The idea of the European User Group Meeting (EUGM) is to create a discussion forum between leading scientists in structural biology and Rigaku representatives, and will cover both X-ray techniques and automated protein crystallization. This is the first time that we hold such a meeting, so all of us at Rigaku are excited about the EUGM!

We already have a great list of speakers, including Oliver Einsle from University of Freiburg, Jeremy Moore from Imperial College London, Dmitri Svergun from the EMBL in Hamburg, Samar Hasnain from University of Liverpool, Klaus Fütterer from University of Birmingham, and Michael McDonough from University of Oxford. All participants are invited to share their research in our poster session.

Wladek Minor will discuss HKL-3000 and solve real-world data sets at the EUGM. Don't forget to bring your data!

Wladek Minor from University of Virginia will give an introduction to structure solution in HKL-3000 alongside hands-on sessions using lab data provided by the attendees. Everybody is invited to bring their own data to take advantage of this exciting opportunity.

The EUGM will take place in Frankfurt, Germany, Feb 23^rd – 24^th . If you are interested in joining us, find out more at http://www.rigaku.com/protein/eugm.html or go directly to our registration page.

The new nano-head. Most noticeable from this perspective: The longer needle

We wanted to let you know about an important change to our Phoenix RE drop-setters, namely that from today onwards all Phoenix RE units will be equipped with a new protein nano-dispense head. The change might be subtle in terms of overall appearance, but the new nano-dispense head has some very distinctive advantages.

Conceptually, the biggest change is a flow through wash which improves wash speed and efficacy. A pump connected to two wash bottles allows for different wash strategies, e.g. water plus detergent or water plus buffer. The wash works by flushing fluids from behind the dispense head into the wash fountain.

The flow through design also allows for more precise aspiration and dispensing of small volumes using an isolated liquid mode where water (or any other liquid for that matter)  is used as a system fluid together with an air gap. Of course the new head can also run in air backed mode which matches the dispense characteristics of the previous model dispense head.

The pump of the new nano head, together with the wash bottles. Both can be conveniently stored on a shelf mounted behind the Phoenix RE, reducing the overal footprint relative to the old style pump.

Within recommended operational parameters for the previous dispense head model, dispense accuracy is virtually identical between old and new style nano heads, yielding coefficients of variation (CV) of 3.5% for 200 nL drops. Still, the new nano-head performed very favorably at 100 nL (CV of 3.2%) and 50 nL (CV of 5.1%), both using isolated liquid mode.

The new pump also improves the overall behavior of the dispense characteristics: It eliminates bubbling during wash, allows for more protein sample to be recovered after dispensing because of reduced aerosolization, and generally samples are pushed out more gently from the dispense head. All this is thanks to a syringe driven soft purge.

Alongside the new pump comes a completely new way of defining liquid classes: Aspirate and dispense classes are now combined, simplifying definition and referencing. Also, the dependency of the liquid classes as a function of dispense volume is now modeled by a functional relationship which allows for dynamic changes for a wide range of dispense volumes.

Last but not least, the actual nano-nozzle is completely redesigned. As show in the picture on the left, the new nozzle is significantly longer and thicker. The longer needle allows for more sample to be accessed from the sample tubes and the larger diameter reduces the chances of clogging the needle, and opens as well, opportunities for exciting applications such as microseeding.

Many thanks to Matt Lundy and of course everybody at Art Robbins Instruments!

Figure1: 1951 USAF resolution test chart. Source: Wikipedia

No conflict, right? This could be the end of the post, if it wasn’t for one small detail: Protein drops are three dimensional. And an optical resolution is generally measures with two dimensional test patterns. Consider the 1951 USAF resolution test chart in figure 1. A series of horizontal and vertical lines are used to determine the smallest size object – or line separation – that could be resolved by a given optic. In contrast, when a three dimensional object is to be imaged, concessions need to be made between depth of field, DOF, and highest optical resolution at a specific focal plate. The DOF is generally determined by the relative aperture or f-number. Increasing the DOF implies lower overall optical resolution of the image since resolution drops off gradually around the maximum resolution any optic can achieve (again, a good summary is given in Wikipedia). Figure 2 illustrate this behavior: The maximum line pair resolution is only achieved in a single point in the focal plane, increasing the depth of field increases the focal plane, but as a consequence, line pair resolution is reduced for points away from the optimum focal plane.

Figure 2: Line pair resolution as a function of depth of field.

For protein drop imaging, especially in automated fashion, the DOF should be adjusted to match the height of the drop. This way, the content of the drop can be adequately represented by a single image. Otherwise, if the DOF is smaller than the height of the drop, various images need to be taken at different focal planes and either inspected individually, or assembled into a composite image with adequate focus across the drop using a software application. This technique is commonly referred to as “slicing” and comes at a significant cost in terms of imaging acquisition time and CPU requirements.

