Testing a Point Grey Camera for Fluorescence Microscopy

About two years ago, I mentioned Point Grey cameras. These are cameras sold to the machine vision and industrial inspection market, and are much cheaper than typical microscopy cameras – most are <$1000. Point Grey puts out very nice spec sheets listing all of their cameras, and the specifications for some are pretty impressive – cameras with < 3e- read noise for ~$500. Nico Stuurman has recently written a Micro-manager driver for these cameras, and was kind enough to let me test one of these cameras. We mounted it opposite a Hamamatsu Flash4.0 (an older Flash4.0, with ~72% QE), and did a qualitative comparison by taking sequential images of the same test slide on both cameras.

The Point Grey camera we tested was a Chameleon3 CM3-U3-31S4M. This uses a Sony IMX265 sensor, which has 2048 x 1536 3.45 μm pixels, with 71% QE, <3e- read noise, and sells for ~$500. It can run at up to 55 fps. On paper, this camera should perform almost as well as the Flash 4.0. The images below are of a Texas red-phalloidin stained cell, captured with a 20x / 0.75 NA objective and a 10 ms exposure on both cameras. Click on the images to see the full size image.

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The Flash 4.0 camera, 10 ms exposure. Click for full size.

ptgrey-cy3-20x-10ms

The Point Grey camera, 10 ms exposure. Click for full size.

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Testing the Prime95B – a back-illuminated sCMOS camera with 95% QE

A few weeks ago I mentioned that Photometrics had released a new camera, the Prime95B, featuring a back-illuminated sCMOS sensor with 95% peak QE. I got a chance to play with it last week, and I’m pleased to say that it performs as well as you would expect. We compared it to an iXon 888 EMCCD mounted on our CSU-W1 spinning disk confocal. We had purchased this EMCCD for imaging those samples that were too dim to get good images with the Zyla 4.2 sCMOS camera we also have mounted on the confocal. (You can see a sketch of how everything is configured in this previous post). For testing the Prime95B, we replaced the Zyla 4.2 with the Prime95B, allowing us to directly compare the Prime95B and the iXon 888.

Before I get to the data, however, what performance do we expect? To get a sense of what to expect I wrote a Matlab script that calculates the theoretical performance for a number of different cameras, using their quantum efficiency, read noise, and excess noise factor (for EMCCDs). You can get the script here.   You can read more about how to calculate the signal-to-nosie ratio for a camera in this Hamamatsu white paper. Here, I’m ignoring the different pixel sizes of the various cameras by assuming that they all receive the same photon flux per pixel, as if the magnification had been adjusted to produce the same effective pixel size at the sample.

Theoretical performance of different cameras.

Theoretical performance of different cameras. Ideal is a theoretical ideal camera with QE=1 and no read noise. EMCCD assumes a high EM gain, ~200x; 82% QE sCMOS is a Flash4.0v2 or Zyla 4.2 Plus; 72% QE sCMOS is a Flash 4.0 or Zyla 4.2; ICX285 is a Coolsnap HQ2 or similar interline CCD camera.

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95% QE, Back-illuminated sCMOS camera

Photometrics has just announced a new sCMOS camera, the Prime 95B, featuring a back-side illuminated sCMOS sensor with 95% peak QE and over 90% QE from about 500 – 650 nm. It’s using a version of this 4 MP sensor from Gpixel. It’s a 1200 x 1200 pixel sensor, with 11 μm pixels and 1.3 e read noise, so it should be substantially more sensitive than a conventional sCMOS camera, and close in performance to an EMCCD camera.

If it performs as well as the specs indicate, this should be a real game changer for cameras, and could displace EMCCDs from all but the lowest light applications. Tucsen had previously released a back-side illuminated sCMOS camera based on the Gpixel sensor, but earlier versions used a sensor with peak QE at ~420 nm (it now uses the version with peak QE in the visible), and distribution in the US has been a bit of a mystery (I was not able to get one to demo, although I didn’t try that hard).

I hope to get a chance to test out the Prime 95B soon and will definitely report results from it here once I have a chance to try it out.

New Sony Back-illuminated CMOS camera

Sony announced a new camera today that features a 42 megapixel back-illuminated CMOS sensor. I’m not that interested in the camera, but the sensor sounds pretty intriguing. No back illuminated CMOS sensors have yet made it to the scientific imaging world that I’m aware of, but we’ve all been eagerly awaiting them. It’s not clear how well a monochrome version of this Sony sensor would perform for microscopy, but the prospect of a back-illuminated CMOS sensor is pretty tantalizing. The other sensor that Sony announced, a back-illuminated, stacked sensor, also looks pretty interesting, although I understand the details less well. As far as I can tell there are no data sheets for the sensors themselves.

While trying to learn more about these sensors, I also came across this interesting page from Chipworks, comparing sensors in different Nikon cameras and their specifications.

New Nikon Cameras

A little while ago, Nikon released a new line of CMOS cameras, based on the sensors used in their digital cameras. I hadn’t looked closely at them until now, and it turns out they are quite impressive. There is both a color version (the DS-Ri2) and a monochrome version (the DS-Qi2). Both are based on a 16 megapixel sensor with 7.3 μm pixels. The DS-Qi2 sports a 77% peak QE and 2.2 electrons of read noise. The only apparent drawback to them is the relatively low speed of 6 fps at full frame. For many applications, though, that won’t be a problem and I’m eager to get my hands on one to give it a try.

