Confocal microscopy is also known as confocal laser scanning microscopy (CLSM) or laser confocal scanning microscopy (LCSM). Sometimes, it is also referred to as laser scanning confocal microscopy. It is an optical imaging technique designed to increase the optical contrast and resolution of a microscope sample using a spatial pinhole to block the out-of-focus light during the image process.
In biomedical sciences, confocal microscopy is often used to examine both fixed and live cells and tissues labeled with at least one fluorescent probe. Outside of biomedical sciences, it is used for multiphoton imaging and a variety of deconvolution techniques.
The invention of this type of microscopy is generally attributed to Marvin Minsky, who created a working microscope in 1955. The original design used a pinhole in front of a zirconium arc as the light source. The light point was then focused by an objective lens directed at the focal plane in the specimen. The light that passed through was focused again by using a second objective lens with the same focus as the first. Any light that passed through the second pinhole struck a photomultiplier to generate a signal related to the brightness of the light. The second pinhole stopped the light that came from either above or below the plane of focus from reaching the photomultiplier.
The original design had no way to produce a real image. Instead, the photomultiplier output was translated into an image on the screen of an oscilloscope from military surplus, without the ability to record. The technology to produce what we have today just wasn’t available back then.
Technological advancements have made today’s generation of instruments much better in every aspect of the image generation process. Newer instruments also have better usability because it is now easier than ever to change or adjust filter combinations, alignment, and laser power. These things are generally software controlled so it doesn’t take as long to create images of samples.
What Does a Confocal Microscope Do?
When you hear the word “microscope”, chances are what first comes to mind is actually fluorescence microscopy. It is an optical technique that relies on fluorescent light as the light source. After fluorophores are applied to the sample in the area of interest, the sample is exposed to electromagnetic energy at a certain wavelength, which excites the fluorophores to cause them to light up. The sensors capture this and create an image, leaving out anything that isn’t lit up on the image plane.
While this is highly useful, it is limited because when part of a sample is tagged with fluorophores, the entire area will light up equally. That makes it impossible to distinguish between the fluorescence on the surface of a thick sample and the background fluorescence, which in turn, makes it difficult to observe anything useful about the sample.
Another issue with fluorescence microscopes is photobleaching. When a fluorophore loses fluorescence because of light-induced damage, the sample loses fluorescence signals while imaging.
That’s where confocal microscopes are helpful. Instead of fluorescence, this configuration uses lasers as the microscope objective and pinhole filters to eliminate the issues with fluorescence, while also increasing the resolution and contrast of the image to improve it. It’s possible to focus the laser on specific parts of the sample, which ensures that only a specific part of the field of view will light up, making it much easier to observe.
Using a confocal microscope builds images from thin sections of a sample. By scanning several thin sections through the sample, it’s possible to create a clean 3D image of the sample, visualizing sections of thicker samples that would otherwise be impossible to observe using conventional fluorescence microscopy.
How Does A Confocal Microscope Work
In principle, a confocal microscope works using a laser excitation source and two pinhole apertures. The excitation light provided by the laser passes through an initial pinhole that is situated in a conjugate focal plane with a scanning point directed at the sample of interest. The second pinhole filter is positioned in front of the detector, or photomultiplier tube. The laser light, having passed through the first aperture, is reflected off a dichroic mirror, a type of thin-film filter which selectively passes a small range of color while deflecting others, that redirects the light (excitation source) towards the sample. The sample is scanned, or excited, by the redirected light which causes secondary fluorescence. This re-emitted light passes back through the dichroic mirror, becoming focused as a confocal point as it passes through the second filter, or detector pinhole aperture. The light that passes through is measured by a photomultiplier tube, also known as a PMT. The filter eliminates anything that’s out of focus, producing a higher quality image than standard widefield microscopes, in comparison.
The PMT is attached to a computer that builds the image, one pixel at a time, as there is never a complete image of the sample. At any moment, only a single point of the sample is observed. The limitation of the confocal microscope is in the scanning mirrors.
The laser light intensity can be adjusted by neutral density filters. The scanning mirrors move quickly. One mirror tilts the laser beam in the X-direction, while the other tilts the laser beam in the Y-direction. This is different from conventional microscopy in the sense that with a conventional microscope, light only travels as far as it can penetrate into the specimen. With confocal, it travels at a narrow depth, one level at a time, to achieve a limited, yet controlled depth of focus.
With the majority of software options, optical sections can be collected at the perpendicular lateral (x and y-axis) as well as transverse plans (x and z-axis) and the y and z-axis.)
Types of Confocal Microscope Machines
There are three types of confocal microscopes available: laser scanning microscopes, spinning disk confocal microscopes, and programmable array microscopes (PAM.)
- Laser Scanning Microscopes: These machines rely on a precisely focused laser to scan over the sample.
- Spinning Disk Confocal Microscopes: These machines use a disk that has pinholes cut into it. The pinholes are arranged in a spiral shape.
- PAM: These machines are highly similar to the spinning disk models, but the user can open and close the pinhole apertures.
There are two different techniques used for beam scanning. Single-beam scanning is used in the majority of laser scanning confocal microscopes. It used a set of galvanometer mirrors that are controlled by a computer to scan the specimen in a raster pattern at about one frame per second.
Multi-beam scanning systems feature a spinning disk with pinholes and microlenses. Often, these units rely on the use of arc-discharge lamps instead of lasers for their source of illumination. This aims to reduce specimen damage while making it easier to detect low fluorescence levels during the real-time image collection process. Multi-beam configurations can also capture images using an array-detector.
Though all three types produce an image of the sample, the spinning disk and PAM models provide a higher frame per second image. Another difference is that those models do not allow for programming of the sampling density, but the laser scanning microscope does.
To determine the best one for your lab, first consider which of the three types of confocal microscopy you’ll be conducting. From there, you’ll need to determine whether you want to use fluorescent dyes on your samples, as well as what types of dye and how many. Other considerations include allowing for more than one type of fluorophore to be excited at the same time and the imaging speed provided.
Leasing vs. Buying Confocal Microscope Machines
Confocal microscope setups can be incredibly expensive, with used units ranging from $19,000 to well over $70,000 used, and new units often starting at $200,000. For the startup on a budget, spending this kind of money at the beginning just isn’t practical. Leasing the equipment through Excedr gives you access to the confocal microscopes you need for your lab, without a hefty upfront investment. And, you can save even more money and time compared to buying because there’s no need to worry about service contracts since maintenance and repairs are included in the equipment lease.
Contact us today to learn more about how our equipment leasing program can help you.