Researchers have been fascinated with the microscopic world for millennia. Centuries ago, scientists like Robert Hooke and Antoine van Leeuwenhoek developed the first light microscopes. These devices evolved into intricate technologies that, through magnification, give researchers access to the billions of cells comprising our bodies and the living world. Among these contraptions is the confocal microscope.

Produced in the 1950s, the confocal microscope has since produced crisp, colorful images that depict living cells’ morphologies. The high resolution that confocal microscopes afford has, in turn advanced cell biology, biochemistry, and molecular genetics research. Insights from these fields have helped researchers develop novel therapeutics that improve health outcomes and quality of life.

But to best harness confocal microscopy, researchers must consider the many confocal microscopes available and develop workflows that generate clear images and reproducible data. At its core, confocal microscopy involves multiple steps, starting with tissue cultures and ending with image analysis. 

In this article, we will cover confocal microscopy and the process of preparing cell cultures for visualization. Then, we will discuss what researchers should consider when conducting a confocal microscopy study and show how our leasing program helps you acquire the equipment you need to visualize all kinds of cell lines and cell types.

What Is Confocal Microscopy?

Confocal microscopy is a fundamental visualization technique that observes tissues and samples in a three-dimensional (3D) space, facilitating 3D imaging. Marvin Minsky invented the device to address the primary shortcoming of widefield microscopy: its pinhole introduces out-of-focus light while capturing images, reducing the image’s resolution. 

Confocal microscopy is a kind of optical sectioning microscopy, which allows microscopes to capture high-resolution images from tissue without having to cut the tissue into slices. Two kinds of confocal microscopes exist for confocal imaging:

• Laser scanning confocal microscopy (LSCM): LSCM is the most common type of confocal microscopy. It uses a laser beam that generates light for  taking sample images in all three axes. After sweeping through the x- and y-axes to take snapshots, the microscope moves to the next depth within the z-axis and repeats the process. The process occurs sequentially across multiple regions and depths, producing images that are stacked to form the composite image. 
• Spinning Disk Confocal Microscope (SDCM): SDCMs use a Nipkow disc that contains hundreds of pinholes. The many pinholes and their arrangement allow the microscope to capture images much faster for stacking than other kinds of confocal microscopes.

Like a widefield microscope, confocal microscopy can also be conducted with three kinds of imaging views.

• Brightfield: Brightfield microscopy is the simplest type of microscopy. Here, white light passes through the condenser lens, the sample, and the objective lens to form the image.
• Phase contrast: Phase contrast microscopy is an extension of light microscopy. While it also uses white light, it enhances the contrast of images to make cellular components easier to see.
• Fluorescence microscopy: You’ve seen colorful images of cellular components in research papers. Those were produced using confocal fluorescence microscopy. Here, dyes consisting of fluorophores stain tissues to emit fluorescence that aids in cell visualization.

Sample preparation of cell cultures for confocal microscopy

Before researchers can view cell cultures for confocal microscopy, living cells must first be prepared for visualization. This process features a series of steps and diverse reagents that yield quantitative image data. Here are the steps in order:

