JP4854879B2 - Scanning laser microscope and image acquisition method thereof - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a scanning laser microscope that collects laser light with an objective lens to form a light spot on a sample and detects fluorescence from the sample with a photodetector and an image acquisition method thereof.
[0002]
[Prior art]
A scanning laser microscope condenses laser light with an objective lens to form a light spot on a sample (hereinafter referred to as a sample), and detects fluorescence from the sample with a photodetector. In a scanning laser microscope, a plurality of types of laser light wavelengths are prepared according to the fluorescent dye used, and a plurality of dichroic mirrors are prepared according to the laser light and the fluorescence wavelength. The image is acquired by switching the wavelength of the laser light and the dichroic mirror corresponding to the fluorescent dye.
[0003]
Here, when the dichroic mirror is switched, the scanning position is generally shifted due to an error in the angle component of each dichroic mirror. Therefore, the image before and after the switching of the dichroic mirror is usually shifted. For this reason, in observing multiple-stained fluorescent specimens, the position of the fluorescent image is shifted for each fluorescent dye when the images are superimposed for each fluorescent dye, and accurate observation cannot be performed. In addition, when the acquired images are overlapped later, it is difficult to accurately overlap the fluorescent dyes because different portions of the sample emit fluorescence.
[0004]
JP-A-11-52252 discloses a fluorescence microscope (scanning laser microscope) that solves this problem. In the apparatus disclosed in Japanese Patent Laid-Open No. 11-52252, an image shift amount for each dichroic mirror is obtained in advance and stored in a storage means, and the scanning waveform of the scan mirror is calculated for each dichroic mirror based on the shift amount information. The displacement of the fluorescent image at the time of switching the dichroic mirror is corrected by performing change control, changing control of the sampling timing of light detection, or changing control of the position of the stage on which the sample is mounted.
[0005]
[Problems to be solved by the invention]
In the scanning laser microscope disclosed in Japanese Patent Application Laid-Open No. 11-52252, an adjustment process is performed in which an image for each dichroic mirror is acquired with an image such as an actual reference graphic pattern, an image shift amount is obtained, and information is stored in the apparatus. is required. However, later, when a laser light source and a fluorescent dye to be used are added and a new dichroic mirror is added, the above adjustment process must be performed, which is very troublesome.
[0006]
Furthermore, for the dichroic mirror that is known at the time of shipment, this adjustment process can be performed by the manufacturer at the time of shipment or when setting up the delivery device, but as described above, after the dichroic mirror has been delivered for a while, When added by the user himself / herself, the user himself / herself who uses the apparatus performs the adjustment, which may cause an adjustment error or forgetting the adjustment. If the device is used with these poor adjustments (adjustment mistakes or forgotten adjustments), it is not possible to accurately observe the multiple stained image.
[0007]
In addition, changing the waveform control of the scanning mirror, changing the sampling timing of light detection, and the like complicate the apparatus, and there are doubts about reliability.
[0008]
The present invention has been made in order to solve such a problem, and the object thereof is a simple configuration that does not require a complicated adjustment process or complicated control, but a plurality of fluorescent dye images are misaligned. It is an object of the present invention to provide a scanning laser microscope capable of acquiring a superimposed image and a method for acquiring the image.
[0009]
[Means for Solving the Problems]
One aspect of the present invention is a scanning laser microscope, which selects laser light source means for oscillating laser light of a plurality of types of wavelengths and laser light of at least one type from among the laser beams of a plurality of types of wavelengths. Laser light selection means, an objective lens for condensing the laser light on the sample, scanning means for scanning the laser light on the sample, and fluorescence generated from the sample by irradiating the laser light on the sample And a plurality of dichroic mirrors having different wavelength characteristics that are switchably held corresponding to the wavelength of the selected laser beam, and detecting fluorescence from the sample through the objective lens In a scanning laser microscope comprising: a fluorescence detection means for detecting; and a transmitted light detection means for detecting the laser light transmitted through the sample. The laser light to be selected by the user light selection means is switched, and the plurality of dichroic mirrors are switched corresponding to the wavelength, and a fluorescence image is switched by the fluorescence detection means for each of the laser light and the dichroic mirror to be switched. At the same time, the transmitted light image obtained by the transmitted light that has passed through the sample is acquired by the transmitted light detection means, and the transmitted light image acquired for each laser beam is subjected to image processing, thereby being acquired for each dichroic mirror. And a control means for obtaining a positional deviation amount of the fluorescent image and correcting and displaying the positional deviation of the fluorescent image acquired for each of the dichroic mirrors based on the deviation amount.
[0010]
One aspect of the present invention is an image acquisition method for a scanning laser microscope, a laser light source means for oscillating a plurality of types of laser beams, and a laser having at least one type of laser beams of the plurality of types of wavelengths. Laser light selecting means for selecting light, an objective lens for condensing the laser light on the sample, scanning means for scanning the laser light on the sample, and generated from the sample by irradiating the sample with the laser light A plurality of dichroic mirrors having different wavelength characteristics that are switchably held corresponding to a selected laser wavelength, and fluorescence from the sample via the objective lens. Image of a scanning laser microscope provided with fluorescence detecting means for detecting and transmitted light detecting means for detecting the laser light transmitted through the sample In the obtaining method, the laser light selected by the laser light selection means is switched, the plurality of dichroic mirrors are switched in accordance with the wavelength, and the fluorescence image is displayed for each of the switched laser light and the dichroic mirror. Simultaneously with the fluorescence detection means, a transmitted light image by the transmitted light that has passed through the sample is acquired by the transmitted light detection means, and the transmitted light image acquired for each laser beam is subjected to image processing, whereby the dichroic mirror A positional deviation amount of the fluorescence image acquired every time is obtained, and based on this deviation amount, the positional deviation of the fluorescent image acquired for each dichroic mirror is corrected and displayed.
[0011]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the present invention will be described with reference to the drawings.
[0012]
First embodiment
In FIG. 1, the scanning laser microscope of the present embodiment includes a laser light emitting means for emitting laser light for exciting fluorescence, and a condensing optical system for condensing the laser light and irradiating the sample. An XY galvanometer mirror 6 for scanning the laser beam on the sample, an excitation dichroic mirror unit 5 for separating the fluorescence generated from the sample and the laser beam, and fluorescence detection for detecting the fluorescence from the sample Means and transmitted light detecting means for detecting the laser light transmitted through the sample.
