Humans have always been fascinated with things they could not see with the naked eye. The 17^th century saw van Leeuwenhoek and Hooke unveil the microscopic world when they invented the light microscope. Since then, the life sciences community has come a long way. They invented sophisticated microscopes to build on what we know about cellular anatomy and physiology. 

Confocal microscopes stand tall among these contraptions. Since its invention by Marvin Minsky in 1957, scientists have used the confocal microscope to study cell and tissue structures, allowing researchers to produce innovative medications that change lives for the better.

In this article, we will provide a deep dive into confocal microscopy. As most confocal microscopes can also detect fluorescent light, we will focus on fluorescence microscopy in this article. The information from this article will then help you conduct fluorescence microscopy experiments effectively and drive biotech research forward. It can also serve as a starting point in your decision-making process when looking to lease a confocal microscope for your lab.

What Is Confocal Microscopy?

You’ve probably seen a light microscope in your biology classes at school. They create magnified images by shining a beam of light through an objective lens and the eyepiece. The crisp images you observe are formed when the specimen on the microscope stage is adjusted to the lens’s focal point. It is at the focal point where parallel light rays converge and focus the image. 

However, light microscopes struggle to focus samples thicker than 2 micrometers (µm) because their objective lens lacks the sufficient depth of focus to clarify thicker samples. When specimens are too thick, the light microscope passes all light from below and above the focal plane to the eyepiece, causing a blurry image.

Minsky conceived the confocal microscope to address the issue of thick specimens in light microscopy. The following features have allowed confocal microscopes to visualize thicker specimens clearly:

• Fluorescent dyes: Samples for confocal fluorescence microscopy are stained with fluorescent dyes. These dyes, through the photoelectric effect, emit light when exposed to light of a specific wavelength. In the photoelectric effect, electromagnetic radiation like a light source excites electrons on a surface into a higher energy state. When the electron returns to its original state, a fixed amount of light energy called a photon is released. The fluorescent dyes emit photons at different wavelengths during a confocal microscopy experiment, allowing researchers to distinguish between different kinds of stained molecules with multiple dyes.
• Laser beam: Confocal microscopes produce light with a focused laser beam instead of a visible light source, causing an intense, narrow beam of light with a single wavelength to be produced. The beam travels from its source to fill an aperture and focus light on a small spot of the specimen with high brightness. On spots across the specimen, the laser beams excite the dyes staining the specimen. As a result, the dyes emit light that the microscope uses to form an image.
• Dichroic mirrors: Dyes can only emit light when exposed to light at specific wavelengths. These wavelengths are referred to as the excitation peaks for a dye. The dichroic mirrors in a confocal fluorescence microscope allow light of specific wavelengths to be passed while reflecting the other wavelengths, providing confocal microscopes with the precision needed to alight the dyes and detect specific cellular features.
• Galvanometric mirrors: The narrow beams of light that a laser emits do not cover the whole sample. Galvanometric mirrors address the beam’s small range by directing the laser beam over the sample point-by-point. The confocal microscopes then take pictures of specific sections of the specimen to form a complete image.
• Pinhole aperture: This component is located between the fluorescence filter and the microscope’s light sensor. It acts as the key feature of Minsky’s invention by preventing out-of-focus light from being detected. In doing so, the final images of 3D specimens become focused.
• Sensors: Once the dye emits light from the specimen, there needs to be a way to process the signals and form a final image. All confocal microscopes are equipped with sensors that convert the emitted light into an electric signal that allows a complete image to be formed. These sensors also amplify the excited light as the latter can lose some of its intensity after passing through the pinhole.

Types of Confocal Microscopes

While the typical confocal microscope contains several common essential components, many manufacturers like Leica produce microscopes with features suitable for different applications. Even so, you will encounter two common types of confocal microscopes: 

• Laser Scanning Confocal Microscope (LSCM): LSCMs are the most common type of confocal microscope. These microscopes sweep a laser beam across the sample using a pair of galvanometric mirrors. The setup allows the microscope to generate a series of images along the x- and y-axis. Once the x- and y-axes have been covered at a given depth, the microscope selects a new focal point to stack the z-axis. After imaging the specimen’s entire depth, the z-axis images are stacked together to form the final 3D image.
• Spinning Disk Confocal Microscope (SDCM): SDCMs combine confocal microscopy with wide-field microscopy to scan multiple points of an image at the same time. This is accomplished using a Nipkow disc comprising hundreds of pinholes. The disk rotates along an axis to allow every part of the specimen to be imaged as it is spun. Changing the speed of the spinning disk also allows you to change the image’s brightness, contrast, and quality.