• First processing steps: Researchers typically prepare cell cultures under two formats. First, researchers can grow free-floating cultures. Suspension cultures allow researchers to produce specific biologics in bulk. Alternatively, researchers can prepare adherent cell cultures, where cells are grown on a suitable substrate to facilitate adhesion. Adherent cell cultures are used to study cellular morphology and evaluate biopharmaceuticals as drug candidates.
• Live cell imaging: Researchers can first choose to profile live cells or fix them. In live cell imaging, researchers keep the cells alive during the sample processing for the real-time characterization of cellular behavior. These protocols typically use fluorescent protein tags that light up when they bind target proteins. Keeping the cells alive allows researchers to scan protein kinetics with diverse substrates in vivo. Nonetheless, researchers must keep the cells alive and maintain their cellular physiology while processing and visualizing the cells. For example,  strong temperature control is essential for keeping cells active, whether at room temperature or any other temperature.
• Fixation: Alternatively, researchers can analyze cells at fixed time points through fixation. Fixation prevents any further cellular activity, preserving cells in a fixed state. Researchers have many fixatives to use that are divided into two classes: cross-linking and precipitating fixatives.‍
• Cross-linking fixatives: Glutaraldehyde and formaldehyde comprise the cross-linking fixatives. These are aldehydes that create covalent bonds with lysine residues to form methylene bridges. These bridges halt cellular activity and hold cells in place. Researchers fixing samples with cross-linking fixatives will have to add an extra step to permeabilize cells for immunofluorescence assays. This helps primary and secondary antibodies bind to their intended targets for fluorescent visualization.‍
• Precipitating fixatives: These fixatives comprise ethanol, methanol, and acetone, organic solvents that replace the water inside cells with the fixative. They degrade the proteins inside the cells and can cause cells to shrink, disrupting their structure.‍
• Fluorescent staining: After processing the cell cultures, researchers can then stain the cells with fluorescent dyes, followed by an incubation period. These dyes add color to specific cellular features during the image acquisition process. Many dyes exist for researchers to stain their cells. Each dye has a distinct excitation and emission maximum, wavelengths where the dye’s electrons are most excited and the photons released are most intense respectively. The excitation occurs using the microscope’s excitation light source: a laser that excites the dye’s electrons at the excitation maximum and emits photons once they return to baseline energy levels. Here are some of the most common dyes that researchers can use:‍
• Fluorescein Isothiocyanate (FITC): FITC was one of the first fluorescent dyes used for fluorescence microscopy. FITC is commonly used for labeling proteins, peptides, antibodies, and other amine-containing biomolecules. The dye reacts with amine groups found in proteins to form covalent amide bonds. Although the dye is commonly used in confocal microscopy, it demonstrates a few shortcomings, such as photobleaching, being sensitive to changes in the sample’s pH, and a broad emission spectrum.‍
• Alexa Fluor: Alexa Fluor is a family of fluorescent dyes modified from multiple well-known fluorescent dye families such as rhodamine and cyanine. Now produced by Thermo Fisher Scientific, these dyes cover the entire visible wavelength spectrum from blue to far-red and near-infrared wavelengths.‍
• DAPI: DAPI, also known as 4′,6-diamidino-2-phenylindole, is a dye that binds to regions of DNA with high abundances of thymine-adenine (T-A) nucleotides. When bound, it emits a blue color underneath a fluorescence microscope. DAPI does not penetrate living cells because it cannot pass through intact cell membranes. For that reason, the dye is useful for distinguishing viable and non-viable cells within a tissue sample.‍
• Image collection and analysis: Once the samples have been processed, confocal microscopes will scan the sample across all three axes to obtain a high-resolution image. The resolution of the final image depends on various factors, such as the pinhole size and the confocal microscope’s scan speed. After capturing the image, processing pipelines use image analysis software tools to generate quantitative insights into cellular behavior. While companies have proprietary software, open-source software such as Fiji can perform similar functions with its many modules.

Along the pipeline, researchers must make sure to use strong microscopy techniques in their experiments. Doing so will help researchers retain cellular morphology and activity as they visualize their samples.

Applications of confocal microscopy

Confocal microscopes are ideal for studying cellular processes, from the molecular level to the level of the whole organism. With tissue cultures, researchers can characterize different kinds of tissues and cells from diverse specimens:

• For one, researchers have used fluorescent phalloidin, a highly specific bicyclic peptide dye, to stain actin filaments. Using phalloidin has shed insights into musculature development in clams and helped characterize treatments against muscle amyloidosis as people age in animal models.
• Scientists can also profile immune responses through confocal microscopy. For instance, researchers can characterize THP-1 monocyte cells to profile immune responses through confocal microscopy.

