Limitations of Hamamatsu ImagEM "Real-time Image Processing" Features

Technical Note: 447
Reads: 5321
Creation Date: 07/12/2009
Modification Date: 07/12/2009

Abstract

The Hamamatsu ImagEM cameras (C9100-13 and C9100-14) include real-time image processing features. This technical note discusses the limitations of some of these features when used for fluorescence imaging.

Background Subtraction and Shading Correction

The "real time background subtraction" and "shading correction" tools on the ImagEM work by processing a single frame. These tools are safe to use for any type of acquisition as they do not depend on successive frames being related.

Spot Noise Reducer

The "spot noise reducer" tool works by comparing each frame to previous ones and eliminating small bright signals that only appear in a single frame. This tool does not work well where successive frames bear little or no relation to each other - it is very likely that spurious events will either not be rejected, or that small bright features will be incorrectly eliminated.

Unfortunately, many acquisition protocols involve moving or reconfiguring hardware between frames and the "spot noise reducer" is therefore not suitable for use with these protocols. In practice this means that this feature can only be used for single channel, single Z protocols where the camera is running at maximum speed.

Recursive Filter

The "recursive filter" tool reduces image noise by calculating a weighted rolling average of each frame against previous frames. Like "spot noise reduction", this tool does not work well where successive frames bear little or no relation to each other. Depending on the exact protocol, this tool may create artefacts including:

- Digital "bleed-through" between channels

- Blurring of Z stacks

- Reduction of intensity where images captured with the shutter closed are averaged against images captured with the shutter opened

Like "spot noise reduction", the "recursive filter" tool should only be used for single channel, single Z protocols where the camera is running at maximum speed.

Additional Information

For additional information on the ImagEM real-time image processing features, see the Hamamatsu ImagEM technical note which is available here:

http://sales.hamamatsu.com/assets/pdf/hpspdf/e_imagemtec.pdf