It is similar to wide-field fluorescence microscopy in that fluorescence optics are used. Unlike traditional fluorescence microscopes, however, confocal microscopy focuses a laser light source onto a defined spot at a specific depth within the sample. This creates the emission of fluorescent light at the exact point defined and, using a pinhole aperture inside the optical pathway, out-of-focus light is removed from the field of view. This leads to much clearer images being produced.
The term Confocal is derived from the conjunction of two focal planes: the objective lens focus point and the focus point where the aperture is placed.
Confocal imaging increases resolution and contrast through the use of the pinhole spatial filters. One pinhole is placed in a conjugate plane with a scanning point on the specimen, and a second pinhole is positioned in front of the detector (e.g., photomultiplier tube).
This effectively removes out-of-focus light and provides a crisper image with the maximal resolution possible for the objective lens being used.
Modern confocal microscopes offer high-power, high-resolution, and multicolor capabilities. They are commonly used to collect high-quality multispectral images of biological and non-biological samples across the visible range. Their maximum magnification level, in general, is 1000x when combining a 10x ocular with a 100x objective lens.
In addition, specialized confocal microscopes can image living cells, and work at very high speeds to perform live-cell imaging without causing severe photo damage, photobleaching, or phototoxicity. Confocal microscopes are even used to assist with in vivo preparations.
Furthermore, modern units are often designed as completely integrated confocal systems. In these systems, an optical microscope works together with a detector or multiple detectors, a computer, multiple laser systems, a wavelength selection device, and beam scanning assembly to produce images and obtain valuable information about a sample. These integrated systems are typically referred to as digital or video imaging systems.
Basic confocal microscopes and more robust systems are used extensively in the life sciences (e.g., biology and cell biology), materials science, and semiconductor inspection. When acquiring one, it’s essential to understand your needs, as this will inform whether or not you’ll want to lease a fully integrated system or something a little less complex.
Confocal microscopes offers several advantages over conventional optical microscopes (e.g. light microscopes) including shallow depth of field, elimination of out-of-focus glare, and the ability to collect serial optical sections from thick specimens. Optical sectioning provides important structural information regarding both biological or non-biological samples.
Multiple subtypes of confocal microscopes have been developed.
Despite their differences, each subtype relies on fluorescence, or the generation of photons using controlled light sources and fluorophores, to produce an image. We’ll cover some of the subtypes below.
Imaging of a sample’s surface provides a lot of information about that object but does not provide a detailed picture of the entire object.
Confocal laser scanning microscopes (CLSM), also known as laser scanning confocal microscopes (LSCM), are able to provide 3D reconstructions of a sample. CLSM adds optical sectioning to standard or wide-field confocal microscopy by allowing for depth selectivity.
This is done by passing the laser beam through a light source aperture that allows for depth selectivity. Multiple depths of the object can then be imaged and each depth’s images can be stitched together to achieve a true 3D rendering of the specimen. This imaging technique is also known as optical sectioning.
Due to the multiple layers that are needed to be imaged in CLSM, it is required that the sample remains still, which is relatively time-consuming. Spinning disk laser confocal laser microscopy (SDCLM) is able to mitigate these shortcomings by using multiple pinholes to project multiple parallel excitation light beams.
These pinholes are arranged on a rotating Nipkow disk that is located within the microscope plane. Due to the spinning of the disk and the multiple pinholes used, images can be captured at a rate of thousands per second.
One disadvantage to this, however, is that due to the holes being larger, the images obtained cannot block out as much background fluorescent light and the result is not as detailed.
PAM utilizes a similar methodology to SDCLM but is able to have programmable pinholes. It uses a digital micromirror device that is used as a spatial light modulator.
This allows that user to tune their microscope light source to match the specific experimental parameters that they are looking at. The microscope can be changed quickly if the experimental conditions require it.
The major advantages that PAM has over other confocal microscopy techniques are that it is controlled by software, has no moving parts, and offers complete flexibility in its lighting and detection methods.
Serotonin, one of the better-known neurochemicals, contributes to wellbeing and happiness, however, it may also be vital to understanding how our brains function in more extreme conditions.
Recent researchers found that serotonin levels may vary depending on the specific emergency situation that an animal is in. For example, if a mouse is in an exposed field and a hawk is diving down to eat it, the fight-or-flight response kicks in and tells the mouse to run.
If however, that same mouse sees the hawk first and the hawk is unaware of the mouse, the more appropriate response is to freeze. Researchers have found that serotonin neurons trigger escape mechanisms in events of high danger. In lower-danger environments, these same neurons trigger a pausing reaction. If true, this discovery could completely change how we view our brains during these emergency situations.
Observing these types of reactions is not easy and requires highly accurate and powerful microscopes. Confocal laser scanning microscopes aid researchers in imaging and understanding how these neurons are triggered and work.
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