GOING LIVE! How to gain an advantage for live cell imaging

Dynamic imaging can reveal so much about cell biology, but which camera system is best for your lab? Here we pit CCD and sCMOS together in a battle royale…

Performance. Price. Data management. These are a few of the most important factors cell biologists consider when purchasing a scientific-grade camera for their laboratory’s dynamic imaging experiments.

Life science researchers need a tool that will allow them to track rapid movements within a cell – with extreme detail and sensitivity, even in low-light conditions. But the device also must be within their budget and have the ability to capture and interpret the large quantity of valuable data derived from each experiment.

Unfortunately, buying decisions don’t usually come easy, especially for those who aren't well versed on the benefits and drawbacks of the different camera sensors available today. To make the evaluation and purchasing process easier, here we examine two popular imaging technologies – Charge-Coupled Device (CCD) and scientific Complementary Metal–Oxide–Semiconductor (sCMOS) cameras.

CCD cameras have dominated the scientific imaging market since the 1970s, but new sCMOS cameras are gaining ground, offering some unique selling points for life scientists.

Those familiar with sCMOS in the technology’s early days will remember how first generations of CMOS devices suffered from image quality issues. But recent improvements in sensor design have largely erased these deficiencies. New sCMOS technology is faster and thus able to better capture time-sensitive cellular events. Increased sensitivity allows these cameras to detect low luminescence signals in short exposure times, and greater resolution results in clearer depiction of small cell structures over a large field of view. Now, researchers are faced with determining which solution best suits their imaging needs: CCD or sCMOS?

CCD and sCMOS sensors perform a similar basic function: They gather light and turn it into electronic signals. Any relative strengths and weaknesses of the two technologies stem from the way they read the signal accumulated at a given pixel.

CCD cameras often use a global shutter so that every pixel in an image is exposed and captured at a precise moment in time. To package that information in a digital form, the pixel signals are sequentially streamed through a single output node to the analog-to-digital converter (ADC) in a process known as digitisation. This data is then processed to computer where it can be analysed and stored. Because every pixel is exposed simultaneously at the same moment in time, global shutter is particularly beneficial when the image changes drastically from frame to frame. However, CCD frame rates are limited by the rate at which individual pixels can be transferred and digitised – the more pixels that need to be transferred, the slower the total frame rate of the camera.

This design causes a bottleneck as millions of pixels stand in a single queue waiting for conversion. Before the next exposure can begin, every pixel in the existing frame must be processed. CCD cameras capture reliable static and time-lapse images for studies with moderate-to-long exposure times. However, the subsequent delay in charge transfer slows the camera’s total frame rate.

The pixel bottleneck of CCD technology isn't an issue for researchers whose microscopy focus is restricted to assays with longer exposures, such as slow cell migration and western blot gels. But frame rates do impact the user’s ability to rapidly study fast-moving cell phenomena, including vesicle formation, protein transport and calcium wave propagation. To capture these intracellular events, cell biologists need frame rates approaching 100 frames per second (fps) or faster. With CCD microscopy, they may be able to see tiny cell structures and measure electrochemical signalling, but accurate data about direction and speed will be lost. Unwanted artifacts such as motion blur and temporal aliasing also appear when frame rates are too slow for the task.

Some researchers find a solution for this with sCMOS chips, which position an ADC at the end of each pixel column. With this design the number of queues for conversion is multiplied – by the thousands when large numbers of pixel columns are involved. With sCMOS, digital information for each frame is rapidly generated, the caveat being that only one row of pixels can be digitised at a time by the row of ADCs at the edge of the sensor.

To avoid a drag on frame rate caused by waiting for all sensor rows to be digitised at the completion of an exposure, sCMOS cameras employ a rolling shutter design. Rather than waiting for an entire frame to complete its readout, rows that are digitised first can begin exposure of the next frame while the image sensor digitises signals from later rows. With regard to time, the camera pans across the image from top to bottom. Short time delays develop between neighbouring rows in the order of the sensor readout.

The advantage of a rolling shutter design is that frames can overlap and the overall frame rate is increased. This enables sCMOS sensors to provide frame rates that are 10 times faster than high-end interline CCD cameras. The down side is that the slight time difference between rows could possibly skew the data.

There is a countermeasure: Some sCMOS cameras offer a custom triggering mode that can achieve a global exposure with a rolling shutter readout to maximise the performance of sCMOS sensors. This triggering mode allows rapid shuttering of a high-speed light source so that the light source is pulsed only when all the rows in the frame are exposing at the same moment in time, thus achieving a global exposure. Meanwhile, the camera is kept in rolling shutter mode for digitising charge in order to maintain high frame rates and low read noise.

Researchers have long relied on CCD cameras for scientific imaging due to their quantitative performance and focused sensitivity. However, the scope of these devices is severely challenged as temporal resolution requirements increase. Recent advances in sCMOS sensors make sCMOS cameras ideal for high-speed, high-resolution cell biology, biophysics and ion transport physiology experiments.

The author:

James Joubert is an applications scientist at QImaging.

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