[0013]
The laser light emitting means is a laser light source unit 1 that can selectively output laser light of a plurality of types of wavelengths, an optical fiber 3 for transmitting laser light output from the light source unit 1, and emitted from the end face of the optical fiber 3. And a collimating lens 4 for collimating the laser beam.
[0014]
The laser light source unit 1 selects at least one type of laser beam from among an argon (Ar) laser 1a that oscillates laser beams of a plurality of wavelengths and a plurality of types of laser beams emitted from the Ar laser 1a. And an acousto-optic device (AOTF) 2.
[0015]
The Ar laser 1a simultaneously oscillates laser beams having wavelengths of 458 nm, 488 nm, and 515 nm. The AOTF 2 can select at least one type of laser beam from the above-described three types of laser beam according to the wavelength of the sound wave input thereto. In other words, the AOTF 2 can select any combination of laser beams from the above-described three types of laser beams.
[0016]
The laser beam emission means is selected as described above, and the laser beam collimated through the optical fiber 3 and the collimating lens 4 is emitted toward the dichroic mirror unit 5.
[0017]
The excitation dichroic mirror unit 5 has a turret 5f that is rotationally driven by a motor 5e. The turret 5f has holes for additionally mounting two excitation dichroic mirrors 5a and 5b and other types of excitation dichroic mirrors. 5c is provided on a concentric circle.
[0018]
As shown in FIG. 2, the excitation dichroic mirror 5a has a characteristic of reflecting light having a wavelength of 458 nm and transmitting fluorescence having a wavelength longer than 458 nm emitted from a sample excited by light having a wavelength of 458 nm. . As shown in FIG. 3, the excitation dichroic mirror 5b has a characteristic of reflecting light having a wavelength of 515 nm and transmitting fluorescence having a wavelength longer than 515 nm emitted from a sample excited by the wavelength 515 nm.
[0019]
The excitation dichroic mirrors 5a and 5b are selectively arranged on the optical path according to the wavelength of the laser light emitted from the laser light emitting means by rotating the turret 5f with the motor 5e.
[0020]
The XY galvanometer mirror 6 has two reflecting surfaces that can swing around two different axes (for example, orthogonal to each other), and scans the laser light reflected by the excitation dichroic mirror unit 5.
[0021]
The condensing optical system has a pupil projection lens 7, an imaging lens 8, and an objective lens 9, condenses the laser light that passes through the XY galvanometer mirror 6 and irradiates the sample (double-stained sample) 10. To do.
[0022]
Sample 10 is stained with fluorescent protein CFP excited at 458 nm and fluorescent protein YFP excited at 515 nm (fluorescent proteins CFP and YFP express a gene that emits fluorescence in the sample cells. Therefore, although it is different from staining to be precise, in the case of a general fluorescent sample, the sample is stained with a fluorescent reagent. Use the expression said to be stained.) The fluorescence wavelength characteristic 41c of CFP and the fluorescence wavelength characteristic 41y of YFP are as shown in FIG.
[0023]
The fluorescence generated from the sample 10 by irradiation with the laser light passes through the condensing optical system and the XY galvanometer mirror 6 and is arranged on the optical path according to the wavelength of the selected laser light and the fluorescence wavelength from the sample 10. It passes through the excitation dichroic mirror and is separated from the laser light.
[0024]
The fluorescence detection means includes a mirror 13 that reflects the fluorescence that has passed through the excitation dichroic mirror unit 5, a confocal lens 14 for converging the fluorescence, a pinhole 15 disposed at a position conjugate to the objective lens 9, a pin A spectroscopic dichroic mirror unit 16 for spectroscopically analyzing the fluorescence that has passed through the hole 15, a pair of band-pass filters 17 a and 17 b, and a pair of fluorescence detectors 18 a and 18 b are included.
[0025]
The pinhole 15 is arranged in a confocal relationship with the sample 10 with respect to the optical system constituted by the condensing optical system and the confocal lens 14, and the image formation position shift due to the angle error at the time of switching of the excitation dichroic mirror Can be moved in a plane perpendicular to the optical axis by the XY electric stage 15a.
[0026]
The spectroscopic dichroic mirror unit 16 has a turret 16f that is rotationally driven by a motor 16e, and the spectroscopic dichroic mirror 16a, the reflecting mirror 16b, and the air holes 16c are provided concentrically on the turret 16f.
[0027]
As shown in FIG. 5, the spectroscopic dichroic mirror 16a has a transmittance of 50% with respect to light having a wavelength of 520 nm, reflects light having a shorter wavelength, and transmits light having a longer wavelength than that. It has characteristics. Thereby, the spectroscopic dichroic mirror 16a reflects the fluorescence of CFP emitted from the sample 10 and transmits the fluorescence of YFP emitted from the sample 10.
[0028]
The spectral dichroic mirror 16a, the reflecting mirror 16b, and the air hole 16c can be selectively disposed on the optical path by rotating the turret 16f with a motor 16e. In the present embodiment, the spectral dichroic mirror 16a is always disposed on the optical path.
[0029]
As shown by a solid line in FIG. 6, the bandpass filter 17a has a characteristic of cutting laser light having a wavelength of 458 nm for exciting CFP fluorescence and transmitting light in the CFP fluorescence wavelength range. . That is, the band pass filter 17a selectively transmits the CFP fluorescence. The fluorescence detector 18a detects the fluorescence of CFP that has passed through the bandpass filter 17a.
[0030]
As shown by a broken line in FIG. 6, the band-pass filter 17b has a characteristic of cutting a laser beam having a wavelength of 515 nm for exciting the fluorescence of YFP and transmitting the fluorescence wavelength range of YFP. That is, the band pass filter 17b selectively transmits the YFP fluorescence. The fluorescence detector 18a detects the fluorescence of YFP that has passed through the bandpass filter 17b.
[0031]
Here, it is assumed that a double excitation type reflecting 458 nm and 515 nm as shown in FIG. 7 is used for the excitation dichroic mirrors 5 a and 5 b as shown in FIG. In this case, 50% or more of the fluorescence of CFP to be acquired up to about 530 nm on the long wavelength side is cut by the excitation dichroic mirror in the wavelength range of 505 nm to 525 nm. However, when a non-double excitation type is used as in the present embodiment, since the long wavelength side is determined by the reflection characteristics of the spectroscopic dichroic mirror 16a, it is possible to acquire 50% or more fluorescence up to 520 nm, and bright fluorescence. Observation becomes possible.
