“Lightsheet Readout Mode” is a unique and patented feature of Hamamatsu sCMOS cameras which can improve signal to noise ratios in Lightsheet microscopy.
Utilizing selective plane illumination, Lightsheet microscopy is a fluorescence method that places a thin sheet of excitation light across only the focal plane being imaged. The sheet of light is swept in very precise synchronization to the rolling shutter of the camera. The scan height of the rolling shutter effectively becomes a slit, enabling less background signal and scattered light. By using this combination of illumination and camera readout, very fast acquisition of optically sectioned images is enabled. Additionally, there is significantly reduced photobleaching and phototoxicity to the sample as compared to many other methods.
Lightsheet microscopy is used for live cell imaging at scales that range from small groups of cells to whole organisms. The rapid increase in the use of this technique over the past several years has been remarkable.
Exposure and readout methods of CMOS image sensors are classified into two types; Rolling shutter and global shutter. Rolling shutter uses sequential exposure and readout with one pixel or one line as a single unit. Thus, the exposure timing is different within one image capture. Global shutter performs exposure and readout of all pixels at the same time, the exposure timing within one capture is simultaneous. (See Fig 1.)
To take advantage of the nature of the sweeping light in this application Hamamatsu Photonics K.K. developed Lightsheet Readout Mode using the intrinsic characteristics and speed of rolling shutter readout.
Fig 1. Readout timing of rolling shutter and global shutter
The standard mode of operation in sCMOS cameras is to readout each line from the center of the sensor outwards upper half and lower half simultaneously, row after row. (See Fig 2.)
With Lightsheet Readout Mode, the user can scan the sensor in one continuous sweep. (See Fig 3.)
Fig 2. Standard mode
Fig 3. Lightsheet Readout Mode
Lightsheet Readout Mode provides selectable rolling shutter operations from top to bottom and bottom to top readout. (See Fig 4. and Fig 5.)
Fig 4. Top to bottom readout
Fig 5. Bottom to top readout
The rolling shutter mechanism used in Lightsheet Readout Mode enables exposure reset right before exposure of the next line starts. This feature provides sharper and more resolved images by eliminating scattered light.
By comparison, using global shutter all of the pixels are exposed simultaneously and scattered light from the illuminating lightsheet can fall onto neighboring lines, causing blurred images. (See Fig 6. and Fig 7.)
Fig 6. Exposure reset
Fig 7. Effect of exposure reset
“The gentle illumination schemes of light-sheet microscopes allow for imaging thick samples with unprecedented speed and resolution. However scattering within the sample can impair image quality. Electronic confocal detection in modern sCMOS cameras allows for rejection of scattered photons, resulting in better image quality."
Lars Hufnagel, PhD
In order to synchronize a variety of laser widths and scanning speeds with the sensor, Lightsheet Readout Mode offers a flexible readout interval and the ability to set exposure times independently. The user can easily adjust the readout interval from 10 us to 100 ms for each line. (See Fig 8.)
Fig 8. Flexible adjustment of readout interval and exposure time
External trigger output has been built into Lightsheet Readout Mode whereby the camera generates an external trigger along with each line or frame, which enables the user to control peripheral devices such as a stage or light source. (See Fig 9.)
Fig 9. External trigger output
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