WO2017079902A1

WO2017079902A1 - Random scan system

Info

Publication number: WO2017079902A1
Application number: PCT/CN2015/094214
Authority: WO
Inventors: 屈军乐; 严伟; 邵永红; 叶彤; 田蜜
Original assignee: 深圳大学
Priority date: 2015-11-10
Filing date: 2015-11-10
Publication date: 2017-05-18

Abstract

A random scan system comprising a dispersion pre-compensation unit (1), a random scan unit (2), and an imaging unit (3). The dispersion pre-compensation unit (1) is configured to perform dispersion pre-compensation and optical path adjustment on incident light to enable the incident light to reach the random scan unit (2) perpendicularly, thereby implementing scanning of any region. The random scan unit (2) is further configured to collect fluorescent signals generated after the scanning of any region, and process the fluorescent signals and then enable same to reach the imaging unit (3) for imaging. The use of dual acousto-optic deflector based scanning technology avoids a mechanical offset of galvanometer scanning, and provides high scanning precision and a fast scanning speed. In addition, the acousto-optic deflector based scanning technology can further implement quick selection, scanning and imaging of a region of interest, greatly reduce photodamage while ensuring the resolution unchanged, and cause almost no photodamage in a region of non-interest in a field of view.

Description

Random scanning system

Technical field

The invention belongs to the field of microscopic imaging, and in particular relates to a random scanning system for STED super-resolution microscopic imaging.

Background technique

Since 1994, German scientist S.E. Hell first proposed stimulated emission microscopy (Stimulated emission) Depletion After the concept of super-resolution of microscopy, STED, super-resolution microscopy has been paid close attention. Especially after the Nobel Prize in Chemistry was obtained in 2014, the research on super-resolution imaging has pushed the research of super-resolution imaging to a climax. STED, STORM and PALM are the main methods to overcome the diffraction limit. STORM and PALM need to collect a large number of pictures and then reconstruct them to form super-resolution imaging. Therefore, the imaging speed is very slow and not suitable for living imaging, and STED is utilized. The optical method achieves super-resolution imaging beyond the diffraction limit, so it is very suitable for the application of living cell super-resolution imaging, but STED super-resolution imaging has a disadvantage that the intensity of the loss of light is large, and there is a certain damage to living cells. .

In order to promote the application of STED super-resolution in living cells, the commonly used method is to reduce the intensity of the loss of light to reduce the damage of light to living cells. Although this reduces the light damage to the cells, it greatly reduces the resolution of the system and limits its wide range of applications.

technical problem

The technical problem to be solved by the present invention is to provide a system for random scanning, which aims to ensure that the living cells are damaged by light damage based on the resolution of the STED super-resolution microscopic imaging system.

Technical solution

The present invention is achieved by a random scanning system including a dispersion pre-compensation unit, a random scanning unit, and an imaging unit;

The dispersion pre-compensation unit is configured to perform chromatic dispersion pre-compensation and optical path adjustment on the incident light, and then vertically incident to the random scanning unit to implement scanning of an arbitrary region;

The random scanning unit is further configured to collect a fluorescent signal generated after performing an arbitrary area scan, and process the fluorescent signal and then incident on the imaging unit for imaging.

Further, the incident light includes STED loss light and excitation light.

Further, the dispersion pre-compensation unit includes a first prism, a second mirror, a first mirror group, and a second mirror group;

The STED loss light is pre-compensated by the first prism and then incident on the first mirror group, and is optically adjusted by the first mirror group and then vertically incident to the random scanning unit;

The excitation light is pre-compensated by the second prism and then incident on the second mirror group, and is optically adjusted by the second mirror group and then vertically incident to the random scanning unit.

Further, the first prism and the second mirror are both placed at an angle of 45° in the horizontal direction.

