Application Note: Cameras for Single-Molecule Imaging

Author: Dan Croucher, Applications Team Manager, Photometrics

Prime 95B Scientific CMOS (sCMOS)

Figure 1: Photometrics Prime 95B

Single-molecule imaging is typically a very low-light technique so one of the main challenges is to collect as many of the emitted photons as possible to maximize signal to noise ratio and minimize excitation light intensity to lessen the impact of photobleaching and photodamage on samples.

Single-molecule imaging has historically been performed with high-performance EMCCD cameras. However, with the advent of back-illuminated CMOS cameras, EMCCD cameras are being used less and less for this technique. The Photometrics Prime 95B (Figure 1) is a high sensitivity sCMOS camera designed for imaging at extremely low light with high speed and low noise.

The almost perfect, 95% quantum efficient (QE) sensor of the Prime 95B has equivalent sensitivity to an EMCCD camera but with the much larger field of view (1200x1200 pixels, 18.7 mm diagonal) and high speed (82 fps, full frame) expected of a CMOS device.

It also doesn’t suffer from the complications associated with an EMCCD camera such as EM-gain degradation, where the effectiveness of the EM gain process is reduced with usage. This results in EMCCD cameras needing to be regularly calibrated which is not only an annoyance, but there eventually comes a point when no more can be done and no signal improvement can be achieved when using EM-gain.

The EM-gain process also introduces excess noise factor, an extra source of uncertainty caused by the probability of impact ionization creating an electron. Excess noise factor multiplies other camera noise characteristics by a factor of √2, reducing the overall signal-to-noise ratio. CMOS devices such as the Prime 95B do not suffer from any of these issues. The signal-to-noise improvement of the Prime 95B can be clearly seen in Figure 2 where a comparison was performed between the Prime 95B and 1024x1024 EMCCD camera, respectively. The Prime 95B image looks cleaner, details are sharper and structural elements are identified that can’t be seen with the EMCCD camera.

The Prime 95B also has large, 11x11 µm pixels which provide a large 80,000 e- full well capacity whilst maintaining a low 1.6 e- read noise, giving the 95B a very high dynamic range ideal for performing high contrast imaging. Larger pixels also fit perfectly with high magnification objectives, achieving Nyquist sampling without the need for any additional optics with 100x magnification typically used for single-molecule imaging.

Furthermore, as microscopy equipment moves towards larger-scale approaches, there is an increasing demand for higher throughput capabilities. To this end, larger format versions of the Prime 95B has been created with 22 mm and 25 mm diagonal fields of view. The larger field of view options offer a significantly increased field of view over EMCCD devices. This can be seen in Figure 3 which shows how much more of a sample can be imaged by taking advantage of a larger field of view.

Figure 2: Comparison of image detail visible with the Prime 95B and a 1024x1024 EMCCD camera, highlighted with light blue circles.
Left Image captured using a 100 ms exposure time with the Prime 95B
Right Image captured using a 100 ms exposure time with a 1024x1024 EMCCD camera with 1200 EM-gain
Figure 3: Field of view comparison between a 512x512 EMCCD with an 11.6 mm diagonal, the Prime 95B with an 18.7 mm diagonal and the Prime 95B 25 mm with a 25 mm diagonal

Prime BSI Scientific CMOS (sCMOS)

Figure 4: Photometrics Prime BSI

The Prime BSI (Figure 4) presents an alternative option for single-molecule imaging where higher resolution imaging is required.

Similar to the Prime 95B, the Prime BSI is back-illuminated with an almost perfect 95% quantum efficiency but with a smaller, 6.5 µm pixel. This allows the Prime BSI to deliver the highest amount of signal whilst matching Nyquist sampling with 60x magnification.

The Prime BSI retains the signal to noise ratio advantages over EMCCD cameras because, as a CMOS device, it doesn’t suffer from EM-gain decay or excess noise factor. It also delivers a larger 18.8 mm field of view (2048x2048 pixels) and a fast 63 fps full frame framerate.

When compared to conventional sCMOS devices, which also typically feature a 6.5 µm pixel, the higher quantum efficiency of the Prime BSI means that the exposure times or excitation light intensity could be reduced further, preserving the physiological conditions of the sample.

We would encourage anyone performing single-molecule imaging to request a demonstration of the Prime 95B or Prime BSI to see the advantages these cameras can provide.