One of the basic and general questions about each confocal microscope setup is: which are the key steps to combine optical modules in the best way and, if there is a failure, what will go wrong?
We will briefly show here the criteria that guide the design, the installation, and the alignment of such modules, why these steps are important to give high quality images and how easy the entire process is.
Easiness of the process and stability
A guiding principle for the installation of these systems is: easiness, speed, efficiency and stability.
An easy alignment will take a very short amount of time for the installation (< 1 hour) and will lead to the nominal efficiency that the system can offer.
3 key steps of the alignment are:
- Internal alignment of the confocal spinning disk module
- Alignment to the microscope
- Conjugation of spinning disk pinholes to camera
Internal alignment
The importance of this kind of alignment lies in the fact that the alignment of the illumination axis from the fiber to the output optical axis of the confocal unit must be guaranteed.
So the excitation light will impinge on the filters, on the spinning disk and on the microscope’s optics with the correct angles, and the light throughput will be maximized through the microscope.
CrestOptics spinning disk unit
In this way, the spinning disk confocal unit is ready to be coupled to any other optical system.
One of the features of CrestOptics units is that they are internally pre-aligned at the factory and the alignment is preserved for the entire lifetime of the unit without the need of doing periodic corrections that would involve technical personnel. Hence the stability.
Alignment to the microscope
Every microscope is an optical system with its own optical axis. When the spinning disk unit is combined with it, the output optical axis of the spinning disk must be aligned to the optical axis of the microscope.
This goal is reached by centering the illumination beam with respect to a target mounted in place of the objective, at the back aperture.
With a simple tool it is possible to adjust the illumination beam and center it in a very short time.
Figure 1: photo of a centered illumination beam at the objective back aperture (X-Light V3)
The difference between images acquired with centered and off-centered illumination beam is evident from the following figure: when the illumination beam is off-centered the intensity levels can be 50% lower than the optimal values (or worse).
Figure 2: A) imaging with off-centered beam; B) same field of view with illumination beam correctly centered
Disk pinholes vs camera
The last step, the most important from the perspective of the spinning disk microscopy principle, is to guarantee that the spinning disk pinholes and the camera detector are optically “conjugated” to each other. The spinning disk is by construction positioned on a plane which is conjugated to the sample plane (either on the side port of the inverted microscopes or on the top port of the upright microscopes).
A conjugate plane P’ of a given plane P is that plane such that objects on P are imaged on P’.
In this way, the spinning disk lets the light coming from the objective focal plane go through and rejects the undesired light coming from the bulk of the sample (out-of-focus planes). The camera detector, being conjugated to the disk pinholes, collects all the desired light coming from layer filtered by the disk, improving the final axial resolution.
The difference between images acquired with wrong and correct focus of the pinholes is evident from the following figure:
Figure 3: A) image with pinholes focus NOT correctly set; B) same field of view but in this case with pinholes focus correctly set.
Let’s summarize the outcome of the main steps
- When the illumination beam is correctly aligned to the microscope, the intensity levels are optimal meaning that the sample is uniformly illuminated and quantitative data can be extracted from the images.
- When the pinholes are correctly focused, images are sharp and at the best confocal resolution.
Figure 4: Retinal Ganglion Cells at full spinning disk resolution with 40x water objective.
Figure 5: Drosophila image at full spinning disk resolution with 40x silicon objective.
