Image at Speed with the Sona-6 Back-illuminated sCMOS

The Sona-6 back-illuminated camera offers a very wide performance envelope from a sensor that gives an excellent balance of sensitivity, resolution and speed. This makes it a highly applicable and effective imaging solution for many different applications from routine fluorescence imaging to localisation based super resolution like DNA-PAINT. The updated Sona-6 introduces an additional new High-Speed Mode. This further extends the suitability to applications that demand the highest possible speeds- but maintain a low noise floor and quantitative accuracy e.g. calcium flux and ion imaging, FRET, FRAP, blood flow.

Important Requirements for Imaging at Speed

The ability of a camera to run at very high frame rates only tells part of the story. There are other criteria that must be met to make high-speed imaging possible for real-world imaging applications. Failure to meet some of these requirements would mean that the camera may offer very impressive imaging performance on the specification sheets, yet will not translate across into practice. These requirements are outlined below:

• High Signal to Noise. Short exposures mean less photons. High 95% QE sensors can help make the most of the small signal that there is, but the noise floor becomes very important. Normally running sensors faster means higher noise – with read noise and fixed pattern noise effects increasing. If the noise floor cannot be kept low, then the signal to noise ratio becomes low and we either need to extend the exposure to collect more signal, but reduce temporal resolution, or we may need to use another mode in a smaller ROI to keep the signal to noise high. By carefully optimising the high-speed mode of the Sona-6, the noise floor is only increased by 0.3e- over the High Dynamic Range mode thus when combined with the high QE, it is possible to attain high signal to noise at high speeds.
• A suitable Dynamic Range. Fast imaging applications often involve a moderate change in signal intensity. To measure changes in intensity it is therefore necessary to have a suitably large well depth that allows changes in signal intensity to be measured without saturation or loss of linearity. For example, a very small well depth of 200-400 electrons would not be useable for all but a few imaging scenarios. It would also be very difficult to capture the signals within this small window of measurement. The Sona-6 has a well depth of 1,800e- in High-Speed mode and this is found to provide a sufficiently wide signal handling capacity for many measurements. For exceptionally wide dynamic range, you can switch to HDR mode for a full 16-bit data range.
• Quantitative Accuracy. Many high-speed measurements seek to measure changes in intensity over short time intervals and derive rates, velocities etc. These measurements are inherently based on the ability to make accurate and precise measurements of intensity. The detector must thus have sufficient temporal resolution but also the precision and accuracy – otherwise we cannot infer meaningful measurements. The Sona-6 High Speed mode maintains a high quantitative accuracy of >99.5% that ensures confidence in image data. Additionally, a digitization range of 11-bit allows precise measurement over the available dynamic range. A further reduction to 8-bit mode would further limit the practical use of high-speed operation to a smaller number of applications where quantitative accuracy is not important.
• Stable data transfer: Kinetic series and burst acquisitions place great demands on data handling throughout the readout chain to the data storage e.g. SSD drive. Deficiencies in data throughput efficiency can lead to data loss and drop-outs within longer experiments. Sona-6 features a number of key technologies to ensure stable data transfer. These include large on-camera memory buffers, efficient data pipelines and stable, high data rate capable connections. The Sona camera platform has a 2-lane coaXPress data interface. This maximises the frame rate possible so that the sensor readout is the limit, and transmission of data is not a bottleneck. The combination of high-speed mode and coaXPress of the Sona enables higher sustained data rates than other cameras.
• Temperature Stability: sCMOS cameras are calibrated at specific temperature set-points. If the temperature drifts from this set temperature the response of pixels starts to deviate. Running the sensor faster generates more heat. Cameras that have limited cooling capacity cannot regulate the sensor temperature, leading to increased noise and potentially interruptions in the imaging experiment. Sona-6 has a vacuum sealed, high-capacity thermo-electric cooler that locks sensor operating temperature to the set point.

