Simply put, confocal microscopy is a microscopy technique that acquires images with out-of-focus light optically removed from the resulting image at the focal plane. The resulting images have less blur, higher contrast, and better resolution, which is the primary purpose of confocal uses. Since each image shows fluorescent samples in a specific focal plane, it is possible to construct three-dimensional images, by acquiring multiple images and sequentially changing z-axis focal plane positions. Such images can be used to accurately determine the three-dimensional positioning of the sample.
Figure 1: Comparison images between confocal microscopy and standard widefield microscopy.
Figure 2: Example of a 3D image acquired with a confocal microscope.
From this point forward, the principles of confocal microscopy are explained in an easy-to-understand manner, showing how to remove light from outside the focal plane.
In order to measure fluorescence, it is necessary to emit an excitation light to the samples. In this section, we will describe the principle of confocal microscopy by following the path of the excitation light including where it generates fluorescence.
In confocal microscopy, a laser beam, is used primarily as the excitation light source. Since the laser beam is a point light source with a good straight-line performance, its focus is on the focal plane through the objective lens forming a light spot.*1 In contrast to conventional widefield fluorescence microscopy, which excites the entire sample area at a time, confocal microscopy excites only one single point on the focal plane at a time.
*1: The combintion of objective lens magnification, NA, and excitation wavelength, determine the smallest spot size that can be focused. The higher the NA and the shorter the wavelength, the smaller the spot size and the higher the resolution.
Figure 3: Conceptual diagram of the laser beam focus onto the focal plane.
Observing the light path travel through the air is challenging. For this reason, we placed a block of fluorescent material above the objective lens. As the excitation light from the objective lens passes through the block, the fluorescent material in the light path emits fluorescence in all directions. By observing the fluorescence, the path of the excitation light is traced as shown in Figure 4. The beam forms a spot at the focal plane, and on the other, when using conventional widefield microscopy, the excitation light remains spread out over the whole sample.
Figure 4: Comparison image between laser excitation light and standard widefield microscope excitation light.
As mentioned above, the laser beam is focused to a point on the focal plane forming a spot; however, in the z-direction, the laser beam is converged and diverged by the objective lens with a width corresponding to this process. As a matter of course, the laser light travels up and down the focal plane and when there is a fluorescent material, it emits fluorescence. In Figure 5, we can see that fluorescence is emitted above and below the focal plane in the fluorescent block.
This out-of-focus fluorescence also occurs in real samples and is the cause of blurred images. In confocal microscopy, the fluorescence emitted outside the focal plane is blocked from reaching the detector. This makes it possible to produce images using only the light from the focal plane.
Figure 5: This figure shows how the laser beam converges and then diverges from the focal plane.
How does confocal microscopy eliminate fluorescence outside the focal plane?
The principle of confocal microscopy may be explained by tracing the light path from the fluorescence generated at, above and below the focal plane directly to the detector.
Figure 6: Conceptual diagram of the detection of fluorescence emitted from the focal plane.
The fluorescence generated at the objective focal plane passes from the objective lens through the imaging lens, and forms a focused image at the detector. A pinhole is placed at this position of the imaging system.
In this confocal optical scenario, the following three objects are usually placed at the conjugate points:
A. The light source (more precisely, the point source of light)
B. The focal plane of the objective lens
C. The pinhole in front of the detector
If points A, B and C are in a conjugate positional relationship, light emitted from any one of them will be focused on the other two points. When light emits from point A, it will focus on both point B and C. When light emits from point C, it will focus on both point A and B, and vice versa.
Therefore, in confocal optics, how does fluorescence emitted from the out of focus light source travel in the light path?
Figure 7: Conceptual diagram of the elimination technique of fluorescence emitted outside the focal plane.
Light emitted from the light source is focused at the objective lens focal plane and at the pinhole in front of the detector.
The newly generated fluorescence light at the focal plane of the objective lens is then focused at the pinhole in front of the detector. Excitation light emitted from the light source is rejected (blocked) by a bandpass filter, therefore it is not detected in a practical confocal system (in the blue circle on the left of Figure 7).
As shown in Figure 7, fluorescence emitted above the focal plane is focused above the pinhole and is largely blocked from reaching the detector because the light spreads out at the pinhole.
Secondly, fluorescence emitted at the bottom of the focal plane, as shown in the blue circle, is focused at the bottom of the pinhole and is therefore largely blocked from reaching the detector.*2
In this manner, only the fluorescence emitted from the focal plane can be directed to the detector.
*2: As the light spot size on the sample varies with the objective lens, so does the fluorescence spot size at the pinhole position. In confocal microscopy, the pinhole size is usually adjusted to match the objective lens. In our confocal unit MAICO, three different pinhole sizes (S, M and L) can be selected according to the objective lens.
Excitation light will produce fluorescence anywhere in the light path if there is a fluorescent material present. However, by selecting only the fluorescence emitted from conjugate positions in the pinhole, it is possible to obtain an image originating only from the focal point. This is called optical slice image. The main advantage of confocal microscopy is the ability to obtain this optical slice from a thick sample.
The excitation light spot is fixed at a single point in the description so far, but in practice, the light spot is moved in a two-dimensional (x, y) direction with an optical scanning device to produce a single confocal fluorescence image. In our confocal unit MAICO, a MEMS mirror is used for this optical scanning.*3
Figure 8 shows the MEMS mirror changing its angle in the optical path and the associated lateral movement of the excitation spot on the sample. Figure 9 shows a schematic diagram of the successive optical scans of the sample plane.
*3 For more information on MEMS mirrors, please see the Features.
The scanning of the excitation spot has another advantage compared to widefield excitation. Adjacent fluorescent molecules are not excited, and are less likely to produce stray fluorescence.
Figure 8*: Conceptual diagram of the movement of the MEMS mirror and the excitation light spot movement.
* This is different from the actual image acquisition operation.
Figure 9: Conceptual diagram of planar scanning of the focal plane by the excitation light spot
PMTs (photomultiplier tubes), which are photodetectors, output an electrical signal over time according to the amount of fluorescence incident on the light-receiving surface but do not have positional information.
On the other hand, an optical scanning device scans a fixed position in the XY direction for a fixed time.
The signal output by the PMT is reconstructed from the location of the signal at any given time. In other words, the temporal information of the signal intensity is transformed into spatial information, and the image is generated by assigning brightness information to the XY position of the image.
Figure 10: Example of conversion from PMT signal output over time to 2D image.
As mentioned in the introduction, the optical section image obtained by confocal measurement reflects the specimen at a particular focal plane, so it is often possible to construct a three-dimensional image.
Figure 11*: The focused laser spot moves with the vertical movement of the objective lens and the acquired image changes accordingly.
Figure 11 shows that by moving the objective lens vertically, the focal position on the sample also moves vertically. As a result, the confocal image position also changes as the focal point moves vertically. The three-dimensional image in Figure 2 can be created by three-dimensionally reconstructing the individual confocal images derived from each height position acquired on the right side of Figure 11.
*In the image on the left of Figure 11, the light spot is not scanned, but in the actual measurement, it is scanned in the XY-plane by the scanning device. The focal point is moved up and down in successive steps.
MAICO®'s superior features, including its subunit structure, are introduced here.
MAICO® enables imaging with reduced bleed-through between wavelengths, which is an issue in multi-wavelength simultaneous observation. We will introduce how we have achieved a reduction of bleed-through.
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