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What is Fluorescence Microscopy and 3D Confocal Imaging in Life Sciences Vision?

What is Fluorescence Microscopy and 3D Confocal Imaging in Life Sciences Vision?Fluorescence microscopy is widely-used in life sciences to detect particles below the resolution of a light microscope. By the use of staining and 3D imaging software, researchers are able to visualize substances that can’t be seen with conventional microscopy.

How Fluorescence Microscopy Works

Fluorescence microscopes work much like conventional light microscopes. But unlike a  conventional microscope that only uses visible light, fluorescence microscopes label samples with a fluorescent substance called fluorophore.  The sample is then illuminated through the lens with a higher energy source.

The light is absorbed by the fluorophores in the sample and they emit a longer, lower energy wavelength light. This light can be filtered out from the surrounding radiation so the viewer only sees the fluorescence.

The main task of the microscope is to let the excitation light radiate the specimen and sort out the weaker emitted light. The microscope has a filter that only lets through radiation with the wavelength that matches the fluorescing material, resulting in a clear image that wouldn’t typically be seen with a conventional microscope.

Why Fluorescence Microscopy Is Used

Fluorescence microscopy helps to image the specific features of tiny specimens. It can also enhance 3D features at small scales. Attaching fluorescent tags to antibodies that adhere to the targeted features or staining the sample are two methods used to produce an image. Researchers can then more easily visualize organelles or unique surface features.

These microscopes are often used to:

  • Image structural components of small specimens like cells
  • Conduct viability studies on cell populations
  • Image genetic material within a cell (DNA/RNA)
  • View specific cells within a larger population

Just as important to the gathering of this data is the time spent processing and understanding the results. Post-processing software is used to perform image and data analysis. Fluorescence microscopes usually come with a bundled software platform for imaging and image processing.

3D Confocal Imaging Improves Fluorescence Microscopy

Confocal imaging was developed to overcome limitations in traditional wide-field fluorescence microscopy. In a traditional fluorescence microscope, the entire specimen is flooded with light evenly. All parts of the sample are excited at the same time, including the background.

Confocal microscopy is used to highlight the 3D aspects of a sample. Powerful light sources, such as lasers, are used to focus on a pinpoint. This focusing is done repeatedly throughout one level after another. Then, image reconstruction software assembles the multi-level image data to form a 3D reconstruction of the sample.

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