Further, the random scanning unit includes:

a first two-dimensional acousto-optic deflector, a first data acquisition card connected to the first two-dimensional acousto-optic deflector;

a second two-dimensional acousto-optic deflector, and a second data acquisition card connected to the second two-dimensional acousto-optic deflector;

a mirror, a first dichroic mirror, a second dichroic mirror, and an objective lens;

The dispersion-precompensated STED loss light is incident perpendicularly to the first two-dimensional acousto-optic deflector, and the first two-dimensional acousto-optic deflector loses light to the incident STED under the control of the first data acquisition card Performing beam modulation and deflection, and the modulated and deflected STED loss light is redirected by the mirror reflection, projected by the first dichroic mirror, and incident on the objective lens;

The dispersion-pre-compensated excitation light is incident perpendicularly to the second two-dimensional acousto-optic deflector, and the second two-dimensional acousto-optic deflector beams the incident excitation light under the control of the second data acquisition card Modulating and deflecting, the modulated and deflected excitation light is incident on the objective lens after being projected by the second dichroic mirror;

The objective lens is used for focusing the incident excitation light and the STED loss light to be incident on the sample, thereby exciting the sample to generate a fluorescence signal exceeding the diffraction limit; and also for collecting the fluorescence signal generated by the sample and irradiating the second dichroic mirror;

The second dichroic mirror is further configured to transmit the fluorescent signal to the imaging unit after being reflected by the first dichroic mirror.

Further, a distance between the first prism and the first two-dimensional acousto-optic deflector is 35 cm; a distance between the second prism and the second two-dimensional acousto-optic deflector is 35CM .

Further, the imaging unit includes: a beam splitter, a photomultiplier tube, an image sensor, and an imaging module;

The beam splitter is configured to divide the incident fluorescent signal into a first fluorescent signal and a second fluorescent signal according to a ratio;

The first fluorescent signal is amplified by the photomultiplier tube, and then transmitted to the imaging module for processing and imaging;

The second fluorescent signal is optically adjusted by the image sensor, and then transmitted to the imaging module for real-time display.

Further, the beam splitter splits the incident fluorescent signal into a first fluorescent signal and a second fluorescent signal in a ratio of 9:1.

Beneficial effect

Compared with the prior art, the invention has the beneficial effects that the invention uses the scanning technology based on the dual sound deflector, avoids the mechanical offset of the galvanometer scanning, has higher scanning precision and faster scanning speed, and at the same time The scanning technology of the acousto-optic deflector can also realize the selection of the fast region of interest and scan imaging, which greatly reduces the optical damage while ensuring the resolution is unchanged, and realizes substantially no photo damage in the non-interest area in the field of view. .

DRAWINGS

FIG. 1 is a schematic structural diagram of a random scanning system according to an embodiment of the present invention.

2 is a schematic structural diagram of a random scanning system according to an embodiment of the present invention.

Figure 3 is a diffraction pattern of the AOD at the center frequency.

Figure 4 is a schematic illustration of the overlap of two beams of light after passing through the AOD.

Figure 5 is a schematic diagram of a 4 x 4 dot pattern formed by excitation light passing through AOD.

FIG. 6 is a schematic diagram showing the formation of a 4×4 dot matrix superposition after the excitation light and the STED loss light respectively pass through the respective AODs.

FIG. 7 is a schematic diagram of a method for finding two sets of AOD frequencies by using fixed lattice coordinates to realize coincidence of two light-matrix arrays.

Figure 8 is a schematic diagram of STD super-resolution arbitrary addressing scanning using two sets of two-dimensional AOD techniques.

Figure 9 is a schematic diagram of the letter "A" STED super-resolution scan imaging using the AOD technique.

Embodiments of the invention

The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It is understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

At present, the commonly used STED super-resolution system uses point scanning, and its imaging speed is slow. The scanning system used is galvanometer scanning. This scanning method requires scanning and imaging of the entire field of view, so the live STED super-resolution is displayed. During microimaging experiments, cells in the entire field of view are subject to photodamage. In order to reduce or remove unnecessary light damage, the present application invents a dual two-dimensional acousto-optic deflector (Acousto-optic) in a STED super-resolution imaging system. Defector, AOD) any selection scanning technique that has the advantage of fast and arbitrary region-of-region scanning imaging, which will greatly reduce optical damage and non-interest areas in the field of view while maintaining the same resolution. There is basically no light damage.

