LED excitation light source for quantitative FRET microscopy imaging of claim 1, wherein an aspheric lens is provided in front of each LED bead for converging divergent light to achieve near collimation effect, reducing losses during light transmission.

3. The LED excitation light source for quantitative FRET microscopy imaging of claim 1, wherein the light guide adapter is positioned at the focal plane of the focusing mirror and fixedly connected to the liquid light guide for providing an adjustable stroke for more accurately finding the focal plane;

4. The LED excitation light source for quantitative FRET microscopic imaging of claim 1, wherein the main control circuit is designed with constant current sources, each constant current source independently controls the corresponding LED lamp beads, and the LED lamp beads are driven to switch and intensity by controlling the way of outputting constant current.

5. The LED excitation light source for quantitative FRET microscopic imaging according to claim 1, further comprising a temperature sensor and a heat dissipating fan, wherein the temperature sensor monitors the internal temperature of the housing of the light source in real time and feeds back the temperature to the main control circuit, and when the temperature is normal, the control circuit outputs a PWM signal with a low duty ratio to the heat dissipating fan, and when the ambient temperature is higher than a preset value, the control circuit outputs a PWM signal with a high duty ratio, and when the ambient temperature is higher than a preset value, the heat dissipating fan is operated at a high speed, thereby increasing the heat dissipating effect.

6. The LED excitation light source for quantitative FRET microscopic imaging according to claim 1, wherein the housing is provided with a heat radiation port, and a black sponge is provided at the air port to prevent internal light from leaking out while ensuring ventilation.

7. The LED excitation light source for quantitative FRET microscopy imaging of claim 1, wherein the external controller is further configured with host computer software;

8. The dimming method for the LED excitation light source for quantitative FRET microscopic imaging according to any one of claims 1 to 7, wherein a photosensor is used for monitoring the current output intensity of the light source, and the output voltage of the photosensor is changed along with the detected change of the intensity of the light and is electrically connected with a main control circuit;