Exposure Time Settings Considerations

Guidelines on how to set up exposure times for CODEX® experiments such as CODEX® Antibody Screening & Validation and CODEX® Multicycles.

General Considerations

In fluorescence microscopy, optimal Exposure Times (E.T.s) are crucial to the success of the experiment. Optimal values are highly dependent on the camera, microscope and objective performances as well as on the sample characteristics such as intensity of the fluorescence signal, autofluorescence signal, and of the background noise. Hence optimal E.T.s need to be assessed by the user case by case. Potential starting values for CODEX® Antibody Screening & Validation and CODEX® Multicycles performed with Leica and Keyence microscopes are reported in the following table for FFPE and fresh-frozen tissues.

Starting exposure times values for CODEX

Microscope	Sample	DAPI	AF 488	ATTO 550	Cy5/ AF 647	AF 750
Keyence	FF	10 ms	250 ms	350 ms	350 ms	-
Keyence	FFPE	10 ms	-	350 ms	500 ms	500 ms
Leica (5+ Z-slices)	FF	50 ms	400 ms	400 ms	300 ms	-
Leica (3 Z-slices)	FF	50 ms	400 ms	400 ms	400 ms	-
Leica (5+ Z-slices)	FFPE	50 ms	-	400 ms	300 ms	700 ms
Leica (3 Z-slices)	FFPE	50 ms	-	400 ms	400 ms	700 ms
Zeiss (50% LED)	FF	5 ms	500 ms	500 ms	400 ms	-
Zeiss (50% LED)	FFPE	5 ms	-	350 ms	500 ms	500 ms

Parameters influencing exposure times

The Microscope

The camera acquisition settings such as Gain and Binning influence the values of optimal E.T.s (e.g. the higher the gain, the lower the exposure time), however for CODEX® experiments, no binning is generally performed and the Camera Gain is fixed at the lowest value. The camera-specific performance also has a strong impact on required E.T.s, however microscopes for tissue imaging from Keyence and Leica have shown very similar performances within the same model at a given magnification.

The Light

Additional important parameters are the wavelengths of the different Fluorescence Channels (it impacts the background noise - auto-fluorescence - and the camera sensitivity) and the fluorophore of choice (each one has a specific brightness). Generally, the camera sensitivity decreases at higher wavelengths (moving from the blue to the red and then to the NIR), while the auto-fluorescence is maximum in the FAM (FITC/GFP) channel and decreases going towards the red. CODEX has only one or two different fluorophores per channel, which makes E.T. optimization easier.

The Sample

Important factors to consider are the specific antibody and the tissue quality. Abundant antibodies are likely to give a strong signal, while using high-quality tissues minimizes auto-fluorescence and imaging artifacts.

Optimization of exposure times

E.T.s must be optimized for defined combination of microscope, fluorophore, antibody and tissue. The rule of thumb for E.T. optimization is to maximize the signal to noise ratio (SNR) while avoiding saturation of the camera (see Appendix C for more details about SNR).

The signal intensity needs to be maximized while making sure it does not reach the saturation threshold, for example, using a 16-bit Camera, maximum values should always be about 20% lower than 65535, the saturation value. At the same time the background noise needs to be kept as low as possible. The optimal exposure time reflects the best combination of these leading factors.

Optimization procedure

We recommend capturing images with different E.T.s and comparing the corresponding SNRs. On Keyence microscopes, this can be done by going to the "Image" tab of the acquisition software. By moving the mouse in different regions of the image you can obtain signal intensity values in areas where the antibody staining is abundant. The noise intensity can be observed by moving the mouse to regions where the tissue does not present the targeted antigen. The best exposure time can be identified as the one giving images that are as close as possible to the following parameters:

• Maximum signal intensity that is still lower than the saturation value (65535 for 16-bit images) by at least 20%.

• Highest possible ratio between the signal and the background intensities (SNR).

Example: The following fluorescence images of DAPI nuclear staining on a FFPE human-tonsil show how increasing the exposure time influences the image intensity distribution in the overall field of view, eventually leading to saturation. The corresponding intensity histograms in a region on interest (ROI) are also reported.

Exposure Time Optimization for Antibody Screening & Validation

For Antibody Screening and Validation, start with exposure times illustrated in Table 1. Depending on the SNR and the occurrence saturation, E.T. can be increased or decreased following the guidelines reported in this page.

Exposure Time Optimization for CODEX Multicycle Experiments

The determination of E.T.s for multicycle experiments could be time consuming as it cannot be done in real time during multicycles, but only afterwards, however E.T.s optimized in screening and validation experiments usually work well for multicycles. Alternatively, E.T.s from Table 1 represent a good starting point: E.T.s can be then further optimized performing multiple multicycles on the basis of the guidelines reported in the Optimization Process.

FAQ

Can I simply use the longest E.T. that does not saturate my signal?

No, the downside of long E.T.s is that they also increase the background intensity (e.g. the noise), so exposure times should be finely tuned to maximize the signal intensity while keeping the background level low.