The basic idea of STED super-resolution is to use the stimulated radiation effect to reduce the effective fluorescent light-emitting area. In a typical STED microscopy system, two beams are needed, one for excitation and the other for depletion. When the excitation light illuminates the fluorescent sample, the fluorescent molecules in the range of the diffraction spot are excited, and the electrons therein will transition to the excited state, and then the circular depletion light is superimposed on the excitation light, and the light is exhausted. The electrons in the overlapping partial excited state return to the ground state in the form of stimulated radiation, and the other excited state electrons located at the center of the laser spot continue to be fluorescently returned to the ground state in the form of spontaneous radiation because they are not affected by the depletion light. Since the direction and wavelength of fluorescence emitted during the stimulated and spontaneous radiation are different, the photons received by the detector after filtering are generated by the autofluorescence of the fluorescent sample located at the center of the excitation spot. The light-emitting area of such effective fluorescence is reduced, thereby increasing the spatial resolution of the system.

Based on the above, the embodiment of the present invention provides a system for random scanning as shown in FIG. 1, including a dispersion pre-compensation unit 1, a random scanning unit 2, and an imaging unit 3;

The dispersion pre-compensation unit 1 is configured to perform chromatic dispersion pre-compensation and optical path adjustment on the incident light, and then vertically incident to the random scanning unit 2 for realizing scanning of an arbitrary area;

The random scanning unit 2 is further configured to collect a fluorescent signal generated after scanning an arbitrary region, and process the fluorescent signal and then incident on the imaging unit 3 for imaging.

The embodiment of the present invention is further explained below with reference to FIG. 2:

The dispersion pre-compensation unit 1 includes a first prism 1 and a second prism 2. The first mirror group and the second mirror group, the first mirror group includes a mirror M2 and a mirror M1, and the second mirror group includes a mirror M5 and a mirror M4.

The random scanning unit 2 includes:

a first two-dimensional acousto-optic deflector 2D-AOD 1, and the first two-dimensional acousto-optic deflector 2D-AOD 1 phase connected first data acquisition card DAQ1;

Second two-dimensional acousto-optic deflector 2A-AOD 2, and said second two-dimensional acousto-optic deflector 2D-AOD 2 phase connected second data acquisition card DAQ2;

a mirror M3, a first dichroic mirror DM1, a second dichroic mirror DM2, and an objective lens;

The imaging unit 3 includes a beam splitter BS, a photomultiplier tube PMT, an image sensor CCD, and an imaging module; the imaging module is a computer in this embodiment.

The following is a detailed description of each component:

AOD-based random scanning system for STED super-resolution microscopy, including:

First Mirror (Prism 1), mainly to pre-compensate the dispersion of STED loss light, the purpose is to offset the dispersion generated by the rear AOD, note that the prism here should be placed in the horizontal direction by about 45°;

Second Mirror (Prism 2), mainly to pre-compensate the dispersion of the excitation light, the purpose is to offset the dispersion generated by the rear AOD, note that the prism here should be placed in the horizontal direction by about 45°;

a first two-dimensional acousto-optic deflector (2D-AOD 1) for performing random arbitrary selection scanning of red STED loss light;

a second two-dimensional acousto-optic deflector (2D-AOD 2) for performing random arbitrary selection scanning of green excitation light;

The first mirror M1 and the second mirror M2 are mainly used to adjust the STED loss light after passing through the mirror to ensure that it enters the 2D-AOD vertically. 1;

Mirror M3 for pairing 2D-AOD The (1,1) diffracted light of 1 undergoes reflection redirection and has a fine adjustment effect during the experiment;

The fourth mirror M4 and the fifth mirror M5 are mainly used for adjusting the excitation light after passing through the prism to ensure that it enters the 2D-AOD 2 vertically;

a first dichroic mirror (DM1) for transmitting 780 nm STED loss light, reflecting fluorescence of about 680 nm;

A second dichroic mirror (DM2) is used to reflect excitation light at 633 nm and to transmit fluorescence at about 680 nm.

A beam splitter (BS) is used to split the signal collected by the objective into two parts (10%: 90%), 10% of the fluorescent signal enters the CCD for adjusting the optical path, and 90% of the fluorescent signal enters the PMT for super-resolution imaging.

The data acquisition card (DAQ1 and DAQ2) is used to generate control signals to realize real-time control of the two pairs of acousto-optic deflectors. At the same time, in order to maintain synchronization, a clock signal is shared between the two DAQs, so that two DAQs can be realized. Two sets of AODs are synchronized to produce different scanning frequencies.

An objective lens for focusing excitation light and STED loss light to excite fluorescence that exceeds the diffraction limit while collecting fluorescent signals generated by the sample;

A photomultiplier tube (PMT) amplifies the fluorescent signal and transmits the amplified signal to a computer for imaging.

