Confocal microscopy

What is confocal microscopy?
Point-scanning type
Spinning disk type
Example images
Highlighted products
Other imaging techniques

What is confocal microscopy?

Confocal laser scanning microscopy is a powerful optical imaging technique designed to improve the resolution and contrast of microscopic images. It achieves this by using a spatial pinhole to block out-of-focus light, enhancing the clarity of the final image. This technology is especially valuable in biological sciences for visualizing detailed structures within cells and other biological materials.

In the broader field of microscopy, there are three main branches: optical microscopy, electron microscopy, and scanning probe microscopy, with the emerging field of X-ray microscopy adding to the range of techniques available. In biological imaging, fluorescence dyes and proteins are often used to provide contrast and specificity, allowing researchers to examine fine cellular structures in detail.

A laser scanning microscope (LSM) is an essential tool in this area. Many LSMs utilize a confocal optical system, where a pinhole at the focal point eliminates out-of-focus light, providing superior optical resolution in the sample’s depth compared to wide-field microscopes. This selective detection of fluorescence only from the focal point leads to images with minimal blurring, high contrast, and high resolution.

However, when the pinhole blocks light emitted outside the focal plane, it also reduces the amount of signal light reaching the detector. To compensate for this, high-sensitivity detectors such as photomultiplier tubes (PMTs), hybrid photodetectors (HPDs), and multi-pixel photon counters (MPPCs or SiPMs) are often used. These detectors, with their high quantum efficiency and low dark current, allow for a strong signal-to-noise ratio, providing clear contrast and a high dynamic range in the resulting images.

A key advantage of confocal microscopy is the ability to capture precise fluorescent images at specific focal planes. By acquiring a series of images at different focal depths, researchers can reconstruct three-dimensional representations of the sample, accurately depicting spatial relationships within the structure. This capability is crucial for studies that require a detailed understanding of biological samples in three dimensions.

Comparison between confocal microscopy and standard widefield microscopy

Figure 1: Comparison of images between confocal microscopy and conventional epifluorescence microscopy.

Figure 2: Optical schematic diagram of confocal microscopy

Point-scanning type

The point-scanning confocal microscope acquires images by scanning a laser in a two-dimensional direction using mirrors called galvanometer mirrors. A scanner using this mirror is called a galvanometer scanner.

In addition to the galvanometer scanner, there is also a method that uses a resonant scanner (which utilizes a mirror resonating at a constant speed). A resonant scanner generally allows faster scanning than galvanometer scanners, enabling high-speed imaging. However, a drawback of resonant scanners is that they resonate at a fixed speed and angle, limiting flexibility for changing the field of view or selectively illuminating specific areas with a laser.

A resonant scanner is faster than galvanometer scanners, which means that the laser exposure time per point is shorter, resulting in weaker fluorescence intensity. If you want to achieve the same fluorescence intensity as a galvanometer scanner, you can either increase the laser intensity or perform multiple scans. Fluorescent molecules are less likely to fade when excited repeatedly with weaker excitation light compared to a single strong excitation. Therefore, using a resonant scanner for imaging within the same time frame as a galvanometer scanner offers the advantage of reduced fading.

The detectors commonly used in point-scanning confocal microscopes are photomultiplier tubes (PMTs) and silicon photomultipliers (SiPMs).

Figure 3: Schematic diagram of a point-scanning confocal microscope

Spinning disk type

The spinning disk confocal microscope is a method for acquiring images by rotating a disk with countless holes, known as the spinning disk. In contrast to point-scanning confocal microscopes, which irradiate lasers point by point and scan using mirrors, the spinning disk confocal microscope simultaneously scans multiple points by rotating the disk, allowing for faster image acquisition.

For detection, CMOS cameras are primarily used, and their sensitivity and noise performance significantly impact the signal-to-noise ratio of the images.

The spinning disk confocal microscope has the advantage of minimizing sample fading due to weak excitation light during multipoint scanning, similar to resonant scanner confocal microscopes. However, it tends to have lower sensitivity compared to point-scanning confocal microscopes.

Figure 4: Schematic diagram of the spinning disk confocal microscope

Example images

Confocal images using ORCA^®-Flash4.0 V3 Digital CMOS camera

Spinning disk confocal, pollen Z-scan images

High-speed intracellular Ca²⁺ gradient driven by UTP stimulation

Confocal images using MAICO^® MEMS confocal unit

4-color live cell confocal Z-section imaging

High-speed confocal intracellular Ca²⁺ imaging of spontaneously beating hiPS-cardiomyocytes

Z-section imaging of mouse brain

Highlighted products

Photomultiplier tube module
H1620X series

Photomultiplier tube module with a highly sensitive crystalline photocathode compared to alkaline photocathodes. It contributes to acquiring high signal-to-noise ratio (S/N) images in confocal microscopy.

H16200-40

H16201-40

MPPC module

Photon-counting module equipped with an MPPC enables ultra-weak light detection. Its excellent multiplication reproducibility contributes to high-contrast visualization of deep biological tissues.

MEMS mirror

An electromagnetic-driven mirror utilizing MEMS (micro-electro-mechanical systems) technology. It achieves low power consumption, wide optical deflection angles, and high mirror reflectivity. It is used as a mirror for point-scanning confocal microscopes.