Figure 1: The grey line indicates an ideal camera that is perfect in every parameter. The new High-Speed mode of the Sona-6 has been configured and optimised to provide excellent performance for a wide range of high speed imaging applications.

High Speed Mode: Principle of Operation

In normal operation modes the sensor reads in a rolling shutter fashion, 1 row at a time. In high-speed mode, the sensor runs both of the readout channels available, simultaneously in 11-bit. The even and odd rows are thus read at the same time and has the effect of doubling the speed for each frame to be read out. After readout the 11-bit datastream pairs from each of the odd and even readout channels are re-combined. Finally, the data is packaged into a 12-bit format for downstream compatibility.

Frame Rate Comparisons

The frame rates for each of the three operational modes are outlined below for both USB and CoaXPress operation. The higher data throughput of the 2 lane coaXPress interface available on the Sona can be seen for high-speed mode operation as well as the higher ROI sizes for High Dynamic range mode. For USB 3 operation, the maximum frame rate supported by the interface will be ~40 fps regardless of mode in full frame. Burst rates for a series of frames will be higher, until the data reaches that of the maximum transfer rate of the interface. Note that with sCMOS sensors, including Sona, the frame rate is dependent on the height of rows. Cropping the row width does not impact speeds. This is because each row must be read out by the sensor as a complete row, and then the unneeded row information removed before it is sent on to the control PC.

Note that frame rates are given using Solis Imaging Software. Frame rates will vary with different software and other overheads of complete imaging systems.

Technical Parameters of Different Operational Modes

The 3 different modes available on the Sona-6 series offer exceptional imaging flexibility. Each mode has been carefully configured and optimised to best match the intended applications. Refer to the following table to help you determine which imaging mode is suitable for your intended use.

Note that frame rates are given using Solis Imaging Software. Frame rates will vary with different software and other overheads of complete imaging systems.

Applicability and Compatibility

The updated Sona Extreme specification which includes the High Speed mode, has been applied to all Sona-6 cameras from November 2022. This also applies to Marana-6 cameras featuring the same GS2020BSI sensor.

The updated Sona will share software compatibility with the original Sona-6 in Low Noise and High Dynamic Range modes. Camera features are controlled at the camera level meaning that updates do not impact the SDK level and interactions with the acquisition software. Note that CoaXPress and High-Speed modes may not be validated in certain 3^rd party software. Contact your software vendor for further information.

Guidelines for use of High-Speed Mode and other modes for fast imaging applications

It should be noted that general applications will be well covered by the low-noise and high dynamic range modes since typical fluorescence imaging applications require exposures in the region of 50-250 milliseconds or longer. It is possible to crop the sensor in these modes and increase frame rates while keeping the main benefits of that mode:

• Use Low Noise mode at reduced ROI sizes for maximum sensitivity of weak signals e.g. low abundance labels, or weakly emitting labels
• Use High Dynamic range mode at reduced ROI sizes for fast imaging of high contrast images

When these modes are not sufficiently fast, or over as wide a field of view as desired, high-speed mode will provide a much higher speed capacity. Please refer to the following useful information on running at high speeds:

• For very fast operation it is recommended to run at 0°C temperature. The dark current is a minimal component of overall noise under very short exposure conditions. What is important is tight regulation of the sensor temperature over time, and across the sensor when operating at high speeds. This is achieved by the optimised cooling system implemented on the Sona cameras.
• The Sona camera has been designed and tested to ensure minimal vibration, (fan speed is also adjustable) however some applications are very sensitive to vibration e.g. localisation based super resolution, high-speed, high-magnification and electrophysiology. To eliminate vibration induced by air cooling in such sensitive applications it is therefore recommended to use the liquid cooling option of the Sona and consider other measures within the full imaging system to isolate vibrations.
• High speed operation will result in large volumes of data fast. Ensure that the disc that data is written to is a high-speed drive (SSD or RAID SATA) that will handle the sustained data rates and there is sufficient storage capacity for the duration of a given experiment.