The image sensor CCD is used to collect the fluorescent signal when adjusting the optical path, and transmits the signal to the computer for real-time display.

Computer is used to control the data acquisition card and the photomultiplier tube, and simultaneously process the signal of the photomultiplier tube to form a complete super-resolution image.

The detailed process of the present invention is as follows: Firstly, it is assumed that both the excitation light and the STED loss light have been adjusted to meet the STED imaging requirements when the random scanning system is reached, and the adjusted excitation light is first subjected to dispersion pre-compensation through the mirror 2, and then through the mirror ( M4 and M5) adjust the height of the beam, and then enter the two-dimensional acousto-optic deflector. At this time, the dispersion generated in the acousto-optic deflector is exactly offset by the dispersion compensated by the prism, and the excitation light emitted by the acousto-optic deflector should be It is a circular spot, which is then reflected by the dichroic mirror DM2 and then focused by the objective lens. The STED loss light, which has also been adjusted, is pre-compensated by the prism, and then the height of the loss light is adjusted by the mirrors (M1 and M2), and the horizontally incident two-dimensional acousto-optic modulator passes through the acousto-optic modulator. The pre-compensated dispersion of the prism should cancel out the dispersion generated by the acousto-optic modulator. Finally, the light emitted from the acousto-optic deflector should be a circular spot after being focused by the objective lens. Since the data acquisition cards DAQ1 and DAQ2 controlling the two sets of AOD signals share one clock. Therefore, the excitation light and the STED loss light are scanned synchronously. However, in the pulse interval, the excitation light reaches the sample about 180ps faster than the STED loss light, so that a better super-resolution image can be obtained.

During the implementation process, the AOD scanning system needs to be fine-tuned first, because the diffraction angle of the AOD will change slightly due to the different wavelengths. Therefore, after the system is adjusted, the spectrometer is placed at the exit of the light source of the excitation light and the STED loss light, and is monitored at any time. The change in wavelength minimizes the effect of wavelength on the scanning system. When fine-tuning the system, first use the software Scan designed for the present invention. The imaging control data acquisition card (DAQ) allows the AOD to be fixed at the center frequency when the two beams of light pass through the AOD (AOD frequency range is 6000 Hz-9000 Hz, center frequency is 7500 Hz). Therefore, the AOD at this time is equivalent to a fixed grating, and the diffracted spot appears at the AOD light exit after the two beams pass through the respective AODs. In the present invention, the (-1, -1) point is selected, and then the fluorescent light is selected. The CCD is used to collect the spot on the road for real-time imaging. At this time, the fine-tuning mirror and the two-color mirror are used to ensure that the (-1, -1) points of the two beams are completely coincident. Then look for the one-to-one correspondence between each pixel coordinate and the AOD scanning frequency. First, find the relationship between the pixel point coordinates of the excitation light and the AOD scanning frequency, and then fix the coordinate position of the corresponding pixel point of the excitation light spot after determining the relationship. Reverse looking for the relationship between the control loss light AOD frequencies corresponding to the positions of the same coordinates to ensure that the two beams are completely coincident at each pixel.

Specifically, the excitation light and the STED loss light of the embodiment of the present invention have been adjusted by default, and the work of the present invention is to ensure the high overlap of the excitation light and the STED loss light at any time, and construct the optical path according to the method of FIG. . Firstly, the excitation light path is established, the excitation light is kept horizontal, the position of the prism is adjusted, and the excitation light is incident at about 45°, and the prism itself is placed at an angle of about 45° with the horizontal plane, and the excitation light passes through the mirror dispersion. After pre-compensation, it is combined by a double mirror to adjust the height and direction of the excitation light. Adjust the position of AOD to keep the position of AOD to the mirror about 35 cm, then pass AOD diffraction (the frequency of AOD work is the intermediate frequency), project the diffraction point on the paper screen, and see four on the paper screen. Diffraction point (as shown in Figure 3), where the (-1,-1)-order diffraction point is the desired spot, and the shape of the diffraction spot is observed. If it is an ellipse, it can be adjusted by tilting the angle of the prism, AOD, or AOD. The distance to the mirror is changed to change the shape of the spot until the spot is round, as shown in Fig. 3, which is the modulated diffraction pattern. The same operation is also performed for the STED loss light. After the two beams are adjusted, the object with better reflectivity is used as the sample (the slide in this embodiment) for reflection imaging. First, the excitation light is turned on, and a circular spot can be seen on the CCD. The CCD allows the spot to be located in the center of the CCD, then turns off the excitation light to turn on the STED loss light. A circular spot can be seen on the CCD. By adjusting the mirror M3, the ring spot is located at the center of the CCD, and finally the excitation light is turned on. Observe the degree of coincidence of the two beams. Make sure that the two beams are completely coincident by fine-tuning the mirror M3 and the dichroic mirror DM2, as shown in Figure 4.

