Selecting the right camera to suit your research can be a daunting task; there are many camera choices available, so what should you consider when choosing a digital camera for fluorescence imaging? Understanding your imaging requirements and the technology balance between features can allow you to allow you to put together the right system for your research needs.
Imaging Cells and Tissues
For wide-field fluorescence microscopy the typical camera will be used to image fixed cells and tissues. This example illustrates Alexa 488-labelled actin filaments in fixed endothelial cells imaged with two different cameras. There are a few key technical differences between the two cameras, resulting in a four fold cost differential. Using standard imaging conditions both cameras produce sharp, clear images of the actin filaments with no difference in noise or background levels. So what are the differences between the two cameras and what are the conditions when a higher spend is necessary?
The main difference between the two images is that the one on the left is taken with an uncooled camera and the one of the right is from a cooled camera. Historically cooling was necessary for fluorescence imaging, however, advances in camera sensor technology over the last 25 years mean that cooling is no longer required for routine fluorescence imaging. Cooled cameras are ideal for challenging fluorescence samples that require the use of more sophisticated microscopes such as Confocal microscopes. For most fluorescence imaging situations cooling is not required. Uncooled cameras can provide excellent, low noise images for general fluorescence applications at a lower cost than a cooled camera.
Noise
In fluorescence microscopy noise can impact signal to noise ratio and dynamic range. The signal to noise ratio represents a ratio of measured light to combined noise. A high signal to noise ratio will result in an image with a sharp contrast and good definition, as the signal to noise ratio decreases, that is the background noise increases, image definition and contrast decreases. Pixel binning can be used to reduce noise and improve the signal to noise ratio, but impacts spatial resolution. The main source of noise in fluorescence microscopy with shorter exposures is read noise which is typically much higher than dark current noise.
Dark current, another source of noise, does depend on the temperature of the sensor. Cooling the camera reduces dark current noise and should be considered for situations requiring long exposures or if light intensity will be extremely low, in such applications as in vivo bioluminescence or astronomy. In general, dark current will not have a major impact on typical fluorescence imaging situations. So unless you are imaging under situations requiring long exposures or low light intensity, dark current will have minimal impact on your imaging.
Other Considerations
Let’s not forget that there are a number of other imaging variables that are inherent to microscopy that will influence image quality. To maximize image quality, one should use a calibrated and uniform light source, high NA objectives, and filter sets that are in good condition. Combining the right camera technology with your microscope system will ensure that you get excellent images that properly convey the information required for proper analysis and research.
There are a number of specific and challenging applications, such as confocal microscopy, calcium signaling imaging, NIR fluorescence and absorbance measurements that have tougher camera requirements in order to achieve the results required. However, for general fluorescence imaging, high quality, sensitive images can be taken with lower cost, high quality uncooled scientific cameras, allowing you to invest in other equipment or personnel.
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