AAT Bioquest

Confocal Microscopy

Principle of spinning disk confocal microscopy
Simplified principle of spinning disk confocal microscopy, showing the interaction between laser, pinhole disks, specimen, and generated signal. Figure made in Biorender.
Confocal microscopy can eliminate common fluorescence microscopy issues that arise from out of focus illumination and allow thick specimens (up to roughly 200 nm) to be viewed with high resolution. In confocal microscopy, only one diffraction-limited region of the specimen is illuminated at a time, and a single signal is accepted from that specific region. This illumination technique overcomes issues present in wide field (WF) microscopy where much of the emitted light may appear out of focus.

In design, the confocal microscope is composed of one or more electronic detectors, a multi-laser system, a beam scanning assembly, and an adapted computer and software for sample imaging, analysis, and data storage. Spatial filtering techniques remove blurred components and glares, common elements in images from thicker specimens.

Some advantages of confocal microscopy lie in the ability to finely, optically, section samples, where unwanted light can be removed above and/or below the focal plane. Confocal microscopy can provide better contrast and allow 3D reconstruction by combining the image data from a stack of images using appropriate software. This technique also gives the user the ability to control the depth of field, and to remove or reduce background noise. For these reasons it has become the premier choice of microscopy in the field of immunofluorescence.


Limitations of Confocal Microscopy

Human lung adenocarcinoma tissue
Confocal Microscopy Imaging: FFPE human lung adenocarcinoma tissue was incubated with an anti-EpCAM primary antibody, and an HRP conjugated anti-mouse secondary antibody. TSA signal was developed by incubation of tissue section with iFluor® 430 tyramide. Images were acquired with a Violet filter set.
Confocal microscopy, however, does not come without limitations. Even though techniques offer less signal degradation, photobleaching is still a significant issue, especially for samples with weaker fluorescent signals. Resolution of images is less than other techniques, and the cost may be 2-7 times more than WF microscopy. The best resolution possible remains around 0.2 μm on the x-axis (worse than WF microscopy) and 0.6um on the y-axis. High resolution of the Z-axis can only be achieved by sacrificing the level of light that reaches the detector.

Similar to WF microscopy, confocal microscopy is also limited by diffraction effects. Image capture is, as well, determined by a number of variables including the technology of the imaging system, image brightness, and the speed of the laser raster that scans the specimen. Because of this, traditional confocal microscopy can only capture 1-10 images per second where dwell time, or the time that excitation light remains in one location in the specimen, may increase considerably.


Subcategories of Confocal Microscopy

Laser Scanning Confocal Microscopy (LSCM)

Laser scanning confocal microscopy
Laser scanning confocal microscopy: Intracellular free Ca2+ was detected by red fluorescent probe dihydrorhod-2 AM (Rhod-2 AM). The nuclei were stained with DAPI (blue). The pEZ-LV203 vector harboring the eGFP reporter gene produced green fluorescent protein. Source: Csseverin inhibits apoptosis through mitochondria-mediated pathways triggered by Ca2+ dyshomeostasis in hepatocarcinoma PLC cells by Shi M et al., PLOS, Nov. 2017.

In LSCM, a focused laser beam controlled by two high-speed oscillating mirrors scans across a sample region in a defined area via a raster pattern. Next, the laser light is scattered and reflected and any fluorescent light from the illuminated spot is received by an objective lens. From here, a device known as a beam splitter separates a portion of the received light and sends it through a filter thereby selectively allowing emission, but not excitation, light to pass. The filtered light passes on to a photodetection device, which then transforms the light into electrical signals, recorded by a computer.

As LSCM sequentially scans excitation at specified points, fluorescence intensities across the specimen can be collected and whole images can be sequentially generated. LSCM has a number of advantages, in that the technology offers programmable sampling densities at very high resolutions. Out-of-focus light is blocked, which increases image resolution and prevents non-target elements from appearing in the background.

Spinning Disk Confocal Microscopy

To eliminate some of the most common issues of standard confocal methods, spinning disk confocal microscopy was created. Spinning disk confocal microscopy is a multi-point scanning system developed to capture images at high speed. In this technique, the sample is both illuminated and viewed through a spinning disk with rows of pinholes. As the disk spins, each pinhole on the disk provides a point source of light that scans across the specimen. Emitted light passes through each pinhole before being separated by a dichroic mirror. Then, images can be captured with an array detector, like a charge-coupled device.

Compared to traditional confocal microscopy, frame rates can be obtained at greater than 50 images per second, an increasingly beneficial attribute for the dynamic observation of live cells. When compared to other techniques, namely LSCM, spinning disk confocal microscopy provides a faster image acquisition rate with fewer light requirements.

FAQs:Digital Catalogs:

Product Ordering Information

Table 1. iFluor® and Related Products

Cat No.
Product Name
Unit Size
Ex (nm)
Em (nm)
16860FITC goat anti-mouse IgG (H+L)1 mg492515
16876FITC goat anti-rabbit IgG (H+L)1 mg492515
16520iFluor® 350 goat anti-mouse IgG (H+L)200 µg345442
16670iFluor® 350 goat anti-rabbit IgG (H+L)200 µg345442
16524iFluor® 405 goat anti-mouse IgG (H+L)200 µg401420
16674iFluor® 405 goat anti-rabbit IgG (H+L)200 µg401420
16528iFluor® 488 goat anti-mouse IgG (H+L)200 µg491514
16678iFluor® 488 goat anti-rabbit IgG (H+L)200 µg491514
16532iFluor® 514 goat anti-mouse IgG (H+L)200 µg518542
16682iFluor® 514 goat anti-rabbit IgG (H+L) 200 µg518542
16536iFluor® 532 goat anti-mouse IgG (H+L)200 µg542558
16686iFluor® 532 goat anti-rabbit IgG (H+L) 200 µg542558
16537iFluor® 546 goat anti-mouse IgG (H+L)200 µg541557
16688iFluor® 546 goat anti-rabbit IgG (H+L)200 µg541557
16540iFluor® 555 goat anti-mouse IgG (H+L) 200 µg559569
16690iFluor® 555 goat anti-rabbit IgG (H+L)200 µg559569
16541iFluor® 568 goat anti-mouse IgG (H+L)200 µg568587
16692iFluor® 568 goat anti-rabbit IgG (H+L)200 µg568587
16548iFluor® 594 goat anti-mouse IgG (H+L)200 µg592614
16558iFluor® 633 goat anti-mouse IgG (H+L)200 µg638655
16704iFluor® 633 goat anti-rabbit IgG (H+L)200 µg638655
16562iFluor® 647 goat anti-mouse IgG (H+L)200 µg654674
16710iFluor® 647 goat anti-rabbit IgG (H+L)200 µg654674
16566iFluor® 680 goat anti-mouse IgG (H+L)200 µg682701
16574iFluor® 700 goat anti-mouse IgG (H+L)200 µg693713
16714iFluor® 700 goat anti-rabbit IgG (H+L)200 µg693713
16586iFluor® 750 goat anti-mouse IgG (H+L)200 µg753779
16720iFluor® 750 goat anti-rabbit IgG (H+L)200 µg753779
16587iFluor® 790 goat anti-mouse IgG (H+L)200 µg782811
16721iFluor® 790 goat anti-rabbit IgG (H+L)200 µg782811