Since the present invention performs scanning using AOD, each group of AODs is composed of two separate AODs (each AOD can perform one-dimensional line scanning), so that surface scanning can be performed through a set of AODs, in order to achieve random scanning. The function must correspond to the scanning frequency of the two-dimensional AOD and the two-dimensional coordinates on the scanning surface. To this end, in this embodiment, the AOD scanning system is used for dot matrix scanning, and then the coordinates of the centroids of each point are found by using MATLAB, and then the lattice scanning frequency of AOD is increased, and the corresponding formula of lattice coordinates and frequency is found f _k (x , y).

First, take the excitation light as an example: suppose one AOD performs X-direction scanning and the other AOD performs Y-direction scanning. To achieve dot matrix scanning as shown in Fig. 5, the AOD frequency in the X and Y directions is set to [6000, 9000], step size is 750. Then, the dot pattern is collected by CCD, and the centroid coordinates of each point are found by the MATLAB software using the centroid positioning method. Assuming that the points on the coordinates and the frequency of the AOD are linear, the expression of f ₁ (x, y, F1) is as follows Shown as follows:

x=A _1x *λ ₁ *F _1x +B _1x

y=A _1y *λ ₁ *F _1y +B _1y

In the above formula, x, y are resolved as coordinate points, F _1x and F _1y are frequencies in the x direction and y direction, respectively, A _1x and A _1y are coefficients before frequency, and B _1x and B _1y are constant, and known coordinate points Position and frequency distribution, you can find the parameters A _1x , A _1y , B _1x and B _1y through MATLAB . Once these parameters are determined , the equation f ₁ (x, y, F ₁ ) can be determined, and the coordinates can be based on any position. Let the software automatically find the corresponding frequency through the formula f ₁ (x, y, F ₁ ) to realize the arbitrary selection scan imaging of AOD.

Also, the same method is performed on the operation of STED loss of light. However, since the wavelength of the excitation light is 633 nm and the wavelength of the STED loss light is 780 nm, the diffraction angle of AOD to different wavelengths will be somewhat different. Therefore, when overlapping, there will be some shift in the position of the lattice center of the two beams. As shown in Figure 6. In order to make the centroids of the two light lattices coincide, in this embodiment, two sets of AODs (2D-AOD1 and 2D-AOD2) are used to respectively control the excitation light and the STED loss light, in order to make the lattices generated by the two beams coincide, First, let the excitation light generate a set of lattices as shown in Fig. 5. Then, with the position of the centroid coordinates of each point in Fig. 5 as the target, combined with the frequency distribution of AOD, the formula f ₂ (x, y, F ₂ ) can also be found. However, the parameters of the formula at this time are different from the formula f1(x, y, F ₁ ) of the excitation light, and therefore, the expression of f ₂ (x, y, F ₂ ) is assumed here as follows.

