Question

What are the primary differences between a laser scanning confocal and a spinning disk confocal microscope? Consider

(a) the physical design of the microscope

(b) the method of forming an image

(c) the detector that is used to capture the image.

Answer 1

a) Laser scanning confocal

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1.Physical design of the microscope

In laser scanning confocal systems, point laser excitation and the emission pinhole combine to remove most out-of-focus light

The emission pinhole size is adjustable

2) Laser scanning confocal microscopes offer advantages, particularly in imaging thick cells with a high density of intracellular components. These instruments allow extremely thin optical sectioning of specimens, enabling structures and intracellular features deep within the specimen to be viewed, and the laser provides a very intense, small spot of light that can easily be used for purposes other than imaging, such as photobleaching and molecular uncaging.

3) The point acquisition requires point detectors, and photomultiplier tubes are only 30 to 50 percent efficient at converting light energy into a detectable signal. The point-by-point image acquisition can capture the thinnest obtainable sections but at the expense of speed. The system may take between 0.3 and 1.0 s to capture a 1024 × 1024-pixel image and may miss a burst of light that lasts only 0.1 s.

b) spinning disk confocal

1) These systems use a rotating disk with a pattern of slits or holes installed in a microscope plane that is conjugate with the specimen. The opaque areas of the disk block out-of-focus light from both the excitation and emission light paths.

The emission pinhole is fixed

There are two principal types of spinning disks. Nipkow disk systems provide good confocality and improved throughput, but their pinholes do not permit as much light transmission as some experiments require. The Yokogawa modification to this system adds a second disk of microlenses to focus the excitation light and to improve transmission efficiency. Typically configured with laser sources, these systems are limited in their excitation wavelengths based on the laser lines installed.

2) This confocal method is popular for applications that require fast image acquisition. However, the systems’ larger pinhole or slit openings cannot deliver optical sections with the same thinness as those that are provided by their laser-based cousins. Compared with laser scanning systems, confocal systems offer the benefit of reduced toxicity to specimens during time-lapse imaging of living cells.

3) The technique is highly useful for high speed imaging of living cells expressing fluorescent proteins or stained with membrane-permeant synthetic dyes. Photobleaching and phototoxicity are reduced with spinning disk microscopy, but a tighter light budget requires very sensitive camera systems for optimum signal detection.