![]() ![]() A typical setup is illustrated in Figure 4. The light source and cameras are then routed through the disk module. The spinning disk confocal unit is typically a self-contained module that can be added to the camera port of a microscope. The unit pictured can accommodate two cameras observing different wavelength channels, due to the internal dichroic beam splitter and filter wheels. This unit includes the motorized spinning pinhole disk, as well as a microlens disk, and can be mounted to the camera port of a microscope to provide confocal images. Spinning Disk Confocal Microscopyįigure 4: Schematic cut-away diagram showing a Yokogawa Electric Corporation spinning disk confocal unit. Such cameras can operate at quantum efficiencies of up to 95%, in contrast to the 10-30% efficiency of the photomultiplier tube of a laser scanning confocal. But also using array-based rather than point-based scanning means the system can take advantage of the latest state-of-the-art Scientific CMOS and EMCCD cameras in place of the photomultiplier tube. The parallelization of pinhole scanning not only vastly improves the speed of acquisition. The rotation speed of the disk, therefore, determines the maximum image acquisition speed. The holes are positioned so that every part of the image is scanned as the disk is turned.Įach area of the image is scanned by a single pinhole (typically) every 30° rotation of the disk. When spun, the pinholes scan across entire image rows in sequence. The primary means of achieving this is through arranging pinholes in a spiral pattern, etched into an opaque disk (Figure 3). Spinning disk confocal microscopy solves the scanning problem by using multiple pinholes. As the disk spins, the Archimedean spiral causes the pinholes to scan the image. The pinholes are of diameter □ and are separated by a distance □. The axial (z-) resolution of a conventional fluorescence microscope is however poor – on the order of 2.5 μm – much larger than the scale of many biological features.įigure 3: Nipkow-Petran disk with spiral pattern of pinholes. Understanding 3-dimensional structure also requires the ability to resolve distinct details. This is particularly challenging when attempting to observe processes occurring inside cells, which will be obscured by fluorescence from the cell membrane. As most biological samples are 3-dimensional structures with fluorescent signal emission across the whole thickness of the sample, this out-of-focus light can considerably obscure whatever fine details are the microscopist’s target, as is evident in Figure 1. A conventional fluorescence microscope can focus on a particular plane but has no way of preventing out-of-focus light from reaching the detector. The main challenge to accurate and clear 3-D image reconstruction is how to deal with light that comes from above or below the sample plane. This makes it a very common choice for studying 3-D structure, fast dynamic processes, long-term time-lapse or details inside the cell membrane, all possible with live cells. Compared to other optical sectioning techniques, Spinning Disk confocal microscopy is high-speed, high-sensitivity and simple to implement. This process removes the out-of-focus light from other planes. Confocal microscopy uses optical sectioning to take multiple, thin, 2-dimensional slices of a sample to construct a 3-dimensional model from them. Light from out of focus planes, and from bright features above and below the desired plane cannot be blocked with conventional fluorescence microscopy.Ĭonfocal microscopy offers the solution to this issue. This gives no control over where within the light path the returning light comes from. These problems are difficult to overcome due to the need to pass light through the entire sample to illuminate the chosen image plane. Secondly, many processes biologists would want to study occur inside biological structures, but other cell features such as the cell membrane block a clear view. Firstly, biological specimens are 3-dimensional structures so to fully understand them we often need to construct 3-dimensional images. There are two significant challenges in biological imaging that conventional fluorescence microscopy cannot overcome. ![]()
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