x=A _2x *λ ₂ *F _2x +B _2x

y=A _2y *λ ₂ *F _2y +B _2y

In the above formula, F _2x and F _2y are the frequencies in the x direction and the y direction, respectively, A _2x and A _2y are the coefficients before the frequency, and B _2x and B _2y are constants, in which the centroid coordinates and frequencies are known. Therefore, each parameter can be calculated by MATLAB software to determine the equation f ₂ (x, y, F ₂ ), and finally the two sets of equations f ₁ (x, y, F ₁ ) and f ₂ (x, y, F ₂ ) The parameters are respectively input to the corresponding scanning control software Scan imaging, run the software, perform 4×4 dot matrix scanning, and obtain the dot pattern as shown in FIG. 7 to explain the excitation light and the STED loss light in the field of view. The points are coincident. Therefore, the STED super-resolution random addressing scan as shown in Fig. 8 can be performed. The basic idea is as follows: First, determine the point of interest (x _i , y _j ) in the field of view, and then according to the equation f ₁ (x, y , F ₁₎ and f ₂ (x, y, F ₂ ), find the frequencies F ₁ (x _i , y _j ) and F ₂ (x _i , y _j ) corresponding to each point, and then execute the scanning software to realize the point STED super-resolution imaging of (x _i , y _j ). As shown in FIG. 9, the schematic diagram of the letter "A" STED super-resolution imaging is implemented by using the designed software in this embodiment. Therefore, two sets of two-dimensional AOD systems can be utilized to realize arbitrary selection imaging of the STED super-resolution system.

The above is only the preferred embodiment of the present invention, and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection of the present invention. Within the scope.

Claims

A system for random scanning, characterized in that the system comprises a dispersion pre-compensation unit, a random scanning unit and an imaging unit;

The dispersion pre-compensation unit is configured to perform chromatic dispersion pre-compensation and optical path adjustment on the incident light, and then vertically incident to the random scanning unit to implement scanning of an arbitrary area;

The random scanning unit is further configured to collect a fluorescent signal generated after performing an arbitrary area scan, and process the fluorescent signal and then incident on the imaging unit for imaging.
The system of claim 1 wherein said incident light comprises STED loss light and excitation light.
The system according to claim 2, wherein the dispersion pre-compensation unit comprises a first prism, a second mirror, a first mirror group, and a second mirror group;

The STED loss light is pre-compensated by the first prism and then incident on the first mirror group, and is optically adjusted by the first mirror group and then vertically incident to the random scanning unit;

The excitation light is pre-compensated by the second prism and then incident on the second mirror group, and is optically adjusted by the second mirror group and then vertically incident to the random scanning unit.
The system of claim 3 wherein said first prism and said second mirror are each placed at an angle of 45° in the horizontal direction.
The system of claim 3 wherein said random scanning unit comprises:

a first two-dimensional acousto-optic deflector, a first data acquisition card connected to the first two-dimensional acousto-optic deflector;

a second two-dimensional acousto-optic deflector, and a second data acquisition card connected to the second two-dimensional acousto-optic deflector;

a mirror, a first dichroic mirror, a second dichroic mirror, and an objective lens;

The dispersion-precompensated STED loss light is incident perpendicularly to the first two-dimensional acousto-optic deflector, and the first two-dimensional acousto-optic deflector loses light to the incident STED under the control of the first data acquisition card Performing beam modulation and deflection, and the modulated and deflected STED loss light is redirected by the mirror reflection, projected by the first dichroic mirror, and incident on the objective lens;

The dispersion-pre-compensated excitation light is incident perpendicularly to the second two-dimensional acousto-optic deflector, and the second two-dimensional acousto-optic deflector beams the incident excitation light under the control of the second data acquisition card Modulating and deflecting, the modulated and deflected excitation light is incident on the objective lens after being projected by the second dichroic mirror;

The objective lens is used for focusing the incident excitation light and the STED loss light to be incident on the sample, thereby exciting the sample to generate a fluorescence signal exceeding the diffraction limit; and also for collecting the fluorescence signal generated by the sample and irradiating the second dichroic mirror;

The second dichroic mirror is further configured to transmit the fluorescent signal to the imaging unit after being reflected by the first dichroic mirror.
The system according to claim 5, wherein a distance between said first prism and said first two-dimensional acousto-optic deflector is 35 CM; said second mirror and said second two-dimensional The distance between the acousto-optic deflectors is 35 CM.
The system of claim 1 wherein said imaging unit comprises: a beam splitter, a photomultiplier tube, an image sensor, and an imaging module;

The beam splitter is configured to divide the incident fluorescent signal into a first fluorescent signal and a second fluorescent signal according to a ratio;

The first fluorescent signal is amplified by the photomultiplier tube, and then transmitted to the imaging module for processing and imaging;

The second fluorescent signal is optically adjusted by the image sensor, and then transmitted to the imaging module for real-time display.
The system of claim 7 wherein said beam splitter splits the incident fluorescent signal into a first fluorescent signal and a second fluorescent signal in a 9:1 ratio.