One interesting thing is that the sensor is very large (36mm x 24 mm). It’s so large that the camera comes with an F-mount, and in fact, the sensor is larger than the field number of the microscope. I suspect if you used it with a 1x coupler that you would see noticeable vignetting. Nikon mentioned that they have 2.5x couplers for these lenses, and I think something like that is the way to go. If you used a 2x coupler, you would be very close to Nyquist sampling for a 10x / 0.45 NA objective and could bin 2×2 for imaging with a 100x / 1.4 NA objective.

All in all, it looks pretty exciting, and it’s nice to see another option for cameras out there.

A python script for automatically moving and deleting files

On our microscopes equipped with high speed (100 fps) sCMOS cameras, we’ve generally set up a fast SSD RAID 0 array for streaming data to and a slower magnetic disk RAID 1 array for longer term data storage. To simplify data management and keep the SSD from filling up, I wrote a script that moves data every night from the SSD array to the magnetic disk array. It also deletes files on the magnetic disk older than 30 days, and benchmarks the write speed of the SSD array, so we can detect any slowdown. In case it’s useful to other people, I’ve posted it on Github.

If you use it, be careful, as it will happily delete whatever directory you tell it to, so you can easily wipe out your OS if you set it up incorrectly.

Point Grey Cameras

I just came across this interesting PDF summarizing the performance of Point Grey cameras. Point Grey is a machine vision camera manufacturer, and I don’t normally think of using machine vision cameras for microscopy. However, some of their cameras have respectable performance – I wouldn’t want to use them for low light fluorescence work, but for brightfield imaging, and possible for routine fluorescence imaging, they would be fine. Most interestingly, they are very cheap – all are under $2k, and many are under $1k.

For example, the GS3-U3-23S6M-C is a 1920 x 1200 pixel CMOS camera, with 6.8 e read noise, and 76% QE at 525 nm. It has 5.86 μm pixels, and runs at up to 162 frames per second. Best, it’s only $1295.

Or, the FL3-GE-20S4M-C – it’s a 1624 x 1224 pixel CCD camera, wih 8.35 e- read noise, and 59% QE at 525 nm. It has 4.4 μm pixels, and runs at 15 fps.  It’s only $995.

These cameras are uncooled, so noise will be an issue at longer exposure times. Driver support for these in microscopy software is also an issue, but some of the firewire and GigE cameras may work in Micro-manager.  It would be nice to see support for these low cost cameras appearing in microscope software and would open up some interesting possibilities for cheap imaging.

 

Full Field of View Fluorescence Performance

I finally got around to doing something I’ve wanted to do for a while: inspecting the point spread function of our new wide field of view microscope that uses the Andor Zyla camera. If you look back at some of my early posts, you can see that I’ve been wondering for a while what limits the effective field of view we can image through the microscope. As it’s clear that a much bigger field of view is accessible from the objective than makes it to the camera, why is it so hard to access that larger field of view? Once possibility that I’ve suspected is that the image quality is poor at the edges of the field of view.

To test this, I’ve measured point-spread functions (PSFs) for a Nikon Plan Apo VC 100x/1.4 objective using beads distributed across the field of view.  The PSF is an excellent way to see aberrations in your image (a colleague once compared measuring a PSF of your microscope to being naked; both are excellent at spotting imperfections that might otherwise be hidden). These images were recored on our Andor Zyla camera, which captures nearly the full field of view of the eyepieces.  As this is a new lens, the PSF in the center of the field of view is excellent, aside from some modest spherical aberration (see below).

CentralPSF

Z-series montage of the point-spread function of a 100nm bead in the center of the field of view. Sections are space 200 nm apart.

If we look at one of the corners of the image, however, the PSF appears very different. Below is the PSF from the upper left corner of the image. Here we can see that as we go out of focus there is a pronounced elongation of the PSF. The PSF is elongated perpendicular to the vector connecting the location of the PSF to the center of the image.

CornerPSF

The point-spread function from the upper left corner of the image. Otherwise identical to above.

We see similar aberrations elsewhere in the image – at the edges of the field of view the PSF becomes elongated. Fortunately, the aberration is only pronounced at the very edges of the field so that by reducing our image size modestly, we throw away most of the worst parts of the image. For high-resolution work on this microscope, I’m now recommending using the 2048 x 2048 ROI on the camera so that the worst aberrations are eliminated.

High Speed Imaging with the Andor Zyla

We’ve been having some more fun with our Andor Zyla. While it runs at 100 fps when you image the full field of view, if you work with smaller regions of interest (ROIs), it goes quite a bit faster. The fastest we’ve had it running is 2000 fps for a 1024 x 64 ROI, and it runs at 420 fps for a 528 x 512 ROI. Here’s a movie of a swimming Tetrahymena recorded at 420 fps. It’s been slowed down by 10-fold for display here.

Swimming tetrahymena, recorded at 420 fps with an Andor Zyla camera using a 100x / 1.4 NA lens and DIC optics.

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