Figure 3: Slicing, optical resolution and depth of field

Slicing can increase the optical resolution since the depth of field is reduced for each slice, and therefore each image is taken closer at the best line pair resolution level. This technique also mimics how we would use a typical bench top microscope where we would scan through a drop by changing the focal plane. For automated high throughput image acquisition this technique comes at a significant disadvantage, since imaging speed and the sheer amount of imaging data are of major concern.

The correct approach for high throughput imaging is to design optics that match the depth of field of the types of objects we are most likely to image, which would be smaller (sitting) drops and larger (hanging) drops. This approach can only be successful if we pay attention to another optics parameter: The field of view or field of vision (FOV), or the extend of a well in a crystallization we can observe at the time of taking the image. In an interactive scenario this is less relevant since we can move the plate around as part of the investigative process, but in high speed imaging, we better make sure to capture the entire drop. The best way of doing this is by designing optics where DOF and FOV match the dimensions of the objects you are trying to image.

So, how do you measure the optical resolution of your optics at a depth of field that matches our experimental conditions? Actually, there is a simple but elegant solution to this problem, discussed by my colleague Jian Xu during the last ACA. First, it makes sense to measure resolution on objects you are interested in imaging which would be small crystals in protein drops. Second, if you can distribute randomly a large number of these objects throughout the volume of question, i.e. the drop, you will image some of the objects at the maximum line pair resolution limit. In practice, what Jian did was to grow a shower of needles in a drop, snap an image and measure the smallest needle diameter he could find. Figure 4 shows a schematic of this setup.

Figure 4: Schematic of Jian's experimental setup to meassure the optical resolution of our new optics.

Art Robbins hanging drop seal

UV imaging is a great technique to detect protein crystals under difficult imaging conditions  (precipitates, membrane screens, etc), but it requires some extra care when it comes to selecting crystallization plates and seals.

The issue is that plates or seals that are optically transparent can give disappointing results under an UV microscope because of resins and other polymers used in the plate or seal. They either induce background fluorescence that can overwhelm the fluorescence signal from a protein crystal or turn out to be not UV transparent enough to be useful for experimentation.

For hanging drop experiments in particular, choices for UV transparent tape seals were somewhat limited. In that sense we are particularly happy to announce that we have tested the new Art Robbins Instruments 96  hanging drop seal and found  it to be compatible with our Desktop Minstrel UV and Minstrel HT UV imagers.

The brand new Desktop Alchemist: Ready to setup screens!

Well, the day has finally arrived: As our first desktop screen maker rolled of the production floor, we couldn’t resist snapping some pictures and taking some video; we had Mike take a stand in front of the camera and personally introduce the Desktop Alchemist.    

This particular model is special to us as it is going to be one of the stars of our upcoming automated protein crystallization world tour.    

First stage will be the 2010 Meeting of the American Crystallographic Association in Chicago. And if you are wondering if the Desktop Alchemist is too much of a baby to serve up our famous Alchetini, don’t worry, they will be served.  For all of you who are interested in seeing the DT Alchemist in action right now, here is a short video:    

If you have ever worked with an Alchemist you’ll be right at home on the deck of the new  Desktop Alchemist. There are 24 positions for BirdFeeders and the dispense head is identical to the one used in his big brother.    

A view on the Desktop Alchemist deck configuration

The plate nest is also exactly the same as that of the Alchemist, which means that switching between plate types is just as easy.    

Apart from the fewer stock positions, the main difference between both instruments is the compact size and much smaller footprint (30″x30″) of the desktop version.    

Curious? Well, we hope so. If you can’t make it to the ACA this year, you’ll have another chance at the ICCBM13 and AsCA meetings in Dublin and Busan respectively. Incidentally, the Desktop Alchemist will not be the only new instrument we’ll be showing; you’ll also have the chance to take a closer look at our new Minstrel HT UV protein drop imager at the ACA!    

Desktop Alchemist with open hood

Small crystals under heavy precipitate

There are three main scenarios in protein crystallization where visual microscopy fails, independently of the resolution of an optical microscopy: Heavy precipitates, salt formation, and otherwise opaque screens, such as the ones found in membrane crystallization.

The image above shows the effect of heavy precipitate. The visual image is complex to a point where small protein crystals cannot be identified. Under UV light the situation is completely different since the fluorescence of the precipitate is significantly less intense than from the actual protein crystals.

The next picture shows a drop where a large salt crystal obscures small concanavalin A protein crystals. Again, under visible light it is impossible to detect the small protein crystals that are easily distinguishable under UV light.

