Fluorescence Microscopy/Imaging Flow Cytometry Organic Dyes Quantum Dots Fluorogens
A fluorescence microscope is a light microscope used to study properties of organic or inorganic substances using the phenomena of fluorescence instead of, or in addition to, transmitted light. In most cases, a component of interest within the sample is labeled with a fluorescent molecule called a fluorophore (examples include fluorescent proteins (such as GFP and RFP), or organic dyes (such as fluorescein, Alex Fluor 488 and Cy3). The sample is then illuminated with light of a specific wavelength (or wavelengths) which is absorbed by the fluorophores, causing them to emit longer wavelengths of light (of a different color than the absorbed light). The light used to illuminate the sample is separated from the much weaker emitted fluorescence through the use of an emission filter. Typical components of a fluorescence microscope are the light source (xenon arc lamp or mercury-vapor lamp), the excitation filter, the dichroic mirror (or dichromatic beamsplitter), and the emission filter (as illustrated in Figure 1). The filters and the dichroic are chosen to match the spectral excitation and emission characteristics of the fluorophore used to label the sample. In this manner, one single fluorophore (color) is imaged at a given time. Multi-color images of several fluorophores must be composed by combining several single-color images.
The majority of fluorescence microscopes are in use as epifluorescence microscopes (i.e. excitation and observation of the fluorescence are from above (epi) the specimen). These microscopes have become an important tool in the field of biology, opening the doors for more advanced microscope designs, such as the confocal laser scanning microscope and the total internal reflection fluorescence microscope (TIRF).
Confocal laser scanning microscopy (CLSM or LSCM) is a technique for obtaining high-resolution optical images. The key feature of a confocal microscope is its ability to produce in-focus images of thick specimens via a process known as optical sectioning. Images are acquired point-by-point and reconstructed with a computer, allowing three-dimensional representations of topologically-complex objects. The principle of confocal microscopy was originally patented by Marvin Minsky in 1957, but it took another thirty years and the development of laser technology for CLSM to become a widely used technique.
In a confocal laser scanning microscope (as illustrated in Figure 2), a laser beam passes through a light source aperture and is focused by an objective lens into a small (ideally diffraction limited) focal volume within a fluorescent specimen. A mixture of emitted fluorescent light as well as reflected laser light from the illuminated spot is then collected by the objective lens. A dichoric beam splitter separates this mixture of wavelengths by allowing only the laser light to pass through and reflecting the fluorescent light into the detection apparatus. After passing a pinhole, the fluorescent light is detected by a photodetection device (a photomultiplier tube (PMT) or avalanche photodiode), transforming the light signal into an electrical one that is recorded by a computer.
The detector aperture servers to reject the light that is not originating from the focal point, as shown by the dotted gray line in the image. This out-of-focus light is suppressedby the pinhole resulting in far sharper images than those from conventional fluorescence microscopes, and also allows one to obtain images of various z axis planes (also known as z stacks) of the sample.
The detected light originating from an illuminated volume element within the specimen represents one pixel in the resulting image. As the laser is sscanned over the z plane of interest, a whole image is obtained pixel-by-pixel and line-by-line, where the brightness of a resulting image pixel corresponds to the relative intensity of detected fluorescent light. The beam is scanned across the sample in the horizontal plane by using one or more (servo controlled) oscillating mirrors. This scanning method usually has a low reaction latency and the scan speed can be varied. Slower scans provide a better signal-to-noise ratio, resulting in better contrast and higher resolution. Information can be collected from different focal planes by raising or lowering the microscope stage. The computer can generate a three-dimensional picture of a specimen by assembling a stack of these two-dimensional images from successive focal planes.