[0032]
The transmitted light detection means has a lens 11 and a transmitted light detector 12, and is disposed on the opposite side of the condensing optical system with respect to the sample 10. The lens 11 picks up the light transmitted through the sample 10, and the transmitted light detector 12 detects the transmitted light that has entered the lens 11.
[0033]
This transmitted light detection means is attached to a commercially available scanning laser microscope for fluorescence observation in order to observe the cytoskeleton, which is the whole image of the fluorescently stained cell specimen. Since the fluorescent dye is used by labeling a part of the cell, such as a nucleus or a microtubule, for example, the whole image of the cell cannot be grasped only by the fluorescent image. The transmitted light detecting means is prepared for grasping the whole cell image that cannot be grasped by the fluorescence image. Nomarski observation may be applied to the transmitted light detection means because a transmitted image of the cell is observed with good contrast.
[0034]
As shown in FIG. 1, the scanning laser microscope further includes a control device 19 and a display unit 20. The control device 19 controls the rotation drive of the excitation dichroic mirror unit 5, the rotation drive control of the spectroscopic dichroic mirror unit 16, the control of AOTF2, the movement control of the XY electric stage 15a of the pinhole 15, the drive control of the XY galvano mirror 6, and the fluorescence. Control is performed to capture the outputs of the detectors 18 a and 18 b and the transmitted light detector 12 in synchronization with the driving of the XY galvanometer mirror 6. The display means 20 displays the fluorescence images detected by the fluorescence detectors 18a and 18b in a superimposed manner with a pseudo color for each fluorescence.
[0035]
The control device 19 switches and emits laser light of a plurality of types of wavelengths from the laser light emitting means, switches the excitation dichroic mirror unit 5 so that the excitation dichroic mirror corresponding to the laser light is arranged on the optical path, For each of the laser beams emitted after switching, the fluorescence generated from the sample 10 is detected by the fluorescence detection means to acquire the fluorescence image, and at the same time, the light transmitted through the sample 10 is detected by the transmitted light detection means and transmitted light. An image is acquired, a positional deviation amount of a plurality of fluorescent images is obtained based on the obtained plurality of transmitted light images, and a positional deviation of the plurality of fluorescent images is corrected based on the deviation amount. Control to display the fluorescent images in an overlapping manner.
[0036]
Next, the operation of the scanning laser microscope having the above configuration will be described. In the present embodiment, the spectral dichroic mirror 16a in the spectral dichroic mirror unit 16 is arranged on the optical path. An instruction to start scanning is input to the control device 19 by a PC (personal computer) or the like (not shown), and a double-stained fluorescent image corresponding to two types of fluorescent proteins is converted into a laser beam having a wavelength of 458 nm and 515 nm according to the command of the control device 19. The two types of fluorescence are acquired in a time-sharing manner by switching in synchronization with the scanning of the laser beam having the wavelength of.
[0037]
Therefore, a CFP fluorescence image is acquired in the first step and a transmitted light image having a wavelength of 458 nm is acquired as the excitation light, and a YFP fluorescence image is acquired in the second step and the wavelength that is the excitation light is acquired. A transmitted light image of 515 nm is acquired, and the positional deviation between the two fluorescent images is corrected based on the two transmitted light images acquired in the third step.
[0038]
In the first step, the control device 19 performs the following control. The excitation dichroic mirror unit 5 is rotationally controlled, and the excitation dichroic mirror 5a is arranged on the optical path. The XY electric stage 15a is driven to place the pinhole 15 at the pinhole position corresponding to the excitation dichroic mirror 5a. The AOTF 2 is controlled so that laser light having a wavelength of 458 nm out of the laser light emitted from the Ar laser 1 a is selectively incident on the fiber 3.
[0039]
The laser light incident on the fiber 3 travels through the inside thereof, is emitted from the end face of the fiber 3, and is converted into parallel light by the collimating lens 4. The laser beam having a wavelength of 458 nm is reflected by the excitation dichroic mirror 5a, passes through the XY galvanometer mirror 6, is irradiated onto the sample 10 through the pupil projection lens 7, the imaging lens 8, and the objective lens 9, and a light spot on the sample 10 Tie. This light spot is scanned two-dimensionally on the sample by the XY galvanometer mirror 6.
[0040]
The CFP fluorescence generated from the sample 10 by irradiation with laser light having a wavelength of 458 nm travels backward through the irradiation path of the laser light, reaches the excitation dichroic mirror 5a, and is transmitted therethrough. The CFP fluorescence that has passed through the excitation dichroic mirror 5a is reflected by the mirror 13, converged by the confocal lens 14, passes through the pinhole 15, is reflected by the spectral dichroic mirror 16a, and passes through the CFP band-pass filter 17a. , And is detected by the fluorescence detector 18a.
[0041]
Further, the laser beam having a wavelength of 458 nm that has a light spot formed on the sample 10 passes through the sample 10 and is detected by the transmitted light detector 12 through the lens 11.
[0042]
A fluorescence image of CFP is obtained by detecting the fluorescence of CFP by the fluorescence detector 18a while scanning the range corresponding to one image by the XY galvanometer mirror 6. At the same time, the transmitted light detector 12 detects transmitted light having a wavelength of 458 nm, thereby simultaneously obtaining a transmitted light image having a wavelength of 458 nm corresponding to the CFP fluorescence image.
[0043]
In the second step, the control device 19 performs the following control. The excitation dichroic mirror unit 5 is rotationally controlled, and the excitation dichroic mirror 5b is arranged on the optical path. The XY electric stage 15a is driven to place the pinhole 15 at the pinhole position corresponding to the excitation dichroic mirror 5b. The AOTF 2 is controlled so that laser light having a wavelength of 515 nm out of the laser light emitted from the Ar laser 1 a is selectively incident on the fiber 3.
[0044]
The laser light incident on the fiber 3 travels through the inside thereof, is emitted from the end face of the fiber 3, and is converted into parallel light by the collimating lens 4. The laser beam having a wavelength of 515 nm is reflected by the excitation dichroic mirror 5 b, passes through the XY galvanometer mirror 6, is irradiated onto the sample 10 through the pupil projection lens 7, the imaging lens 8, and the objective lens 9, and the light spot on the sample 10. Tie. This light spot is scanned two-dimensionally on the sample by the XY galvanometer mirror 6.
