1. UVP BioImaging Systems Solutions for the Science of Life Digital CCD Cameras 101

2. Camera Definitions CCDCharge Coupled Device (either interline transfer or frame transfer sensor designs) ResolutionHorizontal x Vertical Pixels (determines the minimum resolvable spot size) PixelPhotodiode or Full Well Depth of electrons (measured in µm²) Full Well DepthMeasurement (in electrons) of how much charge a pixel can hold before saturation (typically 1000 times the cross-sectional area of the pixel) Readout SpeedHow fast the charge is digitized off the chip (higher readout speed = higher read noise) 10-30 frames per second is typical and appears like live video

3. Camera Definitions Dynamic RangeAbility to resolve low and high light levels in the same image (measured in maximum gray scales, OD, orders of magnitude, or bit depth) Quantum EfficiencyMeasurement of the CCD’s ability to convert photons into electrical charge (maximum 45-60% in the blue-green, but higher in back-illuminated CCD’s) SNRSignal-to-Noise Ratio SignalPhotons successfully converted to electrons in the digitizer NoiseReadout Noise (Electrical Noise), plus Dark Current Noise (Thermal Noise) plus Photon-Shot Noise (Non-Converted Photons from the Chip) typically 5-12 electrons

4. Camera Definitions Readout NoiseNoise created by the digitization of the electrical charge in the CCD amplifier (faster the readout, the higher the noise) Dark Current NoiseNoise contribution from the thermal affect of heating on the CCD (dark current noise is reduced in half for every 6-9°C cooling) DigitizerSelected to match the SNR of the CCD CCD GradeSurface quality of the sensor (grade has no pixel defects, grade 2 has defects; better grade chips have fewer hot pixels and, therefore, are better for long term integration)

5. Camera Definitions BinningCombines neighboring pixels to increase signal at the cost of resolution (readout rate or frame rate is also increased due to less data being processed from the chip) Bit DepthResolvable amounts of image data (most high-end scientific grade cameras in our market can resolve 10-12 bits of data, but an SVGA monitor can only resolve 8-bits on its screen and the naked eye can only ascertain approximately 6-bits of data) this number is generally the square root of the full well electrons expressed in the maximum number of resolvable gray scales)

6. Camera Calculations SNR=Signal / Total Noise =Full Well Electrons / Read Noise =Dynamic Range =Maximum Gray Scales =2 (CCD Bits)

7. Camera Calculations Example 1: BioChemi Cooled CCD 1.3M pixel CCD with 25,000 electrons/pixel and 7 counts of read noise and binned 1x1 SNR = 25,000/7 = 3571 maximum resolvable gray scales CCD Bit Depth = (log max gray scales/log 2) = 3571/0.3 = 11.8 Bits = 12 Bit Performance = 4096 gray scales

8. Camera Calculations Example 2: BioChemi Cooled CCD 1.3M pixel CCD with 25,000 electrons/pixel and 7 counts of read noise and binned 2 x2 SNR = (25,000 x 2 x 2)/7 = 14,285 maximum resolvable gray scales CCD Bit Depth = (log max gray scales/log 2) = 14,285/0.3 = 13.8 Bits = 14 Bit Performance = 16,384 gray scales Resulting Camera Resolution = 1.3M/(2 x 2) = 325,000 pixels

9. Camera Calculations Example 3: BioChemi Cooled CCD 1.3M pixel CCD with 25,000 electrons/pixel and 7 counts of read noise and binned 4 x4 SNR = (25,000 x 4 x 4)/7 = 57,142 maximum resolvable gray scales CCD Bit Depth = (log max gray scales/log 2) = 57,142/0.3 = 15.8 Bits = 16 Bit Performance = 65,536 gray scales Resulting Camera Resolution = 1.3M/(4 x 4) = 81,250 pixels

10. Common Questions Why do I need more than an 8-bit camera system when I may never realize the full dynamic range because my light levels are so low? By utilizing a higher bit depth camera with lower overall noise and higher signal-to-noise ratio, you will have a better chance of resolving the low light signal.

11. Common Questions Does a system with higher quantum efficiency always have more sensitivity? Not necessarily. Quantum efficiency is not the only variable in the determination of sensitivity. Total noise is also a very important factor. Therefore, a better measurement of sensitivity is the signal- to-noise ratio at the specific wavelength of interest.

12. Competition Competitor tells your researcher they have a true 16-bit camera (Syngene and Alpha Innotech). This means it has a SNR of 65,536 and can resolve all of these gray scales at full resolution. The lowest read noise of any scientific CCD on the market is 5 counts (the OptiChemi CCD). Therefore, the competitor’s CCD would require each pixel well to have 327,680 electrons. The BioChemi CCD has 25,000 full well electrons/pixel and each pixel is 6.7µm. This equates to a 2/3” CCD chip. The competitors CCD chip would have to be 13 times this size (327,680/25,000) for each pixel to hold 327,680 electrons. This equates to an 8-2/3” CCD chip with 87.1µm pixels! This size chip does not exist, and if it did would cost millions of dollars!

13. Competition Competitor convinces your researcher that he has a true 16-bit camera (despite your attempt to invoke reality into his analysis of imaging systems), because he is producing large file formats with 65,536 levels of gray scale. This is truly “smoke and mirrors” and can be accomplished two ways: (1) The competitor (Syngene) has installed a very inexpensive analog to digital converter in the camera to convert a 10 or 12-bit acquired image to 16 bit gray scale. This is highly unethical and gains the user nothing except the management of large file sizes (3-4MB each) with no additional image data. If inspected properly in LabWorks with the “Display Range” tool, the true dynamic range of this camera will become apparent.

14. Competition (2) The competitor (Alpha Innotech) actually does capture a true 10 or 12-bit image, so they can avoid the large file sizes, but automatically convert the saved file to a 16-bit gray scale image. This is also highly unethical and provides the user with no additional image data. If contested on this issue the argument this competitor uses is that 12-bit file formats are not usable in most image processing programs (Adobe PhotoShop, Paint Shop Pro, etc) so they automatically convert the file to the next highest acceptable format, 16-bit. While this argument has some validity, it is still deceitful. What we provide in LabWorks is the ability to operate the BioChemi and OptiChemi CCD’s in 12, 14 or 16-bit performance modes, save them as the truly acquired image and allow the user to convert the image to 16-bit or RGB formats, if desired.

15. Remember Increasing the binning of a camera, increases the sensitivity and frame rate of the camera, but reduces the resolution. Faster Frame Rates produce more read noise and reduces the signal-to-noise ratio and resulting dynamic range of the camera.