Monthly Archives: February 2013

Open Hardware for Microscopy

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Two recent posts on the Micro-Manager mailing list alerted me to the interesting idea of developing open source hardware for microscopy. Open source hardware is analogous to open source software – the schematics, circuit board designs, parts list, etc. are made freely available so that you can easily build your own copy of the hardware.

The first post was about Karl Bellve’s pgFocus – pretty good Focus. A take off on Nikon’s Perfect Focus, as well as similar systems from other vendors, it uses a laser beam to track the position of the coverslip-sample boundary and feedback on the reflection position so as to hold that boundary a constant height above the sample. If you’re interested in more details, links to the designs and documentation are in this post.

In response, Johannes Schindelin posted to announce OpenSPIM, a multi-institution project to develop open hardware plans for building a Selective Plane Illumination system. Their wiki appears to be quite complete and to have just about all the information needed to build your own SPIM system. It’s not trivial – a number of pieces need to be machined – but it seems totally doable to build one.

Open hardware is just getting started but it seems promising- for instance, see Bunnie Huang’s plans for building an open hardware laptop.

Direct fluorescent monitoring of RNA levels

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The same user who was looking for destabilized fluorescent proteins was also looking for ways to directly monitor RNA levels fluorescently. It turns out that two nice reviews on this subject have recently been published: [1] and [2]. Traditionally, this has been done by using RNA binding proteins that bind to a specific RNA sequence, as in the MS2 system. More recently, however, an RNA aptamer has been selected that binds an analog of the GFP chromophore. The chromophore is non-fluorescent in water, but becomes brightly fluorescent on binding to the aptamer [3]. The chromophore, DFHBI, is now commercially available, which means that this system should be pretty easy to use. I’ve not seen anyone use it yet to monitor RNA levels in cells, but I’ll be curious to see if it takes off. Interestingly, this is not the first fluorogenic RNA aptamer synthesized, but none of the others seem to have been that successful.

References

Using Matlab to Find an Optimal Filter

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I ran into a problem today – I’m putting together a quote for a new spinning disk confocal, and I want to find optimal emission filters for the dyes I want to image. For example, my confocal will have a 647 nm excitation laser that I want to use to excite both Alexa 647 and the infrared proteins iFP1.4 and iRFP. So, I need to find an appropriate emission filter. If I go to Semrock, I can look up the filter they recommend for Alexa 647, but not the infrared proteins. If you do this, you will find a large number of filter sets recommended for Alexa 647, of which this one seems to be the most appropriate for laser excitation.  It uses a 676/29 emission filter, which seems pretty narrow for Alexa 647, and certainly doesn’t extend far enough to the infrared to collect fluorescence from the infrared proteins.

I can look through Semrock for a better filter, but what I’d really like to do is automate this process. Ideally, I’d check each filter to see that it blocked the 647 nm laser excitation, and then find the one that has maximal transmission of the dyes I want to image. It turns out that with some computer tricks, this is not that hard to do. Continue reading →

Camera sizes

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Following up on the previous post, I thought it would be worthwhile compiling the pixel sizes and numbers of some existing cameras:

SensorDimensions (mm)Pixel Size (micron)MegapixelsExamples
Sony ICX2858.98 x 6.716.451.45Photometrics Coolsnap HQ2, Andor Clara, many others
Sony ICX6748.8 x 6.64.542.83Photometrics Coolsnap Myo, Raptor Photonics Kingfisher V
Sony ICX69412.49 x 9.994.546.05Raptor Photonics Kingfisher V
Fairchild sCMOS16.6 x 14.06.55.53Andor Neo and Zyla, PCO Edge
Flash 4.013.3 x 13.36.54.19Hamamatsu Flash 4.0

This is a very truncated listing of the cameras out there, and basically only includes cameras I’m directly familiar with.  I’m also excluding EMCCDs and only including cameras 1.4 MP or larger. The vendors mentioned here all many other camera models and there are a number of other vendors out there, some of which make pretty interesting cameras.

Artemis CCD, a company I came across while doing research for this post, appears to make some pretty nice CCDs. I’d be curious to know more about them.

For larger format CCDs there are a number of companies that target both the astronomy and the biology market. Apogee and Finger Lakes Imaging are two I’ve seen a lot. Many of their cameras use Truesense (formerly Kodak) sensors, which come in very large formats, but generally seem to have lower quantum efficiency and higher read noise.

GFP-friendly clearing

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This recent paper describes an improved dehydration and clearing method for clearing mouse brains and preserving GFP fluorescence. The use tetrahydrofuran, instead of ethanol, for dehydration, and dibenzyl ether for clearing, and show superior results to the use of BABB or methyl salicylate.  They claim this works better than the Miyawaki lab Scale protocol in adult mouse brain.

This is figure 3 from their paper – the left side is conventionally cleared; the right side with their new method:

Figure 3

Reference: Chemical Clearing and Dehydration of GFP Expressing Mouse Brains
Becker K, Jährling N, Saghafi S, Weiler R, Dodt H-U (2012) Chemical Clearing and Dehydration of GFP Expressing Mouse Brains. PLoS ONE 7(3): e33916. doi:10.1371/journal.pone.0033916

Cameras, magnification, and field of view

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For my inaugural post, I want to talk about something that I’ve been thinking about a lot recently – how to capture the maximum amount of information from your microscope.  A user came to me recently wanting to maximize the field of view he could acquire at high resolution from the microscope – he was doing an image based screen and wanted to maximize the number of cells he could capture in one field of view.  I immediately realized that our standard 1.4 megapixel, ICX285 based cameras weren’t going to cut it – this was a job for an sCMOS camera, or so I thought

Then I started thinking more about the problem. For his application, he didn’t need high resolution, so we were talking about imaging at 10 or 20x. When I started doing the math for the pixel size you need to acquire a diffraction-limited image from a 10x / 0.45 objective, I realized that our standard ICX285 cameras that are diffraction limited with a 100x / 1.4 oil lens aren’t diffraction limited for a 10x / 0.45 objective. Going from a 100x oil lens to a 10x air lens reduces the magnification by 10-fold, but the NA, and hence resolution, only drops by about 3-fold.  So you either need a 3X magnifier between your scope and your camera, or you need 3-fold smaller pixels.

Illustration of field of view

18 mm side port field of view, with inscribed and circumscribed cameras illustrated.

OK, so all the imaging we’ve done over the years with the 10x objective turns out not to be diffraction limited, and we need a camera with about 3 μm pixels if we want to be diffraction limited.  How many do we need? It turns out the side port of a Nikon Ti has a field of view of 18mm. The eyepieces and the bottom port have a bit larger field of view, 22mm, but since I’ve only ever seen one Ti with a bottom port, I’ll stick with the side port numbers.  If we want to truly maximize the field of view, will want a camera that’s 18 mm on a side. This will have black spaces in the corner, however, because the field of view is circular. If we want to have a camera that doesn’t have any black spaces, say, for tiled acquisition, we can inscribe a square camera in the 18 mm field of view. This gives a camera that’s 12.73 mm on a side, but we only capture 2/π = ~64% of the field of view. Continue reading →