How Images are Generated

The photoelectric effect causes stained samples to emit light after being exposed to a confocal microscopy laser. Nevertheless, any confocal microscope you use will need a sensor. These sensors determine where the emitted light is coming from and how intense the light is. To that end, confocal microscopes can have three kinds of built-in sensors to detect emitted light:

• Photomultiplier tubes (PMT): PMTs contain a cathode with a light-sensitive surface. When this surface is exposed to fluorescent light, a series of photoelectrons are excited through a series of dynodes. By the time the photoelectrons reach the anode, the fluorescent signal becomes amplified. Changes to the voltage at the anode are then used to measure the intensity of the amplified signal. PMTs can also modify gain, the extent to which a fluorescent signal is amplified by the sensor.
• Avalanche photodiodes: Although PMTs can add gain to a signal, they do not amplify the signal as well as an avalanche photodiode. Instead of using dynodes to amplify the signal, a semiconductor amplifies the signal. Photons entering the diode pass through the silicon dioxide layer and a positively and negatively doped region (the pn junction). Once in the depletion region, free electrons and holes are excited to migrate to the cathode and anode, respectively.
• Hybrid detectors: While PMTs have low gain, inducing consistent high-intensity exposure in an avalanche photodiode would destroy the device. Hybrid detectors use a cathode that excites electrons. The excited electron then hits a semiconductor target to enhance the signal through the pn junction found in avalanche photodiodes, mitigating the weaknesses of the individual sensor systems to enhance fluorescent signals.

Imaging Software

Each kind of image sensor has a place in imaging complex microscopic specimens. Nonetheless, tools that analyze these images are needed to generate imaging data. Confocal microscope vendors often develop proprietary imaging analysis software for this purpose. However, several third-party, open-source solutions also enable reproducible analyses for researchers.

• ImageJ: ImageJ is the most popular third-party image analysis software for confocal microscopy. It is also released as Fiji, which comes with plugins that allow 3D images to be rendered and analyzed. ClearVolume is one such plugin for both light and confocal microscopy.
• CellProfiler: CellProfiler is a freeware analysis software that can measure cell phenotypes from confocal microscopy images. Through an automated process, the software can find objects of interest from cells to worms and measure specific properties within those objects.

Regardless of the imaging software used, all analysis tools begin with a grayscale image that measures how intense the emitted fluorescent light is. These tools divide the image into small, equally sized units called pixels. Many factors impact how these pixels are generated, which in turn affects how robust and reproducible the data becomes. Here are just some of the factors to consider:

• Image collection: Parameters such as exposure time affect how bright an image looks. While higher exposure times increase an image’s brightness, too much exposure time can lead to saturation, reducing the accuracy of any quantitative fluorescence data.
• Environmental conditions: The conditions in which images are taken must not cause cell stress and damage⁵. They must also be identical across experiments to ensure reproducibility.
• Data analysis: Whatever raw pixel intensities are obtained must not be altered during data analysis, including using Photoshop to modify raw images and compressing the raw image files. 

Life Sciences Applications

Research groups have allocated significant time and effort to study the cellular world with confocal microscopy. These studies have yielded new insights into how the human body works and how diseases progress, as seen below: 

• Skin cancer: Confocal microscopy is being used to diagnose skin cancer. Reflectance confocal microscopy features using a point source of near-infrared light to reflect tissue structures. This kind of confocal microscopy specializes in identifying structural features based on how much light bends at different features of the skin. Differences in refraction can indicate differences in skin structure that can identify a cancer, such as a lesion or a melanoma.  
• Organoid development: Confocal microscopy can also be used to develop and evaluate 3D organoids. For example, using confocal microscopy helped identify the effects of inflammation on intestinal structure in inflammatory bowel disease (IBD). These features include increased cell death and debris, along with reduced cell size and budding capacity.   
• Microbiome research: How microbes are organized in a human environment impacts how they interact with each other and their hosts. Confocal microscopy can tease apart these dynamics by showing how antibiotic treatment could disturb the gut microbiome. Fluorescent in situ hybridization stains different microbial species by assigning a different dye to each unique taxonomic group. The staining can be used to identify where specific bacterial populations reside within human tissues.   

Speak with Excedr Today

Confocal microscopy is a foundational technique for studying cellular anatomy and physiology. It has recently provided important insights into cancer diagnostics, disease pathophysiology, and human microbiome dynamics. 

If you want to take your research to the next level, speak with us today. Whichever type of confocal microscope you’re interested in, it’s likely we can procure it for you.  Because we do not carry an inventory, you have the freedom to see what kind of microscope works best for you and get that exact instrument in the lab. Harness our technical expertise in confocal microscopy and take advantage of our brand-agnostic leasing program..

Are you interested in leasing a confocal microscope today? Let us know!