Confocal microscopy

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

What is confocal microscopy?

A confocal microscope allows you to acquire images by removing fluorescence generated outside of the focal plane to be observed. As a result, it provides images with minimal blurring, high contrast, and high resolution. Each image precisely captures fluorescent samples positioned at a specific focal plane. By acquiring multiple images with varying focal plane positions, it is also possible to construct three-dimensional images. Such images allow accurate determination of the spatial relationships within the sample.

The key feature of a confocal microscope is the placement of a pinhole in front of the detector. By positioning this pinhole conjugate to the focal point, it selectively blocks fluorescence generated away from the focus, effectively eliminating light that causes blurring (see Figure 2).

There are two main types of confocal microscopes: point-scanning and Nipkow disk (spinning disk). The following is an explanation of the characteristics of each type.

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

Nipkow disk type

The Nipkow disk confocal microscope is a method for acquiring images by rotating a disk with countless holes, known as the Nipkow disk. In contrast to point-scanning confocal microscopes, which irradiate lasers point by point and scan using mirrors, the Nipkow 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 Nipkow 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 Nipkow 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.