Q&A: How should I set up my microscope for creating images for CiliaQ analysis?

Q&A provided in December 2024 based on a Question submitted by Email.

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Question:

"I have some uncertainties regarding the image capture process, which is why I am reaching out to seek your guidance. Specifically, I would appreciate your insights on the optimal parameters for image capture tailored to cilia analysis. This includes considerations such as the number of stacks (or width of each stack), image quality, preferred image size (or zoom), and any other relevant features.

Additionally, I am curious about the preferred settings for z-stack image creation, particularly regarding the use of options such as max intensity.?"

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Answer:

CiliaQ has been created to analyze fluorescence microscopy images of cilia, including cilia in cell cultures, tissues, or organisms (e.g., zebrafish). To detect cilia, a channel is required that clearly shows cilia (e.g., antibody staining for ARL13B (which is enriched in cilia), staining for cilia with a dye like SiR-Tubulin, or ciliary labeling by expression of a cilium-targeted fluorescent construct. When it comes to the microscopy method, confocal microscopy or epifluorescence microscopy can be used. However, since cilia are tiny objects at the diffraction limit of light, it is optimal to image them with high-resolution 3D confocal microscopy.

The advantage of 3D confocal microscopy is that, since cilia often stick out from the cell in angles that are not parallel with the imaging plane, it allows to trace the cilia in 3D giving best length results. Furthermore, especially, when measuring ciliary intensity signals from an image where also signals in the cytosol or other cellular regions are present, 3D confocal microscopy is advantageous since it may allow to at least for cilia sticking out from the cell on the top or bottom to get measurements of intensity without signals from the cell body bleeding into the ciliary "mask". It is however possible and, for many research questions with stark changes, sufficient to analyze the cilia using epifluorescence microscopy without confocal resolution and in 2D.

The choice of imaging method and settings is largely dependent on the research question. In general, most accurate results can be obtained with the highest image and stack resolution. Yet, this requires long imaging times or may not be available in any lab, and - for quantifying large changes (e.g., in length) - high resolution might not be necessary. Thus, it is often required to find a pragmatic compromise for the imaging parameters.

Below you will find a few examples / recommendations / considerations.

Tips from my experience:

We typically image cilia with a 63x water- or oil-immersion objective, z-step sizes of 0.5 to 1 micron, and pixel sizes of 0.07 to 0.4 micron / px. Also 40x objectives can provide a sufficient resolution. CiliaQ analysis was easiest and most accurate at pixel sizes of 0.07 to 0.15 micron / px, and z step sizes of 0.5-0.7 micron / px.

Considerations for imaging settings:

• the finer the pixel size, the more accurate length measurements and intensity profiles along cilia will be, since fine changes in ciliary length may be detectable.
• the worse the image resolution, the harder to optimize the detection parameters and the higher the risk of inaccurate detection (due to reasons explained below)
• the larger the pixel size, the higher the risk for cilia being inaccurately detected or fractionated during segmentation, which in turn leads to detecting cilia in multiple and not one piece. E.g., when a large pixel size is used during imaging (e.g., 0.5 micron per pixel), cilia might only be one pixel wide in the image, meaning that ciliary objects in the segmentation step may be fractionated into multiple pieces if there is a stretch of the cilium that is not very bright.

Considerations for epifluorescence microscopy:

• For strong length changes a 20x or 40x imaging epifluorescence microscopy may be sufficient.
• Epifluorescence microscopy often creates off-focus blurring or artefacts in the image, which makes 3D analysis harder or may deform ciliary masks / detection. Thus, try to suppress such artefacts by using the Subtract Background Method in CiliaQ Prepararator with a relatively small radius that is corresponding to a little bit wider than the width of a sharp cilium in the image (e.g., 10 px in a high res, 4 px in a low res image). Please see this wiki post for details.

Considerations 3D vs 2D and for processing of 3D stacks:

CiliaQ can analyze the cilia from 3D images in 3D with tracing the ciliary skeleton in 3D. Thus, it is usually better submit 3D stacks to CiliaQ than maximum intensity projections of stacks. Rather make use of the 3D aspect to get more precise measurements than removing it by creating maximum intensity projections. The only condition in which a maximum intensity projection may be helpful is if you have very bad signal-to-noise ratio and cannot improve