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Hamamatsu ORCA-Flash4.0

11 March 2020 Tags: microscopy imaging microscope cameras Hamamatsu

When we received our Nikon Ti-U inverted microscope system a few years back it came with a DS-Qi2 camera, which is meant to capture low light fluorescence and large fields of view. However, in the wake of getting the environmental chamber installed, Dr. Leslie Vanderpant from Digital Pixel suggested to try the ORCA-Flash4.0 from Hamamatsu, which comes with a better resolution and also a better peak quantum efficiency than the DS-Qi2. He was very quick to coordinate a trial unit to come so that we could take a look, and the results were indeed quite impressive. It took a little time to get the necessary funds, but with the generous help of Brunel University we were able to buy a unit via Digital Pixel, which was installed by a Hamamatsu engineer.

When comparing the specifications of both systems it seems like the Hamamatsu camera is only a relatively small step up from the DS-Qi2. However, when using these cameras the change is very significant. There is no doubt that the DS-Qi2 is a robust camera that does good work. However, when working with E. coli the resolution really becomes quite crucial. We are using strains in which the proof-reading subunit of DNA polymerase III is fluorescently tagged, which was described in a collaborative project by the Sherratt and the Leake labs (Reyes-Lamothe et al. 2010, Science). Thus, we are visualising complexes with only three copies of the fluorescently-tagged subunit being present. Detectable emission is quite low and especially for time-lapse studies photo bleaching becomes a big problem.

One particular problem of the DS-Qi2 is that the resolution is not good enough to allow for 2 × 2 binning of the sensor to allow for lower exposure times. The images become literally useless. The ORCA-Flash4.0 not only has a slightly better peak quantum efficiency but also a better resolution, and together this allows for a significant leap in the right direction. To illustrate, we have visualised the beta sliding clamp, which is present at higher copy numbers and for this reason gives a more robust signal.

On the left is an image taken with the DS-Qi2, 300 ms exposure, no binning, while the image on the right shows an image taken with the ORCA-Flash4.0 with 2 × 2 binning enabled. Using the binning allows the exposure time to be dropped to 80 ms – almost 25% of the exposure length that was used for the DS-Qi2 image. The resolution is not as good when using the binning, but the information can still be easily interpreted. For conventional snap-shots the resolution of the images without binning is noticeably better than the resolution of the DS-Qi2.

All in all I am impressed with the performance of the Hamamatsu camera and I am delighted that, together with Brunel University, we got the funds to equip our system with it. It has significantly improved our imaging and time-lapse capabilities.

Hamamatsu ORCA-Flash4.0 purchased via Digital Pixel.