Researchers have also used confocal fluorescence microscopy to develop 3D tissue culture models. These structures — spheroids and organoids — have the potential to complement animal models in biomedical research by recapitulating in vivo tissue-like structures and functions. Here are just some of the ways researchers have captured human physiology with microscopy:

• Confocal microscopy can profile 3D co-cultures of cardiomyocytes and cardiac fibroblasts (CFs). Both cell types mediate normal heartbeats in humans. Such a 3D culture would help researchers study medications designed to mitigate heart disease.
• Researchers can also characterize the localization of organelles within cells. For instance, researchers can characterize mitochondrial morphology among pancreatic B-cells. Changes in mitochondrial morphology can indicate stress, which has been linked to diabetes onset.

Considerations for confocal microscopy

As researchers begin considering a confocal microscope for visualizing cells, they must consider several visual phenomena. Each of these occurrences affect the final images and the quantitative data generated from them.

• Autofluorescence: Autofluorescence refers to biological entities that emit light without being stained by a fluorescent dye. Almost all living cells and cellular components (eg. Chlorophyll) demonstrate some form of autofluorescence. Many of these organisms contain polycyclic hydrocarbons, filled with delocalized electrons that photons can excite.
• Saturation: Saturation is a phenomenon that occurs when fluorescent light appears to be brighter underneath a confocal microscope. However, the dye does not cause the saturation. In fluorescence microscopy, photon emission reaches a maximum. Even with additional laser power input, the fluorescence intensity does not increase further. As such, the image may produce brightly fluorescent sections, but the fluorescence would not reflect the increase in the concentration of the biomolecule on which the dye binds.
• Photobleaching: Photobleaching refers to the gradual decrease in emitted fluorescence as a result of the fluorophore getting destroyed. Photobleaching occurs when covalent bonds between the dye and the molecules of interest are destroyed or rearranged with other biomolecules as they are excited. Modifying these dyes reduces the quality of fluorescent images when studying biological specimens.

Excedr leases many kinds of confocal microscopes

Excedr has a long history of leasing confocal microscopes from many different brands. And because we do not carry an inventory, you can select the exact manufacturer and model you want to lease, specific to your experimental needs. Here are just some of the confocal microscopes we have leased in the past:

• Zeiss: The LSM 980 and LSM 900 provide 3D and 4D imaging for fluorescently stained tissue cultures and samples. Both provide high signal-noise ratios and improved multi-color resolution to maximize image quality and produce valuable spatial information through LSM Plus. Both also provide multichannel (the LSM 900 — 3 and the LSM 980 — 36) capabilities to observe a wide array of different fluorescent stains concurrently. Lastly, these Zeiss microscopes take up a small lab footprint, minimizing the lab space and time required for user training.
• Keyence: The company produces two kinds of CLSMs: the VK-X series and the BZ-X series. VK-X harnesses surface metrology to resolve fluorescence structures 12 times faster and 16 times larger than conventional microscopes. The BZ-X is a fully motorized confocal microscope system that supports fluorescence, brightfield, and phase-contrast imaging as well.
• Olympus: Olympus boasts multiple types of CLSMs but the FV3000 is specifically designed for life sciences research. The microscope achieves 3 times more light transmission than traditional spectral detection technologies with the TruSpectral technology. The microscope also comes equipped with lambda scanning mode to tease apart overlapping fluorescent signals. Automation with the FV3000 is also possible thanks to a Macro-to-Micro module in Olympus’s software.

Lease your confocal microscope with Excedr

Confocal microscopes provide researchers with high-resolution digital images of microscopic organisms. Such images provide an unprecedented view into the cells that help our bodies function. 

Understanding cell biology with a confocal microscope also helps researchers develop pharmaceutical and biologics to advance biomedical research. While researchers must consider many factors and refine sample preparation pipelines, doing so will help end users make the best use of the confocal microscopes available.

Excedr’s leasing program can help advance cell biology research for your lab. Speak with our team today to learn how we can help you acquire confocal microscopes that match your needs.