[0045]
The YFP fluorescence generated from the sample 10 by irradiation with laser light having a wavelength of 515 nm travels backward through the irradiation path of the laser light, reaches the excitation dichroic mirror 5b, and is transmitted therethrough. The YFP fluorescence transmitted through the excitation dichroic mirror 5b is reflected by the mirror 13, converged by the confocal lens 14, passes through the pinhole 15, passes through the spectral dichroic mirror 16a, and passes through the YFP bandpass filter 17b. , And is detected by the fluorescence detector 18b.
[0046]
In addition, the laser beam having a wavelength of 515 nm that has a light spot formed on the sample 10 passes through the sample 10 and is detected by the transmitted light detector 12 through the lens 11.
[0047]
A fluorescence image of YFP is obtained by detecting the fluorescence of YFP by the fluorescence detector 18b while scanning the range corresponding to one image by the XY galvanometer mirror 6. At the same time, the transmitted light detector 12 detects transmitted light having a wavelength of 515 nm, thereby simultaneously obtaining a transmitted light image having a wavelength of 515 nm corresponding to the YFP fluorescence image.
[0048]
In this way, the two types of fluorescence images obtained by time division are usually shifted from each other due to the angle error of the two excitation dichroic mirrors 5a and 5b to be switched.
[0049]
In the third step, the following control is performed by the control device 19 in order to correct this displacement. The transmitted light image acquired in the first step and the transmitted light image acquired in the second step are subjected to image processing, and a positional deviation amount between the two fluorescent images is obtained. Based on the amount of deviation, the positional deviation between the CFP fluorescence image obtained in the first step and the YFP fluorescence image obtained in the second step is corrected, and these are superimposed on the display means 20 and displayed.
[0050]
Examples of image processing methods for determining the amount of positional deviation include, for example, a method for finding a pattern having the highest contrast for two transmitted light images and calculating the amount of positional deviation from those positions, or between images. A method is conceivable in which subtraction is performed while shifting the pixels, and the position of the pixel where the absolute value of the luminance of the entire subtracted pixel is the lowest is determined to determine the amount of displacement.
[0051]
The end regions of the two fluorescent images are not necessarily acquired in common for both images due to the positional shift between the two. It is desirable for the display means 20 to display only the area acquired in common for the two fluorescent images, excluding such end areas. For example, as shown in FIG. 8, when two fluorescent images having 512 × 512 pixels are shifted by 20 pixels in the horizontal direction and 10 pixels in the vertical direction, they are acquired in common for both ( A range of 512-20) × (512-10) = 492 × 502 pixels may be displayed.
[0052]
In the present embodiment, the control device 19 automatically performs the first step, the second step, and the third step described above only by a scan start command from a PC or the like (not shown). Therefore, the wavelength characteristics of the excitation dichroic mirror Thus, the two fluorescent images can be overlapped without misalignment without deteriorating the fluorescence acquisition efficiency due to the above, and a double-stained fluorescent image can be easily obtained.
[0053]
Moreover, since the alignment process of the fluorescence image for each dichroic mirror is performed based on the transmitted light image acquired simultaneously with the acquisition of the fluorescence image for each dichroic mirror, a special adjustment step is not necessary.
[0054]
First variation
In the present embodiment described above, the spectroscopic dichroic mirror 16a is always arranged on the optical path. However, in the first step of acquiring the CFP fluorescence image by controlling the motor 16e by the control device 19, the spectroscopic dichroic mirror unit 16 is used. In the second step of obtaining the YFP fluorescent image, the air holes 16c of the spectroscopic dichroic mirror unit 16 may be arranged in the optical path.
[0055]
In this modification, all the fluorescence of CFP is guided to the CFP band-pass filter 17a by the reflection mirror 16b, so that there is no loss when reflected by the spectroscopic dichroic mirror 16a, and the CFP fluorescence is detected more efficiently. Further, since all the fluorescence of YFP is guided to the band pass filter 17b for YFP by the holes 16c, there is no loss when transmitted through the spectroscopic dichroic mirror 16a, and it is detected more efficiently.
[0056]
Second modification
As shown in FIG. 9, the hole 16c of the spectroscopic dichroic mirror unit 16 is disposed on the optical path, and the fluorescence detection means further includes a band pass filter unit 21 disposed in front of the fluorescence detector 18b. ing. The band pass filter unit 21 has a turret 21f that is rotationally driven by a motor 21e. The turret 21f is provided with a CFP band pass filter 21a, a YFP band pass filter 21b, and a hole 21c on a concentric circle. ing.
[0057]
The CFP band-pass filter 21a and the YFP band-pass filter 21b are band-pass filters having the same wavelength characteristics as the CFP band-pass filter 17a and the YFP band-pass filter 17b, respectively. These are the turrets 21f of the motor 21e. It is selectively arranged on the optical path by rotation control.
[0058]
In the first step, the CFP band-pass filter 21a of the band-pass filter unit 21 is arranged on the optical path, and the fluorescence of the CFP that passes through the optical path is detected by the fluorescence detector 18b. In the second step, the band-pass filter The YFP band-pass filter 21b of the unit 21 is arranged on the optical path, and the fluorescence of YFP that passes through this is detected by the fluorescence detector 18b.
[0059]
In this modification, there is no loss of fluorescence due to the spectroscopic dichroic mirror 16a, so that fluorescence can be detected more efficiently. Further, the spectroscopic dichroic mirror unit 16 and the fluorescence detector 18a are unnecessary, and the apparatus can be further miniaturized by omitting them.
[0060]
Third modification
In the present embodiment, the first step of acquiring the fluorescence of CFP, the second step of acquiring YFP, and the third step of correcting and displaying the positional deviation of the two types of fluorescent images by image processing are performed. In response to a single command for starting scanning by a PC or the like (not shown), the control device 19 continuously executes the scan, but the control device 19 executes the process in response to a start command for each process. It may be controlled.
[0061]
In this modification, it is necessary to input a command three times from a PC or the like. However, since a long-flow control program is not required, it is possible to save time for program development.
[0062]
Second embodiment
The configuration of the scanning laser microscope of this embodiment is shown in FIG. In FIG. 10, members equivalent to those already described in the first embodiment are denoted by the same reference numerals, and detailed description thereof will be omitted in the following description.
