What is a confocal fluorescence microscope?

Confocal fluorescence microscopy is an optical imaging technique commonly used in biology, combining fluorescence imaging with confocal microscopy for greater optical resolution. This article will discuss the principles of fluorescence and confocal microscopes, and will describe the stages of fluorophore selection and sample preparation.

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What is fluorescence?

Fluorescence is the process by which a photon is absorbed, and another with a slightly lower energy, and therefore a longer wavelength, is subsequently emitted. Under normal conditions, the electrons of a fluorophore (fluorescence-capable molecule) are in a low-energy earth state, which can be stimulated to a higher energy level when stimulated by interaction with an incident photon .

Some energy is thought to be consumed by non-radiation decomposition processes, and the difference in energy between the event and a released photon is called the Stokes motion. As the system rests quietly the electron returns to the state of the earth, releasing the remaining difference between electron energy levels as a photon. The decay rate usually follows first-order kinetics, with recruited fluorophores typically dispersing within nanoseconds.

Many naturally occurring compounds are fluorescent, and a large library of fluorophores with a special engine is constantly evolving. Organic fluorophores often consist of several double bonds and polyaromatic structures that transmit delocalized electrons throughout the molecule, which are then capable of excitation.

In manual practice, fluorophores are selected by the required excitation and scattering waves, the intensity of the backlight (quantum output), and the fluorescence time. Another consideration that may encourage the use of a particular fluorophore over another involves molecular weight, as in some applications, the presence of other fluorophores may significantly inhibit larger molecules. may give translucent marks, and the specificity of the fluorescent label.

Molecular “lock and key” devices can be used to ensure that fluorophores attach to structures of interest, perhaps simply activating or deactivating when in place. Antibodies form very specific bands with their target structure and are often attached to fluorophores to monitor the number and distribution of the target in situ. Recently, peptide sequences and nucleic acid have been used in the same way in the identification of cell components.

What is confocal microscopy?

In a confocal microscope a laser beam is focused to a certain depth within a sample, and like conventional light microscopes, any light that is reflected or emitted is detected by a properly positioned microscope.

Unwanted visual sound from the sample, as well as light from the spot in focus with the laser, are omitted from the final image by the use of a hole opening opening which prevents scattered light from higher angles passing through. . The pine hole is placed on the same plane as the sample to ensure that only photons traveling in a straight line from the sample to the microscope are detected. This is said to be in a conjugate focal plane, hence the “confocal” portmanteau.

The captured image is generally in the range of hundreds of nanometers, so large samples are scanned to allow a larger image to be stitched together later. Adjusting the focus area of ​​the laser and the microscope on each of the three shots will allow scientists to take a three-dimensional image of the interesting sample.

Depending on the visibility of the sample, and the wavelength of interesting light, samples can be simulated to a depth of a few hundred micrometers. Scanning maintenance over time provides time-lapse images that allow scientists to track the dynamics of molecules or tagged structures over time.

The laser used in a confocal microscope, in addition to illuminating a small section of the sample, can be used instead of stimulating fluorophores, significantly improving detection sensitivity and signal-to-to-aspect ratio. -sound compared to visible light from the sample only.

In addition, significantly lower laser light intensities can be used to obtain sufficient signal, reducing the risks to the sample associated with photon explosion. Other subtypes of confocal microscope are available, according to the application, which limit exposure to photons and improve image collection time by using a slit or spinning disk instead of a pine hole.

However, the availability and diversity of laser types included with laser scanning with confocal microscopy make this the most popular option.

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How are samples prepared for a confocal fluorescence microscope?

As discussed, fluorophores are selected for compatibility with the sample under study and favorable spectrum properties. The high sensitivity of fluorescent probes means that only a relatively small number need to be present in the sample to achieve a sufficient signal.

Too much or too little population with fluorophores can be unbalanced, generating an acoustic signal or incomplete structural illumination, so care must be taken in selecting an appropriate density.

Eventually the activation of fluorophores triggers a phenomenon known as photobleaching, leaving the molecule unable to fluoresce. In time-sensitive studies, this must be taken into account, as the number of bots released decreases after several times of excitation.

Samples are often repaired before a microscope to carefully preserve the structural features of the sample. Formaldehyde has historically been used for this purpose, rapidly penetrating cell organs and creating disulfide bridges between cysteine ​​residues in proteins, preserving even detail structural antigen sites.

In cases where proteins do not need to be preserved, when studying the presence of small molecules for example, a low-temperature alcoholic fixation can be used instead. To ensure that the settling agent is fully absorbed, the cells are exposed to gentle cleansing agents, increasing the permeability of the cell membrane.

Antibodies are the most common target agent in fluorescent labels because of their flexibility and specificity. Specificity can be further enhanced by a process called inhibition, flooding the sample with a protein cocktail that overlaps non-specific binding sites and extracting the protein-crosslinking potential of any formaldehyde that is leave.

After each of these steps, the primary or secondary antibody-bound fluorophore can be added. In some cases, additional counter stains may be introduced to reduce posterior fluorescence.

Fixation, by definition, is inappropriate in dynamic microscopy applications that are expected to observe cell function or tension over time. Living cell imaging raises a number of issues regarding the maintenance of proper cell position within the entire microscopy chamber, including temperature, atmospheric composition of CO₂ and humidity, and pH and cell culture medium content.

Small variations in temperature can cause problems with alignment of the laser and the microscope due to changes in the refraction index of the material, and constant evacuation from the cell culture flask exacerbates this problem. .

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