Tiny protein crystals hidden under crystallized salt - easily detectable with UV

Finally, UV microscopy is great news for membrane protein crystallization. Below are visible and UV images for a thermatoga screen – I am not saying that you can’t see the protein crystals at all under visible light at all, but what a help the UV image is!

Crystal detection in opaque membrane screens

I’d like to thank my colleague Pierre LeMagueres for sharing all above images with us!

Water, DMSO and other stocks with low viscosities are easy to dispense. That’s why we wanted to take a moment and discuss how we can dispense stocks with higher viscosities such as  100% polyethylene glycol 400 (PEG 400) or 50%  PEG 8000 with high precision.

First we dispensed 100% PEG 400 using an unused small syringe without any further preparation into 20 consecutive wells. What happens is that for the first 6 wells no PEG is dispensed at all and after that, a drop of around 6 ul is dispensed (the target dispense volume was 1 ul). Figure 1 shows this behavior over the 20 wells.

Figure 1: Dispensing results for PEG400 with a previously unused small syringe

What happens is that a previously unused syringe aspirates an air bubble when working with stocks with higher viscosities. This air bubble acts as a cushion that prevents the Tapper of doing its work correctly, avoiding tapping off the drops from the syringe tips. The drop increases in size during several dispensing steps until it finally grows to a size where gravity causes the drop to be dispensed.

Luckily the solution to this problem is fairly easy: Wetting the syringe walls by manually aspirating the stocks solution we want to dispense before placing the BirdFeeder into the Alchemist deck. If the syringe walls are wetted, viscous materials can be more easily aspirated which suppresses the formation of air bubbles. Repeating our experiment with a wetted syringe generates quite different results:

Figure 2: The same dispensing experiment using PEG400, but with a manually primed small syringe.

The improvement is dramatic for both medium and small syringes (and should be used for large ones as well). With primed syringes you can achieve coefficients of variation or CV’s of 2% or less, as shown in Table 1.

Table 1: Dispensing accuracies for primed and unprimed syringes for 100% PEG 400 and 50% PEG 800.

So, for which viscosities is priming required?

As it turns out, if you are dispensing stocks with with viscosities of 5 or higher,  you should definitely plan on adding a manual priming step before automatic dispensing. Otherwise you risk drops not being correctly tapped of from the syringe tips, with negative effects on dispensing accuracy. Table 2 shows our test results:

Table 2: Drop removal behavior by tapping for primed and unprimed syringes.

Finally, I want to acknowledge my colleague Matt Lundy for carrying out the experiments and interpreting the results.

UV light has a particular advantage for protein crystal detection as many proteins contain tryptophan and tyrosine which fluoresce for a fairly narrow UV band. The first challenge was to match this band as closely as possible in order to avoid irradiating the crystal to the point where proteins would denaturate. This is a non-trivial problem, so when our engineers came up with a unique solution the US patent office felt that our solution was worth a patent.

Optics is a whole other challenge. Our approach was to take optical and UV images from the same optical train. This would allow us to take UV and visible light micrographs from exactly the same field of view, allowing us to exactly correlate both images without further processing. Because of strongly varying absorption coefficients, the optics had to be highly customized.

Combine everything with an industry leading 5.1 megapixel UV capable camera and voila: We can clearly resolve protein crystals below 2.5 microns. Using UV light.

A needle in a haystack. Sure, there are many, but they give us a very clear idea on the size of nuclei we should be able to detect with this UV microscope. Click on the image for a larger view.

Usually closed, today our doors were open to the public to come and check our our robots!

It’s now the fourth year we are doing this: We try to keep the noise on the production floor down so our customers can come and visit us and learn about what we have been up to over the last year. This time around, the star of the show was our brand new UV microscope.

For me, this was an exciting opportunity to socialize with the experts in the field (after all, I had just been hired a week ago). The coffee breaks were humming of discussions around LCP crystallization for membrane proteins, data management issues, and many more.

Matt and Tom showing how make a fine screen and how to set up a 96-well plate

After brief presentations by Mike and Joe it was finally time for our engineering and scientific teams to talk about our new developments. There was a lot of discussion around UV imaging, both in terms of implications on the actual instrument in terms of UV compatibility, but also on issues around impacts of UV light on the actual protein crystals. But of course everybody also got a chance to take a close look at all the other products, like screen makers and drop setters.

Mike explains the JCSG setup.

The high point of the day was for many a visit to the JCSG at Scripps. Not only did everybody get to see a fully automated protein crystallization robot in action, but also the “protein hall of fame” – about postcard sized images of the now over 1000 structure solved by the JCSG.

If you are interested in joining us in a future open house, please don’t hesitate to contact us – we will be looking forward to your participation!