[0063]
In the scanning laser microscope of the present embodiment, the laser light source unit 1 includes a green helium neon laser (HeNe-G) 1b that oscillates laser light having a wavelength of 543 nm, and a HeNe-G1b in addition to the Ar laser 1a and the AOTF2. A reflection mirror 1c for deflecting the emitted laser light, and a dichroic mirror 1d for coaxially coupling the laser light having a wavelength of 543 nm emitted from the HeNe-G1b to the laser light emitted from the Ar laser 1a. Yes.
[0064]
The excitation dichroic mirror unit 5 includes an excitation dichroic mirror 5d newly attached to the hole 5c in addition to the excitation dichroic mirrors 5a and 5b. As shown in FIG. 11, the excitation dichroic mirror 5d has a characteristic of reflecting light having a wavelength of 543 nm and transmitting fluorescence having a wavelength longer than 543 nm excited by light having a wavelength of 543 nm.
[0065]
In the sample 100, each site in the cell is stained with a fluorescent protein CFP excited by light having a wavelength of 458 nm, a fluorescent protein YFP excited by light having a wavelength of 515 nm, and a fluorescent protein RFP excited by light having a wavelength of 543 nm. This is a triple stained sample. The fluorescence wavelength characteristic 41r of RFP excited by light having a wavelength of 543 nm is as shown in FIG.
[0066]
The bandpass filter unit 21 includes an RFP bandpass filter 21d newly attached to the hole 21c in addition to the CFP bandpass filter 21a and the YFP bandpass filter 21b. This RFP bandpass filter 21d has a characteristic of cutting the laser light having a wavelength of 543 nm for exciting the fluorescence of the RFP and transmitting the light in the fluorescence wavelength range of the RFP, as shown by a one-dot chain line in FIG. It has the characteristics that it has.
[0067]
Next, the operation of the scanning laser microscope having the above configuration will be described. In the present embodiment, the holes 16c in the spectral dichroic mirror unit 16 are arranged on the optical path. A scanning start command is input to the control device 19 by a PC or the like (not shown), and a triple-stained fluorescent image corresponding to the three types of fluorescent proteins is converted into a laser beam having a wavelength of 458 nm, a laser beam having a wavelength of 515 m, Three types of fluorescence are acquired in a time-sharing manner by switching the laser beam of 543 nm in synchronization with the scanning.
[0068]
Therefore, a CFP fluorescence image is acquired in the first step and a transmitted light image having a wavelength of 458 nm is acquired as the excitation light, and a YFP fluorescence image is acquired in the second step and the wavelength that is the excitation light is acquired. Acquire a transmitted light image of 515 nm, acquire a fluorescent image of RFP in the third step, acquire a transmitted light image of wavelength 543 nm as the excitation light, and acquire the transmitted light image of three in the fourth step Based on the above, the positional deviation of the three fluorescent images is corrected.
[0069]
In the first step, the control device 19 performs the following control. The excitation dichroic mirror unit 5 is rotationally controlled, and the excitation dichroic mirror 5a is arranged on the optical path. The XY electric stage 15a is driven to place the pinhole 15 at the pinhole position corresponding to the excitation dichroic mirror 5a. The band-pass filter unit 21 is rotationally controlled to place the CFP band-pass filter 21a on the optical path. The AOTF 2 is controlled so that laser light having a wavelength of 458 nm out of the laser light emitted from the Ar laser 1 a is selectively incident on the fiber 3.
[0070]
The laser light incident on the fiber 3 travels through the inside thereof, is emitted from the end face of the fiber 3, and is converted into parallel light by the collimating lens 4. A laser beam having a wavelength of 458 nm is reflected by the excitation dichroic mirror 5 a, passes through the XY galvanometer mirror 6, irradiates the sample 100 through the pupil projection lens 7, the imaging lens 8, and the objective lens 9, and a light spot on the sample 100. Tie. This light spot is scanned two-dimensionally on the sample by the XY galvanometer mirror 6.
[0071]
The CFP fluorescence generated from the sample 100 by irradiation with laser light having a wavelength of 458 nm travels backward through the irradiation path of the laser light, reaches the excitation dichroic mirror 5a, and is transmitted therethrough. The CFP fluorescence that has passed through the excitation dichroic mirror 5a is reflected by the mirror 13, converged by the confocal lens 14, passes through the pinhole 15, passes through the hole 16c in the spectroscopic dichroic mirror unit 16, and is used for CFP. The light passes through the bandpass filter 21a and is detected by the fluorescence detector 18b.
[0072]
In addition, the 458 nm laser light having a light spot tied to the sample 100 passes through the sample 100 and is detected by the transmitted light detector 12 through the lens 11.
[0073]
A fluorescence image of CFP is obtained by detecting the fluorescence of CFP by the fluorescence detector 18b while scanning the range corresponding to one image by the XY galvanometer mirror 6. At the same time, the transmitted light detector 12 detects transmitted light having a wavelength of 458 nm, thereby simultaneously obtaining a transmitted light image having a wavelength of 458 nm corresponding to the CFP fluorescence image.
[0074]
In the second step, the control device 19 performs the following control. The excitation dichroic mirror unit 5 is rotationally controlled, and the excitation dichroic mirror 5b is arranged on the optical path. The XY electric stage 15a is driven to place the pinhole 15 at the pinhole position corresponding to the excitation dichroic mirror 5b. The band-pass filter unit 21 is rotationally controlled, and a band-pass filter 21b for YFP is disposed on the optical path. The AOTF 2 is controlled so that laser light having a wavelength of 515 nm out of the laser light emitted from the Ar laser 1 a is selectively incident on the fiber 3.
[0075]
The laser light incident on the fiber 3 travels through the inside thereof, is emitted from the end face of the fiber 3, and is converted into parallel light by the collimating lens 4. A laser beam having a wavelength of 515 nm is reflected by the excitation dichroic mirror 5 b, passes through the XY galvano mirror 6, is irradiated onto the sample 100 through the pupil projection lens 7, the imaging lens 8, and the objective lens 9, and a light spot on the sample 100. Tie. This light spot is scanned two-dimensionally on the sample by the XY galvanometer mirror 6.
[0076]
The YFP fluorescence generated from the sample 100 by irradiation with laser light having a wavelength of 515 nm travels backward through the laser light irradiation path, reaches the excitation dichroic mirror 5b, and is transmitted therethrough. The YFP fluorescence transmitted through the excitation dichroic mirror 5b is reflected by the mirror 13, converged by the confocal lens 14, passes through the pinhole 15, passes through the hole 16c in the spectroscopic dichroic mirror unit 16, and is used for YFP. The light passes through the bandpass filter 21b and is detected by the fluorescence detector 18b.
