If you are a bioengineer, you probably use microscopes a lot. But do you know the difference between a confocal microscope and a wide-field microscope? These are two common types of optical microscopes that can reveal different aspects of biological samples. In this article, you will learn what distinguishes them and when to use them.
Optical microscopes use visible light and lenses to magnify images of small objects. They are widely used in bioengineering to observe cells, tissues, organs, and biomaterials. However, they have some limitations, such as low resolution, low contrast, and out-of-focus blur. To overcome these challenges, different techniques have been developed to enhance the quality and specificity of optical microscopy.
Optical microscopes perform three functions. They magnify objects using lenses, they resolve objects, and they can be used to add contrast to otherwise low-contrast biological specimens. Contrast is added most effectively using fluorescence, which captures labeled structures as white on a black background. Other contrasting methods include histological dyes, or transmitted light contrast enhancement methods such as phase contrast, differential interference contrast, or darkfield.
Wide-field microscopy is the simplest and most common technique of optical microscopy. It involves illuminating the whole sample with a broad beam of light and capturing the image with a camera or an eyepiece. Wide-field microscopy is easy to set up and operate, and it can produce high-resolution images of thin samples. However, it also has some drawbacks, such as low signal-to-noise ratio, low depth discrimination, and high background fluorescence.
Confocal microscopy is a more advanced technique of optical microscopy that uses a pinhole to block out-of-focus light and create a sharp image of a thin slice of the sample. It also uses a scanning laser to excite the fluorescent molecules in the sample and a detector to collect the emitted light. Confocal microscopy can produce high-contrast, three-dimensional images of thick samples, and it can also isolate specific wavelengths of fluorescence.
Confocal microscopy has several advantages over wide-field microscopy, especially for bioengineering applications. First, it can eliminate the out-of-focus blur and improve the spatial resolution and clarity of the images. Second, it can reduce the photobleaching and photodamage of the sample by using a lower intensity of light. Third, it can enable the visualization of different layers and structures within the sample by adjusting the focal plane and the pinhole size. Fourth, it can allow the detection and quantification of specific molecules and interactions by using multiple fluorescence channels and filters.
Confocal microscopy also has some disadvantages compared to wide-field microscopy, which should be considered before choosing the best technique for your project. First, it is more expensive and complex to set up and maintain, and it requires more training and expertise to operate. Second, it is slower and less sensitive than wide-field microscopy, because it scans the sample point by point and collects less light. Third, it can introduce some artifacts and distortions in the images, such as speckle noise, pinhole crosstalk, and scan line jitter.
As you can see, confocal microscopy and wide-field microscopy have different strengths and weaknesses, and they can provide different information about your sample. Therefore, you should choose the right microscope depending on your research question, your sample type, your imaging parameters, and your budget. For example, if you want to image a thin sample with high resolution and low background, you might prefer wide-field microscopy. But if you want to image a thick sample with high contrast and depth discrimination, you might opt for confocal microscopy.