Why do I need to use a confocal microscope?
1. The optical microscope has been perfected through the efforts and improvements of our great predecessors. In fact, the usual microscope can provide us with beautiful microscopic images easily and quickly. However, the event that revolutionised the world of nearly perfect microscopes was the invention of the "laser scanning confocal microscope". This new type of microscope is characterised by an optical system that extracts image information only from the surface on which the focus is concentrated, and by changing the focus while recovering the acquired information in the image memory, it is possible to obtain a sharp image with full 3-dimensional information. In this way, it is possible to easily obtain information about the shape of the surface that cannot be confirmed with a conventional microscope. In addition, while "increasing resolution" and "deepening depth of focus" are conflicting conditions for conventional optical microscopes, especially at high magnification, this problem is solved with confocal microscopes.
2. Advantages of Confocal Optical System
Schematic diagram of laser confocal microscope
The confocal optical system is a point illumination of the sample, while the reflected light is also received using a point receptor. When the sample is placed at the focal point, almost all of the reflected light reaches the photoreceptor, and when the sample is out of focus, the reflected light cannot reach the photoreceptor. In other words, in a confocal optical system, only the image that coincides with the focal point is output, and spots and useless scattered light are blocked out.
3. Why use a laser?
In a confocal optical system, the sample is illuminated at a point and the reflected light is received by a point sensor. Therefore, a point light source is necessary. Lasers are very much a point light source. In most cases, the light source for confocal microscopes is a laser light source. In addition, the monochromaticity, directionality, and excellent beam shape of lasers are important reasons for their widespread adoption.
4. Real-time observation based on high-speed scanning is possible.
For laser scanning, an acoustic optical deflection unit (Acoustic Optical Deflector, AO prime) is used in the horizontal direction, and a servo-electronic controlled beam scanning mirror (Servo Galvano-mirror) is used in the vertical direction. Since there is no mechanical vibration in the AO Deflector, high-speed scanning is possible, and real-time observation on the monitor screen is possible. The high speed of this camera is a very important item that directly affects the speed of focus and position retrieval.
5. Relationship between Focus Position and Brightness
In a confocal optical system, the sample is placed correctly in the focal position when the brightness is zui big, in front of and behind it, its brightness will be sharply reduced (Figure 4 solid line). This sensitive selectivity of the focal plane is the principle behind the height orientation of the confocal microscope and the depth of focus expansion. In contrast, the usual optical microscopes do not show any significant change in brightness before and after the focal position (dotted line in Fig. 4).
6. High Contrast, High Resolution
In a conventional optical microscope, the reflected light from the out-of-focus portion of the microscope interferes with and overlaps with the focal imaging portion of the microscope, resulting in a decrease in image contrast. In contrast, in a confocal optical system, scattered light outside the focal point and inside the objective lens is almost completely removed, resulting in images with very high contrast. In addition, the resolving power of the microscope is improved because the light passes through the objective lens twice, sharpening the point image.
7. Optical localisation function
In the confocal optical system, the reflected light is shielded by the micro-aperture at the point other than the focal point. As a result, when observing a three-dimensional sample, the image is formed as if the sample had been sliced with the focal point (Fig. 5). This effect is called optical localisation and is one of the features of confocal optical systems.
