Introduction
The effective action of the spinning disk in enhancing resolution and contrast of a conventional fluorescence microscope, is noticeably related to two geometrical properties only. These are: pinhole size (D) and spacing between pinholes (S).
It is immediate to infer than, that by varying these parameters, one can get different results.
Figure 1: Schematic view of a spinning disk. Pinhole diameter (D) and spacing (S) shown in the inset
Pinhole size
An important relation is the one between the “Airy unit” of a given objective and the pinhole size.
The Airy Unit of the objective is a measure of the objective’s resolution and depends mainly on magnification (M) and numerical aperture (NA) given the same wavelength.
The ratio between disk pinhole diameter and Airy unit can be used as a simple and very informative data to make sure that the disk pattern used is appropriate for a specific setup. Such ratio is usually between 1 and 2:
In this range in fact there is a great improvement of the axial resolution (or confocality) compared to widefield and, at the same time, there is a good trade-off with the throughput of the system, meaning that a sufficient level of light can be collected from the specimen, therefore enabling high-speed and low-phototoxicity in acquisition.
For ratios < 1, the confocality is maximized at the expense of throughput. This situation is not acceptable for most applications and therefore it is not included as a standard choice in commercial spinning disk systems.
For ratios >>2, the throughput is improved but the confocality is too compromised to give a real benefit.
Pinhole spacing
If ideally all samples were thin and sparse, the resulting resolution would be mostly limited by the considerations made above about the pinhole diameter. In reality, every sample analyzed under a microscope is a 3D sample, characterized by multiple layers having different properties and different densities.
The resulting effect is that light coming from remote focal planes will pass through several adjacent pinholes at the same time if the pinholes are too close to each other in relation to the specimen thickness/density. This effect is called pinholes crosstalk.
Crosstalk affects the axial resolution (even in case the pinhole diameter itself is optimal) because light passing through adjacent pinholes contributes to the formation of the image from the wrong points of the specimen (remote points that should have been rejected, see below figure 2).
So it is important to verify the pinhole spacing selected in case of thick specimens.
Figure 2: Crosstalk representation in spinning disk imaging. If the pinholes are too close to each other in relation to the specimen thickness, out of focus contributions become important
Standard and custom disks
In the table below are shown standard pinhole diameters and spacing used in spinning disks:
Typical values | ||
Pinhole diameter | 50µm | 25µm |
Slit aperture | 50µm | |
| Typical values | ||
Spacing | 250µm | 400µm | 500µm |
While most commercial systems offer limited options in terms of disk patterns, CrestOptics offers the possibility to completely customize the disk geometry, tailoring the disk to relevant applications. For example:
- It is possible to have a disk with slits (i.e. continuous spirals instead of pinholes, see paragraph MATCHING YOUR SAMPLE).
- It is possible to realize different pinhole diameters and spacing after careful evaluation for specific projects upon user’s request.
- It is possible to easily swap between different patterns on the same setup by using a multi-pattern disk or by swapping disk boxes
Figure 3: view of the disk box with different disks, pinholes and slits
Matching objectives and disk pattern: typical use cases
We review here 3 typical cases of use and the recommended characteristics of the disk
Case 1
Application | Objective | Suggested Pinhole size |
High magnification, High resolution | 100x, NA>1.3, Oil immersion | 50um |
60x/63x, NA>1.3, Oil immersion |
Case 2
Application | Plan Apo Objective | Suggested Pinhole size |
| Facility with wide range of application | 100x, NA>1.3, Oil immersion | 50um |
60x/63x, NA>1.3, Oil immersion | ||
40x, NA>0.9, Dry | ||
20x, NA>0.7, Dry |
Case 3
Application | Plan Apo Objective | Suggested Pinhole size |
Low magnification, Low resolution | 20x, NA>0.7, Dry | 25um |
10x, NA>0.4, Dry | ||
4x, NA>0.2, Dry |
NOTE: In the specific cases #1 and #3 there is a proper matching between pinhole’s size and objectives properties, while in the case #2 there is a small compromise suggested for the simplicity of use of the setup. This compromise is widely accepted.
Matching your sample
Spinning disk confocal is used to image a variety of specimens: thin or thick, sparse or dense, highly scattering or transparent, etc…
It is then important to understand the effect that pinhole spacing can have on imaging performance depending on the sample properties, which we are separating in three main cases below.
In our discussion, when we refer to standard spacing, we mean a spacing that is typically 5 times larger than the pinhole diameter, while by larger spacing we mean 1.5/2 times larger than a standard spacing.
- Monolayer of cells or thin tissue sections (2-20 of microns). In this case pinholes with standard spacing are perfect to achieve optimal results. In case of very dim samples, it may be advantageous to use slits. They allow to increase the throughput (typically 3-4 times more compared to pinholes) at the expenses of confocality, which is anyway is typically 20%-25% better than widefield (compared to 40% improvement given by pinhole disk). A combination of pinholes and slits on the same disk gives the freedom to automatically switch between these two modes of acquisition when needed so it can represent an advantage for systems used for multiple applications.
- Thick tissue sections (20-200) or a scattering tissue. In these cases, it is very important to have a disk with larger spacing to improve the out of focus rejection. The larger spacing affects the throughput of the system. This means that this kind of disk is not optimal with dim samples.
- Thick clarified tissues. In this case, nevertheless the thickness is large (often >400um), the tissue has a very low level of scattering, therefore it is possible to use the standard spacing.
A
B
C
Figure 4: a) on the left disk with standard spacing, b) in the center same pinholes with large spacing, c) on the right disk with slits
To give an idea of these argument, let’s look at the following images acquired using different disks.
A. Nano-fibers filled with a fluorescent dye. thickness acquired with 60x NA 1.4 oil immersion objectives. These structures are thick and higly scattering.
Figure 5: a) on the left 3D reconstruction acquired with disk standard spacing, b) on the right 3D reconstruction acquired with disk with spacing that is double the standard (Sample Courtesy of Martin Haase, Utrecht University, Department of Chemistry (Nederlands))
Figure 6: XZ and YZ PROJECTION a) on the left with disk standard spacing, b) on the right with disk and a spacing which is double the standard. (Sample Courtesy of Martin Haase, Utrecht University, Department of Chemistry (Nederlands))
B. Fluorescent Beads. thick multilayer array of silica beads ( diameter) embedded in fluorescein solution. The basal layer is collected with Plan Apo λ 60x Oil. It is evident the increase in contrast when a scattering medium is acquired with a larger spacing disk.
Figure 7: a) on the left single plane acquired with disk standard spacing, b) on the right single plane acquired with disk disk and a spacing which is double the standard.
C. Clarified Tissues. thick tissue. Imaged in dual color GFP/CY5 with a 25x NA 1.05 Silicon objective.
Comparison between widefield and confocal shows that standard spacing guarantees the optimal contrast.
Figure 8: Max projection of 250 micron stack: a) on the left widefield acquisition, b) on the right confocal acquisition with standard spacing.
Figure 9: 3D volume a) on the left widefield acquisition, b) on the right confocal acquisition with standard spacing.
In the table below we summarize the considerations provided through the discussion:
| DIAMETER | Confocality | Throughput |
| Large diameter | – | + |
| Small diameter | + | – |
| SPACING | Confocality | Throughput |
| Large spacing | + | – |
| Small spacing | – | + |
