Confocal Microscopy
A Confocal microscope is one type of 3D Fluorescence Microscope in which resolution is increased by rejecting out-of-focus light. This is done by using pinholes with a specific Pinhole Radius and makes it to be an intrinsic 3D microscope: due to the focusing nature of the confocal microscope, it is specially suitable for obtaining 3D images as a collection of intensities at different points of the space.
Confocal images are very well suited for Huygens deconvolution, with the Huygens Confocal Optical Option, because of two main reasons:
1. they still suffer from blurring, which can be clearly seen when recording bead images
2. in general, confocal images are noisier than widefield images, because light from the object is rejected by the pinhole.
In confocal microscopes, the Nyquist Rate is about one half of that in Widefield Microscopes.
How a confocal microscope works
Biological systems are typically three dimensional structures. When the fluorophore labeled sample is flooded with excitation laser light like in conventional widefield microscopy, the resulting image is usually highly disturbed by out-of-focus fluorescent light. This leads to blurring of the image and results in a significant decrease in contrast. Confocal microscopy is a technique to reduce this unwanted out of focus light. In this technique, the excitation light is tightly focused in the sample. The emission light from the focus is collected by the same objective, after which it is focused through a small pinhole and directed towards a photodetector (Figure 1 (a)). The out of focus emission light will be largely blocked by the pinhole (Figure 1 (b)). The result of this method is that only the light from the focal volume is able to reach the detector. The drawback is that also light from other locations in the focal plane is blocked: confocal microscopy is point-wise excitation and point-wise detection. One solution is to raster scan the sample point by point to reconstruct a complete image in which only fluorescent signal from the focal plane is collected. This raster scanning can be repeated for different focal planes to section thick samples and reconstruct a 3D image of the sample.Alternatively, it is also possible to scan the sample with multiple excitation spots in parallel such as with a Spinning Disk microscope.
STED is practically an extension of point-scanning confocal microscopy. The STED beam needs to be focused in the sample to obtain the high intensity needed for stimulated emission, while point-wise detection reduces the out-of-focus light. To make STED work, the excitation focus center and STED intensity null of the doughnut shaped focus need to overlap perfectly. An image can then be obtained by raster scanning the sample, for example with a piezo scanning stage. The size of the pinhole used will determine the depth of focus of the microscope.
Figure 1. Illustration of the confocal microscope principle. A collimated beam of excitation light (blue) is tightly focused in the sample by a microscope objective. The left image shows that the emission light resulting from the focal spot will be focused through the pinhole and most light will reach the detector. The right hand side shows the case where the emitted light arises from a point that is located outside the focal volume. This light will not be focused through the pinhole and only a very small amount of light is able to reach the detector. As a result, the out of focus fluorescent light is mainly blocked by the pinhole and therefore the background signal is significantly reduced.[1]
Deconvolving confocal images
Applying deconvolution on confocal images increases their resolution in x, y and z and restores from noise. Therefore, the images will be easier to visualize and analyze. The default algorithm for Confocal is Classic Maximum Likelihood Estimation (CMLE) and the default SNR value is 20. We suggest to start with the default parameters and explore other SNR values, for example, to optimize the deconvolution results. You should not think about the SNR as a parameter describing your original image, but as a tunable parameter that controls the deconvolved result. Using a too large SNR value might be risky when restoring noisy originals, because you could be just enhancing the noise. A noisy confocal image can have SNR values lower than 20. Another option is testing the Good's roughness Maximum Likelihood Estimation (GMLE) algorithm to improve the results. The GMLE is suited for noisy images, as confocal or STED.Test Huygens for free on your Confocal images
You are welcome to download Huygens via our Download page and request a test license with full options.[1] Image with permission taken from Remko R.M. Dijkstra, Design and realization of a CW-STED super-resolution microscope setup, Master Thesis , University of Twente, 2012