How Does A Confocal Microscope Work

First, What is Confocal Microscopy?

Confocal microscopy is an optical imaging technique designed to increase the optical contrast and resolution of a sample using a spatial pinhole to block out any out-of-focus light in the background of the image. It is also known as confocal laser scanning microscopy (CLSM) or laser confocal scanning microscopy (LCSM), and is also referred to as laser scanning confocal microscopy.

In biomedical sciences, confocal microscopy is used to examine both fixed and live cells and tissues labeled with at least one fluorescent probe, or fluorophore, which causes emission of fluorescent light at a specific depth and point on the sample. Outside of biomedical sciences, confocal microscopy is used for multiphoton imaging and a variety of deconvolution techniques.

The invention of confocal microscopy is generally attributed to Marvin Minsky, who created a working confocal microscope prototype in 1955, which included a zirconium arc as the light source. However, the short-arc lamp’s radiance later proved too low to be useful in modern confocal microscopy techniques. The original design also include pinhole spatial filters, which ultimately allowed the viewer to see a microscopic image with higher resolution.

That said, 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 instruments much better in every part of the image generation process. Newer instruments also have better usability because it is now easier than ever to change filter combinations, alignment, and adjust the 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 imagining.

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 only that 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 formation of the sample. Using confocal microscopy, it’s possible to see visual sections of small structures that would be nearly impossible to section and build 3D structures from the resulting images.

How Does A Confocal Microscope Work?

A confocal microscope uses a laser light source, pinhole spatial filters, multiple objective lenses, and fluorescent dyes to generate a microscopic image. The laser provides excitation light, which reflects off of a mirror. The laser light then hits two mirrors that are mounted. These mirrors redirect the laser light, which effectively scans the sample and emits fluorescent light. The dye and the emitted light are then descanned by the same mirrors that originally scanned the excitation light. 

From there, the emitted light passes through what’s known as a dichroic, a type of color filter used to selectively pass light of a small range of colors. This filtered light is directed toward the pinhole. The light passing through the pinhole is measured by a photomultiplier tube (PMT) or other types of photosensitive detectors, including photodiodes, and solid-state charge-coupled devices (CCDs). The pinhole filter eliminates anything that’s out of focus, producing a higher quality image than standard widefield microscopes.

The detector 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 Microscopes

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 kinds and how many different kinds. 

Other considerations include allowing for more than one type of fluorophore to be excited at the same time, the imaging speed provided, and so on.

Leasing vs. Buying Confocal Microscopes: Which Is Smarter?

Confocal microscope setups are incredibly expensive, ranging from $19,000 to well over $70,000, which are only prices for used devices. New microscopes can be even more expensive.

For the biotech startup on a budget, spending this kind of money when you’re just starting out just isn’t practical. Leasing the equipment through Excedr instead gives you access to the confocal microscopes you need for your lab, without the hefty upfront costs. 

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.