IF imaging: widefield versus confocal microscopy
Immunofluorescence (IF) is a technique used to visualize a protein of interest in its cellular context. It is based on staining cells with antibodies raised against a target protein that is directly conjugated with a fluorochrome or used together with fluorochrome-conjugated secondary antibodies.
How to answer the experimental question in the most efficient manner.
By Dr. Karolina Szczesna, Senior Product Manager & Technical Support Proteintech
- Confocal vs Widefield microscopy
- Main advantages of both types of microscope
- Which microscope is best for my research?
- Microscope setup & appropiate sample control panel
Immunofluorescence (IF) is a technique in biology used to examine the cellular localization of proteins. It is based on antibodies that label specific proteins in cell or tissue specimens. These antibodies are either directly linked with fluorochromes or must be used together with fluorochrome-conjugated secondary antibodies.
Immunofluorescence studies require the application of fluorescence microscopy. This type of microscopy uses fluorescence to create images of the examined samples. Fluorescent probes (e.g., phalloidin conjugates to study actin cytoskeleton), dyes (e.g., FITC, TRITC, Texas Red, Coralite), or proteins (e.g., GFP, RFP, Venus) are used for labeling. They are able to absorb light of a defined spectrum (excitation) and then emit lower-energy light at a longer wavelength (emission). The most common setup of widefield and confocal microscopes is epifluorescence setup – the light emitted from the light source to illuminate the sample shares a path with the fluorescent light emitted by the specimen (Figure 1).
Confocal and Widefield microscopy
In a widefield microscope (Figure 1 A), the most common light sources are mercury lamps, lasers, or LED lights. The chosen light spectrum passes through the excitation filter and is focused on the sample by the objective. Fluorescent light emitted by the sample again passes through the same objective and is registered by the detector. The dichroic mirror ensures that only certain wavelengths of the emitted light pass through to the detector.
A laser is a source of light in a confocal microscope (Figure 1 B). Similar to widefield microscopes, confocal microscopes consist of excitation and emission filters, an objective, detector, and a dichroic mirror. The key difference is the presence of pinholes that block out-of-focus light, allow separation of specimen slices, and ensure that only light that is in the plane of focus passes to the detector.
Widefield microscopes gather emitted light from many focal planes, in-focus but also some out-of-focus planes. The pinhole in a confocal microscope blocks most of the out-of-focus light and allows detection of light only in one focal plane (Figure 2). This enables the generation of sharp, good-quality, and focused images. However, it also means that confocal microscopes require more sensitive detectors compared to widefield microscopes because most of the light is filtered out by the pinhole system and the fluorescent light reaching the detector is much dimmer.
Main advantages of both types of microscope
Widefield microscopes are usually significantly cheaper than confocal microscopes due to their less complicated optics and lower demand on the strength of the light source, and the sensitivity of their detectors. They are easy to use and allow observations by eyepieces directly in the ocular. Because most of the specific emitted light by the specimen contributes to the final image, samples can be imaged with less intense light, which limits photobleaching. It also shortens the exposure times needed to acquire images, thus enabling the observation of fast-moving specimens and more efficient high-content imaging.
On the other hand, widefield microscopes are not suitable for imaging samples that scatter light extensively or are very thick, such as thick tissue slices. Imaging such samples with a widefield microscope would yield blurry images with high background. This can be partly corrected with the deconvolution of images from widefield microscopes. This process is based on digital contrast enhancement of the acquired images by the computational removal of out-of-focus information.
Confocal microscopes, depending on their configuration, are usually suitable for imaging thick samples, creating sharp images that represent only a single focal plane. This not only provides superior image quality but also allows for study of the more detailed structure and spatial resolution of fluorescent dyes/proteins within the specimen, e.g., the subcellular localization of a membrane receptor in a polarized cell membrane. However, image acquisition requires more training and often time due to the longer exposure times needed to gather a sufficient amount of in-focus light to generate images. The image of the specimen seen through the eyepiece of both a widefield and confocal microscope is always widefield. Only digital images processed by the detector of a confocal microscope are true single-plane confocal images.
Which microscope is best for my research?
For many applications, widefield microscopes are sufficient to obtain high-quality images for addressing the investigated phenomena. They are commonly used for analyzing stained cultured cells and thin tissue slices and are recommended for screening assays and during the optimization of staining protocols.
They provide the best trade-off between quality, speed, ease of use, and cost.
Confocal microscopes are indispensable in detailed co-localization studies of two or more fluorescently labeled proteins providing greater image resolution and spatial resolution in the z-axis. They are also superior in analyzing thicker samples. Spinning disk confocal microscopes enable live imaging studies of fast biological processes and overcome the limitations of standard laser scanning confocal microscopes, which require longer exposure times compared to widefield microscopes. This also makes confocal microscopes suitable for more advanced imaging techniques such as FRAP (fluorescence recovery after photobleaching), FLIM (fluorescence-lifetime imaging microscopy), and super-resolution imaging.
Microscope setup & appropriate sample control panel
Irrespective of the choice of microscope, every fluorescence imaging experiment should include appropriate controls:
- The use of specimens stained only with secondary antibodies is an important negative control to examine the background levels and potential non-specific staining. Using samples with no/lower expression of examined proteins (e.g., knock-out cells or siRNA-treated cells) and with overexpressed proteins is highly advised.
- If more than one antibody is used, it is important to have single-stained samples to assess whether there is any cross-channel bleed-through that could lead to misleading results, e.g., falsely claiming co-localization of two proteins.
- It is vitally important to use fluorophores compatible with emission/excitation filter sets of a given microscope. Excitation and emission spectra of common fluorescent dyes and proteins are publicly available.
- Optimization of the staining protocol (e.g., blocking agents, incubation times, antibody dilutions) ultimately helps to reduce background and create high-quality images.
The microscope you choose depends on the nature of the individual experiment and the questions you wish to answer in the most efficient manner.