Observing through Confocal Laser Scanning Microscopy

Problem Background

         Microscopy is term to examine or observe the small thing such as biological tissues, microbacteries, or other particulate matter, that can be seen using naked-eye. The main goal of microscopy is to create image as well as the real object so that resolution of image becomes main consideration. In conventional microscopy, it is still less resolution as we can see 3D-like image. Some of causes are confocal aperture, residual optical aberration and pupil illumination (Figure 1). So, it becomes problem how to filter the ray from object so that on from focal point of objective lens can be process.

Figure 1 Consideration in Microscopy

Reconsideration of Light Characteristics

          Light can be focused. But, not even an optical system of diffraction-limited design can image a truly point-like object as a point. The image of an ideal point object will always be somewhat blurred, or “spread” corresponding to the imaging properties of the optical system. The image of a point can be described in quantitative terms by the point spread function (PSF), which maps the intensity distribution in the image space.

         In order to achieve simple formulae for the range of smallest pinhole diameters, it is practical to regard the limit of PH = 0 at first, even though it is of no practical use. In this case, PSF_det and PSF_ill are identical.

The total PSF can be written as

 

         In fluorescence applications it is furthermore necessary to consider both the excitation wavelength l_exc and the emission wavelength l_em. This is done by specifying a mean wavelength1:

         The diameter of the corresponding half intensity area and thus the optical slice thickness:

axial:

 

lateral:

 

         With: FWHM is Full width at half maximum of an intensity distribution (e.g. optical slice), NA is Numerical aperture of a microscope objective, PH is Pinhole; diaphragm of variable size arranged in the beam path to achieve optical sections, PSF is Point Spread Function, and n is Refractive index of an immersion liquid.

Principle of Confocal Microscopy

         A laser beam is focused into a small point onto a fluorescent specimen. Both reflected light and fluorescent light are detected by a photomultiplier. Reflected light is deflected by a dichroic mirror, and only fluorescent light emitted from the specimen passes through the photomultiplier. Out of focus information is reduced by the placement of a confocal pinhole placed in front of the photomultiplier, which allows only light from the focal plane of the laser beam to pass through. CLSM uses gas lasers, primarily Argon and Krypton, creating a steady stream of photons. The process is destructive, exciting electrons and thereby creating fluorescence both above and below the focal plane, which bleaches the specimen. This limits the time in which images can be collected before significant blurring is inevitable, and also may be toxic to living specimens.

Figure 2 Schematic Diagram of CLSM

(Photonics System Application)

Glossary about Microscope:

顕微鏡（けんびきょう）とは、光学的もしくは電子的な技術を用いることによって、微小な物体を視覚的に拡大し、肉眼で見える大きさにする装置である。単に顕微鏡というと、光学顕微鏡を指すことが多い。

光学顕微鏡は眼鏡屋のヤンセン父子によって発明された。その後、顕微鏡は科学の様々な分野でこれまで多大な貢献をしてきた。その中で様々な改良を受け、また新たな形式のものも作られ、現在も随所に使用されている。顕微鏡を使用する技術のことを顕微鏡法、検鏡法という。また、試料を顕微鏡で観察できる状態にしたものをプレパラートという。

[www.wikipedia.org]