[0077]
In addition, the 515 nm laser light having a light spot tied to the sample 100 passes through the sample 100 and is detected by the transmitted light detector 12 through the lens 11.
[0078]
A fluorescence image of YFP is obtained by detecting the fluorescence of YFP by the fluorescence detector 18b while scanning the range corresponding to one image by the XY galvanometer mirror 6. At the same time, the transmitted light detector 12 detects transmitted light having a wavelength of 515 nm, thereby simultaneously obtaining a transmitted light image having a wavelength of 515 nm corresponding to the YFP fluorescence image.
[0079]
In the third step, the control device 19 performs the following control. The excitation dichroic mirror unit 5 is rotationally controlled, and the excitation dichroic mirror 5d is arranged on the optical path. The XY electric stage 15a is driven to place the pinhole 15 at the pinhole position corresponding to the excitation dichroic mirror 5d. The band pass filter unit 21 is rotationally controlled to place the RFP band pass filter 21d on the optical path. The AOTF 2 is controlled so that laser light having a wavelength of 543 nm emitted from the HeNe-G laser 1 b is selectively incident on the fiber 3.
[0080]
The laser light incident on the fiber 3 travels through the inside thereof, is emitted from the end face of the fiber 3, and is converted into parallel light by the collimating lens 4. The laser beam having a wavelength of 543 nm is reflected by the excitation dichroic mirror 5d, passes through the XY galvanometer mirror 6, is irradiated onto the sample 100 through the pupil projection lens 7, the imaging lens 8, and the objective lens 9, and a light spot on the sample 100 Tie. This light spot is scanned two-dimensionally on the sample by the XY galvanometer mirror 6.
[0081]
RFP fluorescence generated from the sample 100 by irradiation with laser light having a wavelength of 543 nm travels backward through the irradiation path of the laser light, reaches the excitation dichroic mirror 5d, and is transmitted therethrough. The RFP fluorescence transmitted through the excitation dichroic mirror 5d is reflected by the mirror 13, converged by the confocal lens 14, passes through the pinhole 15, passes through the hole 16c in the spectroscopic dichroic mirror unit 16, and is used for RFP. The light passes through the band-pass filter 21d and is detected by the fluorescence detector 18b.
[0082]
Further, the laser beam having a wavelength of 543 nm that has a light spot connected to the sample 100 passes through the sample 100 and is detected by the transmitted light detector 12 through the lens 11.
[0083]
An RFP fluorescence image is obtained by detecting the fluorescence of the RFP by the fluorescence detector 18b while scanning the range corresponding to one image by the XY galvanometer mirror 6. At the same time, the transmitted light detector 12 detects transmitted light having a wavelength of 543 nm, thereby simultaneously obtaining a transmitted light image having a wavelength of 543 nm corresponding to the fluorescent image of RFP.
[0084]
The three types of fluorescent images obtained by time division in this way are usually offset from each other due to the angular error of the three switched excitation dichroic mirrors 5a, 5b, 5d.
[0085]
In the fourth step, the following control is performed by the control device 19 in order to correct this displacement. The transmitted light image acquired in the first step, the transmitted light image acquired in the second step, and the transmitted light image acquired in the third step are subjected to image processing, and the positional deviation amounts of the three fluorescent images are obtained. Based on the deviation amount, the positional deviation between the CFP fluorescence image obtained in the first step, the YFP fluorescence image obtained in the second step, and the RFP fluorescence image obtained in the third step is corrected. These are superimposed on the display means 20 and displayed.
[0086]
In this embodiment, the control device 19 automatically performs the first step, the second step, the third step, and the fourth step described above only by a scan start command from a PC (not shown). Three fluorescent images can be superimposed without misalignment without deteriorating the fluorescence acquisition efficiency due to the wavelength characteristics of the dichroic mirror, and a triple-stained fluorescent image can be easily obtained.
[0087]
In addition, even when a HeNe-G laser 1b or an excitation dichroic mirror 5d is added later as in the present embodiment, a triple-stained fluorescent image with no image misalignment can be easily obtained without any special adjustment. Can do.
[0088]
Modified example
It is expected that the outputs of the three types of laser light for exciting the three types of fluorescence are greatly different. In such a case, the brightness of each transmitted light image is greatly different, and the output of the detector is saturated with respect to specific laser light, and there is a possibility that subsequent image processing cannot be performed accurately.
[0089]
In this modification, the gain of the transmitted light detector for each step is set separately so that the output of the transmitted light detector in each of the first step, the second step, and the third step is not saturated. . The gain is preferably set so that the detector output in each step has the same level of brightness.
[0090]
In this way, the gain of the transmitted light image acquired in each step is set separately according to the laser output intensity of each step, so even if there is a large difference in the intensity of each laser beam, the fourth step Accurate processing can be performed in certain image processing.
[0091]
Third embodiment
The present embodiment is another laser light emitting means applicable in place of the laser light emitting means of the scanning laser microscope of the first embodiment shown in FIG. 1, and its configuration is shown in FIG. In FIG. 12, members equivalent to those already described in the first embodiment are denoted by the same reference numerals, and detailed description thereof will be omitted in the following description.
[0092]
As shown in FIG. 12, in addition to the laser light source unit 1, the optical fiber 3, and the collimating lens 4, the laser light emitting means of the present embodiment is a near-infrared ultrashort pulse for observing fluorescence by two-photon excitation. A laser 22 and a synthetic dichroic mirror 23 for coaxially coupling the laser light to the laser light emitted from the laser light source unit 1 are provided.
[0093]
Since the near-infrared pulsed laser beam cannot be introduced by the same optical fiber as the visible laser beam, the synthetic dichroic mirror 23 is positioned after the collimating lens 4, and the laser beam emitted from the near-infrared pulsed laser 22 is collimated lens 4. Coaxially coupled to the laser beam emitted from.
[0094]
Actually, it is difficult to completely match the optical axis angle difference between the pulse laser beam emitted from the near infrared pulse laser 22 and the visible laser beam emitted from the collimator lens 4 to zero. For this reason, an angle difference occurs between the two laser beams incident on the excitation dichroic mirror unit 5, and the angle difference affects the positional deviation of the fluorescent image. That is, the positional deviation of the fluorescence image acquired by the irradiation of the pulse laser beam is affected by the angle error of the combining dichroic mirror 23 in addition to the angle error between the excitation dichroic mirrors of the excitation dichroic mirror unit 5.
