EMCCD CAMERAS

EMCCDs have an on-chip electron multiplying stage designed to effectively reduce the read noise to zero. One drawback of this kind of multiplication is that it adds a noise factor of 1.4.  In addition to this factor there is excess noise due to spurious charge generated by simply shifting the readout register. More information about EMCCD cameras and how they compares to CCDs can be found here.

The NeuroCCD-SMQ has a read noise of 12 e- at a frame rate of 2000 Hz. A noiseless EMCCD would thus have lower noise than the NeuroCCD-SMQ at photon counts of 100 photons/pixel/frame. With the excess noise of the EMCCD this crossover occurs at even lower photon counts. Most neurobiology and cardiology applications have photon counts of >>100 photons/pixel/frame.

Signal-to-Noise Ratio Comparison of NeuroCCD-SMQ and EMCCD

The following graph compares the singal-to noise ratio (SNR) of the NeuroCCD-SMQ and of an EMCCD camera, at low light levels, at 500HZ :

	Algorithm: Where:      I – Effective Light Intensity,      LI – Light Intensity,      N – Noise (RMS),      Ns – Shot Noise,       Nr – Read Noise,      QE – Quantum Efficiency,      PF – Penalty Factor (shot noise)
Theoretical Limit : QE - 100%, ReadNoise - 0e-, No Shot Noise Penalty factor (PF = 1.0); NeuroCCD-SMQ : QE - 90%, ReadNoise - 6.5-7e- (at 500fps, 30X & 10X gain), No Shot Noise Penalty factor (PF= 1.0); EMCCD : QE - 90% EM on : ReadNoise - 0e- + extraneous noise (0 is used), Shot Noise Penalty factor >= ( is used); EM off : ReadNoise - 51e-, No Shot Noise Penalty factor (PF= 1.0);
A typical example of ArcLight operating range for Drosophila is conceptually placed in the graph based on   the photon measurement and DF/F of 5-10% using the optimal setting of the NeuroCCD-SMQ system.   The Red region shows the S/N advantage of NeuroCCD-SMQ over EMCCD (EM on). EMCCD (EM on) has  advantage over NeuroCCD-SMQ when photons/frame/pixel is less than 100e- (near the origin).

In terms of signal-to-noise ratio the best a camera system can do is to approach the shot-noise limit. Some of the factors in the camera design affect how close the camera approaches shot-noise performance and others affect the range of light intensities over which this performance is achieved. In addition, extraneous factors can make the performance worse than the shot-noise limit.

Shot Noise

The limit of accuracy with which light can be measured is set by the shot noise arising from the statistical nature of photon emission and detection. Fluctuations in the number of photons emitted per unit time will occur, and, if an ideal light source emits an average of N photons/ms, the root- mean-square deviation in the number emitted is the square root of N. The effects of this relationship are indicated by the green line in the Figure which plots the light intensity divided by the noise versus the number of photons measured per ms. At high intensities this ratio is large and thus small changes in intensity can be detected. For example, at 10¹⁰ photons/ms a fractional intensity change of 0.1% can be measured with a signal-to-noise ratio of 100. On the other hand, at low intensities this ratio of intensity divided by noise is small and only large signals can be detected. For example, at 10⁴ photons/msec the same fractional change of 0.1% can be measured with a signal-to-noise ratio of 1 only after averaging 100 trials.

The figure also indicates the performance of the two RedShirtImaging systems, the photodiode array system (NeuroPDA; blue line ) and the CCD camera system (NeuroCCD; red line). NeuroPDA approaches the shot-noise limitation over the range of intensities from 3x10⁶ to 10¹⁰ photons/ms. This is the range of intensities obtained in absorption measurements and fluorescence measurements on in vitro slices and intact brains. On the other hand, NeuroCCD approaches the shot noise limit over the range of intensities from 5x10³ to 5x10⁶ photons/ms. This is the range of intensities obtained from fluorescence experiments on individual cells.

Saturation

The high intensity limit of the NeuroCCD is set by the light intensity which fills the electron wells on the CCD chip. This accounts for the bending over of the camera performance at segment B in the Figure. Even though the NeuroCCD camera has a large well-size compared to other CCD cameras, it will not be optimal for measurements of absorption or fluorescence measurements on in vitro slices and intact brains. The light intensity would have to be reduced with a consequent decrease in signal-to-noise ratio.

The NeuroCCD saturates at light levels that result in resting light intensity readings of 0.3 volts at 1 Gohm on NeuroPDA. In fact, the NeuroCCD saturates at about the light intensity where the NeuroPDA becomes shot-noise limited. Thus, as indicated in the figure, the range of light intensities where the two systems are optimal have very little overlap.

Dark Noise

Dark noise will degrade the signal-to-noise ratio at low light levels. Because the NeuroCCD is a cooled CCD camera, its dark noise is much lower than that of the NeuroPDA. The excess dark noise in NeuroPDA accounts for the fact that segment C (for NeuroPDA) in the Figure is substantially to the right of segment D (for the NeuroCCD). The dark noise of the NeuroCCD is lower than other commercially available CCD's in its performance range.

Extraneous Noise

A second type of noise, termed extraneous or technical noise, is apparent at higher light intensities where the sensitivity of the measurement is high because the fractional shot noise is low. There are several sources of extraneous noise. One type is caused by fluctuations in the output of the light source. Other sources are vibrations and movement of the preparation. Extraneous noise accounts for the break in the NeuroPDA curve at A.