Optical Sectioning & 3-D Reconstruction

In a conventional epifluorescence microscope emitted light from the sample collected by the objective and projected on tube lens by the camera. The fundamental problem of convectional microscopy is out of focus fluorescence that comes from other regions of the sample. That light will not focus onto the camera and thus show up as a blurry object superimposed in-focus light. This will reduce our ability to resolve the in-focus object in the camera.

Confocal microscopy, on the other hand, is a technique that simply removes the out-of-focus part which gives rise to blurry, out of focus light. As a result, we can get clear, in focus crisp details of the sample. How we can take those in-focus images and how to get rid of out-of-focus light? Placing a pinhole in the microscope is a simple trick to prevent out-of-focus light. Instead of a camera, we place a pinhole at the detection plane. Pinhole blocks out-of-focus light that comes from the focal point.

Optical sectioning and confocal microscopy are a family technique that allows us to produce 3-dimensional images of the biological sample. The idea behind optical sectioning is that to take image of slices at different focal planes, stack them up and generate the 3-dimensional structure of the object. For creating the image, we use a laser which collimated and can be focused to a tight spot on the sample. By scanning laser point by point over sample and recording intensity at each spot we can reconstruct an image from all these individual spots. By changing the entrance angle of illumination, we can illuminate a different spot on the sample. One scanning mirror is used to scan different spots on the sample as well as bringing emission light that comes back from the sample. Mirror change the angle at which laser hits the sample and accordingly change the position in the sample we are imaging.

However, optical sectioning and 3-D reconstruction using confocal microscopy has few drawbacks. First, it is very slow because it requires to scan the sample point by point to build up the image. So, it will take more than a second to acquire the image. Second, it is not also very sensitive and thus not the best option for dim samples. Considering these drawbacks laser scanning confocal microscope is not optimal for imaging live cells or fast processes. Using multiple pinholes and highly sensitive CCD, camera, a technique called spinning disk confocal microscopy can solve this problem. In this technique, we can acquire an image from the sample every few milliseconds. However, limited out of focus rejection is the main drawback of this technique. For the small amount of out-of-focus spinning disk exactly perform as the laser-scanning microscope.

For the thick sample, the out-of-focus from far away part of sample significantly contribute to background image were normally blocked by laser-scanning microscope. Tissue penetration depth is a major problem that prevents us from using a confocal microscope to image arbitrarily specimen. Both absorption and scattering of light, which are wavelength-dependent, will prevent light from being a tight focus where can detect the emitted light. Thus, imaging in the near-infrared minimize absorption and also reduce the scattering. One possibility to image in the infrared is using infrared dyes. The other approach is to use two-photon microscopy and image standard dyes in the infrared.

Two photons microscopy is another family of optical sectioning. In this technique, two photons are of half energy to excite dye at almost the same time. By adding up those two half-energy photons we can achieve full excitation. This process depends on the square of the intensity of the light hitting sample. It means that out-of-focus light is much dimmer than in-focus light is not going to excite any species to an appreciable level. In other words, only the excited spot is at the focal spot of confocal. Two photon microscope does not require pinhole because we only get light from in-focus spot there is no out-of-focus light. Due to efficient light collection, this technique works well for thicker samples such as a piece of tissue.

Here we provide a general guideline for choosing the right microscopy technique based on the type sample and its thickness.