[0095]
However, in this embodiment, it is possible to correct a positional shift that combines the effects of both angle errors by performing image processing on a transmitted light image acquired by each laser light irradiation. That is, it is possible to correct without considering the influence of the angle error of the excitation dichroic mirror and the composite dichroic mirror 23 separately.
[0096]
For example, consider a case in which YFP fluorescence is acquired by one-photon excitation and CFP fluorescence is acquired by two-photon excitation. First, a fluorescence image of YFP and a transmitted light image with a wavelength of 515 m are obtained by one-photon excitation using the excitation dichroic mirror 5b and a 515 nm laser, and then the excitation dichroic mirror 5b is an excitation dichroic mirror for a 780 nm pulse laser. To obtain a CFP two-photon fluorescence image and a transmitted light image with a wavelength of 780 nm using a pulsed laser with a wavelength of 780 m. By performing image processing on the transmitted light image for each dichroic mirror acquired simultaneously with each fluorescence image, it is possible to obtain two-photon fluorescence (CFP) and one-photon fluorescence (YFP) images without positional deviation.
[0097]
In the case of two-photon fluorescence, as described in Japanese Patent Laid-Open No. 2000-330029, fluorescence detection is performed by branching the optical path between the XY galvanometer mirror 6 serving as scanning means and the objective lens without returning to the confocal pinhole. May be. In this case, the excitation dichroic mirror unit 5 is equipped with a reflection mirror instead of the excitation dichroic mirror.
[0098]
In this embodiment, not only the angle difference of the newly added excitation dichroic mirror but also the displacement of the fluorescence image due to the effect of the incident angle difference to the newly added excitation dichroic mirror for near-infrared pulsed laser light. It can be easily corrected without the need for special adjustments.
[0099]
Fourth embodiment
The present embodiment is another transmitted light detection means applicable in place of the transmitted light detection means of the scanning laser microscope of the above-described embodiment, and its configuration is shown in FIG.
[0100]
As shown in FIG. 13, the transmitted light detection unit of the present embodiment includes three lenses 11 a, 11 b, 11 c and a field stop 31 instead of the lens 11. The field stop 31 is disposed at a position optically conjugate with the sample 10 in the optical system including the three lenses 11a, 11b, and 11c.
[0101]
Such lenses 11a, 11b, and 11c and the field stop 31 are optical systems that are necessarily configured in a commercially available optical illumination transmission illumination system of an optical microscope. The laser light transmitted through the sample 10 is imaged on the field stop 31 by the lens 11a and the lens 11b, and further detected by the transmitted light detector 12 through the lens 11c.
[0102]
In the embodiments so far, image processing is performed with a transmitted light image by light transmitted through the sample. In general, many cells used as a sample are colorless and transparent, and a slight difference in transmittance on the sample is a contrast on an image. Therefore, depending on the sample, this contrast may be low and accurate image processing may not be possible.
[0103]
In the present embodiment, such a problem is solved, and the field stop 31 is projected on the transmitted light image obtained by the transmitted light detector 12 by narrowing the field stop 31. In the image processing of the transmitted light image, the transmitted light image in which the field stop 31 is reflected is binarized with the inner range of the field stop 31 being bright and the outer range being dark, and the image center of gravity of the bright portion is obtained. Find the position. This difference in the position of the center of gravity of the image corresponds to the amount of displacement of the fluorescence image for each excitation dichroic mirror. Therefore, the position of the fluorescent image may be corrected according to the difference in the image gravity center position.
[0104]
In this embodiment, since the field stop that is always associated with the transmission illumination system of the optical microscope used in the scanning laser microscope is used for image processing, the image processing of the transmitted light image for each excitation dichroic mirror is simplified. Can be done accurately with the configuration.
[0105]
As described above, in each embodiment, the contents described above are applied to one image on one plane, but the present invention is not limited thereto. For example, an XYZ observation in which a plurality of slice images are acquired while moving the sample or objective lens in the direction of the optical axis, which is an observation method usually performed with a scanning laser microscope to observe the three-dimensional structure of the sample. In order to observe temporal changes of the sample over time, the present invention can be applied to XYT observation in which observation is performed at specified intervals. In this case, the transmission light image used for image processing may be acquired at least once for each excitation dichroic mirror.
[0106]
Although several embodiments have been specifically described so far with reference to the drawings, the present invention is not limited to the above-described embodiments, and all the embodiments performed without departing from the scope of the invention are not limited thereto. Including implementation.
[0107]
In the embodiment described above, the sample is stained with a fluorescent protein such as CFP or YFP. However, the present invention is not limited to this, and any fluorescent reagent capable of multiple staining can be applied.
[0108]
In the above-described embodiment, the present invention is applied to a scanning laser microscope. However, the present invention can be modified without departing from the gist of the invention. For example, the present invention can be applied to a fluorescence microscope that does not operate a laser. Is possible.
[0109]
【The invention's effect】
According to the present invention, a transmitted light image is acquired simultaneously with each fluorescent image, a positional shift amount between a plurality of fluorescent images is obtained based on a transmitted light image corresponding to the acquired fluorescent image, and based on the shift amount. A scanning laser microscope capable of acquiring an image in which a plurality of fluorescent dye images are superimposed without a positional deviation by correcting a positional deviation of the plurality of fluorescent images without requiring complicated adjustment or complicated control. An image acquisition method is provided.
[Brief description of the drawings]
FIG. 1 shows a configuration of a scanning laser microscope according to a first embodiment.
FIG. 2 shows transmittance characteristics of an excitation dichroic mirror for CFP fluorescence.
FIG. 3 shows transmittance characteristics of an excitation dichroic mirror for YFP fluorescence.
FIG. 4 shows fluorescence wavelength characteristics of CFP, fluorescence wavelength characteristics of YFP, and fluorescence wavelength characteristics of RFP.
FIG. 5 shows transmittance characteristics of a spectroscopic dichroic mirror for spectroscopically analyzing CFP fluorescence and YFP fluorescence.
FIG. 6 shows transmittance characteristics of a CFP band-pass filter, a YFP band-pass filter, and an RFP band-pass filter.
FIG. 7 shows transmittance characteristics of a double excitation type excitation dichroic mirror.
FIG. 8 shows two fluorescent images with 512 × 512 pixels and a display range suitable for them.
FIG. 9 shows a configuration of a second modification of the scanning laser microscope of the first embodiment.
FIG. 10 shows a configuration of a scanning laser microscope of the second embodiment.
FIG. 11 shows transmittance characteristics of an excitation dichroic mirror for fluorescence of RFP.
12 shows the configuration of another laser light emitting means that is a third embodiment applicable instead of the laser light emitting means of the scanning laser microscope of FIG. 1. FIG.
FIG. 13 shows a configuration of another transmitted light detection means that is a fourth embodiment applicable in place of the transmitted light detection means of the scanning laser microscope of the first embodiment and the second embodiment.
[Explanation of symbols]
1a Argon laser
2 Acousto-optic elements
5a, 5b Excited dichroic mirror
6 XY Galvano mirror
9 Objective lens
10 Double staining sample
11 Lens
12 Transmitted light detector
16a Spectral dichroic mirror
17a, 17b Band pass filter
18a, 18b fluorescence detector
19 Control device
20 Display means

Claims (8)

Laser light source means for oscillating laser light of a plurality of types of wavelengths, laser light selection means for selecting at least one type of laser light from the laser lights of the plurality of types of wavelengths, and a sample of the laser light An objective lens that condenses light, a scanning means that scans the laser light on the sample, and a characteristic that separates the laser light from the fluorescence generated from the sample by irradiating the sample with the laser light. A plurality of dichroic mirrors having different wavelength characteristics held in a switchable manner corresponding to the wavelength of the laser light to be emitted, fluorescence detection means for detecting fluorescence from the sample through an objective lens, and the laser transmitted through the sample In a scanning laser microscope equipped with transmitted light detection means for detecting light,
The laser light selected by the laser light selection means is switched, the plurality of dichroic mirrors are switched corresponding to the wavelength, and a fluorescence image is detected for each of the switched laser light and the dichroic mirror. At the same time, the transmitted light image obtained by the transmitted light that has passed through the sample is acquired by the transmitted light detection means, and the transmitted light image acquired for each laser beam is processed for each dichroic mirror. And a control means for superimposing and displaying at least two fluorescent images obtained by correcting the positional deviation of the fluorescent image acquired for each of the dichroic mirrors based on the deviation amount. A scanning laser microscope. The control means switches the laser light having a wavelength for exciting the fluorescent dyes to the multiple staining sample stained with a plurality of kinds of fluorescent dyes in synchronization with the scanning means by the laser light selection means. The dichroic mirror corresponding to the wavelength of the laser beam is switched in synchronism with the scanning means, and the respective dichroic images corresponding to each laser light and each dichroic mirror are acquired in a time division manner by the fluorescence detection means. Simultaneously with the acquisition of the fluorescence image for each mirror, the transmitted light image by the transmitted light that has passed through the sample is acquired by the transmitted light detection means, and the transmitted light image for each laser beam is subjected to image processing, whereby each dichroic mirror The amount of positional deviation of the fluorescent image is obtained, and the dichroic mirror is based on the amount of deviation. Scanning laser microscope according to claim 1, further comprising a control means for displaying the displacement of the fluorescent image correction to. The control means switches the detection sensitivity of the transmitted light detector to a level at which the output of the transmitted light detector is not saturated with respect to each of the switched laser beams in synchronization with switching of the laser light. The scanning laser microscope according to claim 2 3. The scanning laser microscope according to claim 1, wherein the control unit displays only pixels in a region that is commonly acquired in the fluorescence image for each laser beam in an overlapping manner. 3. The transmitted light detection unit has a field stop, and the control unit performs the image processing by performing image processing on a field stop reflected in a transmitted light image. A scanning laser microscope according to 1. Laser light source means for oscillating laser light of a plurality of types of wavelengths, laser light selection means for selecting laser light of at least one wavelength from the laser lights of the plurality of types of wavelengths, and collecting the laser light on a sample An objective lens that emits light, a scanning means that scans the sample with the laser beam, and a laser that has a property of separating the laser beam from the fluorescence generated from the sample by irradiating the sample with the laser beam. A plurality of dichroic mirrors having different wavelength characteristics held in a switchable manner corresponding to the wavelength; fluorescence detection means for detecting fluorescence from the sample through the objective lens; and detecting the laser light transmitted through the sample In an image acquisition method of a scanning laser microscope provided with transmitted light detecting means for
The laser light to be selected by the laser light selection means is switched, the plurality of dichroic mirrors are switched in accordance with the wavelength, and a fluorescence image is switched by the fluorescence detection means for each of the laser light and the dichroic mirror to be switched. At the same time, the transmitted light image obtained by the transmitted light that has passed through the sample is acquired by the transmitted light detection means, and the transmitted light image acquired for each laser beam is subjected to image processing, thereby being acquired for each dichroic mirror. An image acquisition method for a scanning laser microscope, comprising: calculating a positional deviation amount of the fluorescent image, correcting the positional deviation of the fluorescent image obtained for each dichroic mirror based on the deviation amount, and displaying the corrected fluorescent image. The image acquisition method of the scanning laser microscope synchronizes laser light of a wavelength for exciting each fluorescent dye with the scanning means by the laser light selection means for a multiple stained sample stained with a plurality of types of fluorescent dyes. The dichroic mirror corresponding to the wavelength of the switched laser beam is switched in synchronization with the scanning unit, and each fluorescence image corresponding to each laser beam and each dichroic mirror is time-divided by the fluorescence detection unit. At the time of acquisition, simultaneously with the acquisition of the fluorescence image for each of the dichroic mirrors, the transmitted light image by the transmitted light that has passed through the sample is acquired by the transmitted light detection means, and the transmitted light image for each laser beam is image-processed To obtain a positional deviation amount of the fluorescent image for each dichroic mirror, and based on the deviation amount The dichroic image acquisition method of scanning laser microscope according to claim 6 in which positional shift of the fluorescence image for each mirror correction to the and displaying. In the scanning laser microscope image acquisition method, the detection sensitivity of the transmitted light detector is synchronized with the switching of the laser light so that the output of the transmitted light detector is not saturated with respect to each of the switched laser beams. 8. The method of acquiring an image of a scanning laser microscope according to claim 7, wherein the switching is performed.

Images