From: Harman, Christine
Date: 15 September 2011 20:08
----------
From: Robert Sweet
I'm not going to respond to the larger group, but I know one can buy LEDs that emit strongly at 280 nm, which would give tryptophan fluorescence. They're about $200, and one could build or buy a control circuit for not much more. I think this is about what the commercial tools do. You'd want front illumination. You can get the LED with a convex lens on the front, giving a focus about 1" away. At that point the light is dangerous -- don't shine it into your eye from that distance. You'd want to ask your local safety guys to check it out.
We would use it at the synchrotron with a flash circuit that would be synchronized with a video camera -- I think roughly 20ms would do it.
Let me know if it works.
Bob
=========================================================================
Robert M. Sweet
----------
From: Andrew Purkiss-Trew
Molecular Dimension do such an adaptor which fits to existing microscopes.
See <http://www.moleculardimensions.com/shopdisplayproducts.asp?id=121&cat=X%2DtaLight%3Csup%3E%99%3C%2Fsup%3E+100+%2D+UV+for+Microscope+>
----------------------------------------------------------------
This message was sent using IMP, the Internet Messaging Program.
----------
From: Ed Pozharski
Do you by any chance know the price? I can seemingly "order" it through
the website for the hefty price of $0.00, which is too good to be true.
--
"Hurry up before we all come back to our senses!"
Julian, King of Lemurs
----------
From: Frank von Delft
A while ago I was trying to be cheap, so we played around with it quite a bit in the lab. After rediscovering some of the basics of signal-to-noise and microscope transmission efficiency and that sort of rot, I realised that the commercial systems may not be all that ridiculously overpriced after all. Not if one wants to be able to say something useful about really really small crystals -- the only ones that really matter in the grand scheme of things (big ones are quick to test; little ones must first be optimized = money+time).
But maybe I was just being incompetent. Happens.
phx.
----------
From: Jürgen Bosch
I once tested such a commercial system in Seattle about 4 years ago. It did not impress me. In particular the discrimination between salt and protein did not work for about 10 different proteins from which we already had collected data. sure those were small between 10 and 100 micrometer. Excuse was to few tryptophans
So in theory it is nice but a cheaper variant might be to add Gfp to your protein and screen for something green.
Jürgen
......................
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
----------
From: Andrew Purkiss-Trew
Sorry, I don't know, I think that a new PI at our institute has ordered one from his new equipment budget. But I don't have a price to hand, I can ask though.
--
Andrew Purkiss-Trew
X-ray Laboratory Manager
London Research Institute
Cancer Research UK
----------
From: Bosch, Juergen
----------
From: Edward A. Berry <BerryE@upstate.edu>
Date: 16 September 2011 03:07
To: CCP4BB@jiscmail.ac.uk
A "real" UV microscope requires quartz optics, right?
Probably conventional microscopes use glass.
And you can't see 280 nm (and its not good for your eyes)
so you need some kind of phosphor screen to view the image?
----------
From: Nagarajan V <tatoosh@gmail.com>
Date: 16 September 2011 04:57
To: CCP4BB@jiscmail.ac.uk
Typically, what you image is Trp fluorescence by exciting at around 280 nm and observing at around 350 nm. Standard silicon based detectors do fine at the detection wavelength, although, as you can imagine, increased sensitivity in the UV means increase in the price of the detector. If your excitation and emission light paths do not overlap, you also can get by with standard glass (crown, flint, etc.) optics since they do allow some of the 350-nm light to get through. Therefore, yes, it is possible to build an inexpensive UV imager based on inexpensive excitation light source (Douglas Instruments offers a pen light), and standard lab microscope. Of course, for increased sensitivity and contrast you need a very good light source, optics made of quartz and calcium fluoride that let almost all the UV light through, highly discriminating filters and a sensitive detector.
----------
From: Klaus Fütterer <k.futterer@bham.ac.uk>
Date: 16 September 2011 09:19
To: CCP4BB@jiscmail.ac.uk
From the experience when our (commercial) UV imaging system was set up, I can confirm that signal-to-noise is a non-trivial parameter for imaging in the UV range.
I find the additional info gained from the UV capability very useful, not just to distinguish salt from protein crystals, but also to tell protein from buffer precipitate, buffer phase separation from protein phase separation, etc.
Klaus
=======================================================================
Klaus Fütterer, Ph.D.
Reader in Structural Biology
Undergraduate Admissions
School of Biosciences P: +44-(0)-121-414 5895
University of Birmingham F: +44-(0)-121-414 5925
Edgbaston E: k.futterer@bham.ac.uk
Birmingham, B15 2TT, UK W: http://tinyurl.com/futterer-lab
=======================================================================
----------
From: Shiva Bhowmik <gene1267@gmail.com>
Date: 16 September 2011 18:13
To: CCP4BB@jiscmail.ac.uk
Would be curious to know the current limitations on UV microscopy employed for screening protein crystals - such as content of aromatic amino acids, protein size etc.
----------
From: Patrick Shaw Stewart <patrick@douglas.co.uk>
Date: 19 September 2011 12:35
To: CCP4BB@jiscmail.ac.uk
Hi Christine
patrick@douglas.co.uk Douglas Instruments Ltd.
Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK
Directors: Peter Baldock, Patrick Shaw Stewart
http://www.douglas.co.uk
Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034
Regd. England 2177994, VAT Reg. GB 480 7371 36
Date: 15 September 2011 20:08
Hi All,
I was curious if any of you have tried or even know if it is possible to adapt a stereoscope (in my case an Olympus SZX10 model) so as to view protein crystals with UV illumination. Basically, I want a cheap manual version of what a Rock UV Imager does. I know this is probably a crazy dream. However, I would greatly appreciate any comments, advice or experience any of you may have.
Thanks so much,
Christine
----------
From: Robert Sweet
I'm not going to respond to the larger group, but I know one can buy LEDs that emit strongly at 280 nm, which would give tryptophan fluorescence. They're about $200, and one could build or buy a control circuit for not much more. I think this is about what the commercial tools do. You'd want front illumination. You can get the LED with a convex lens on the front, giving a focus about 1" away. At that point the light is dangerous -- don't shine it into your eye from that distance. You'd want to ask your local safety guys to check it out.
We would use it at the synchrotron with a flash circuit that would be synchronized with a video camera -- I think roughly 20ms would do it.
Let me know if it works.
Bob
=========================================================================
Robert M. Sweet
----------
From: Andrew Purkiss-Trew
Molecular Dimension do such an adaptor which fits to existing microscopes.
See <http://www.moleculardimensions.com/shopdisplayproducts.asp?id=121&cat=X%2DtaLight%3Csup%3E%99%3C%2Fsup%3E+100+%2D+UV+for+Microscope+>
----------------------------------------------------------------
This message was sent using IMP, the Internet Messaging Program.
----------
From: Ed Pozharski
Do you by any chance know the price? I can seemingly "order" it through
the website for the hefty price of $0.00, which is too good to be true.
--
"Hurry up before we all come back to our senses!"
Julian, King of Lemurs
----------
From: Frank von Delft
A while ago I was trying to be cheap, so we played around with it quite a bit in the lab. After rediscovering some of the basics of signal-to-noise and microscope transmission efficiency and that sort of rot, I realised that the commercial systems may not be all that ridiculously overpriced after all. Not if one wants to be able to say something useful about really really small crystals -- the only ones that really matter in the grand scheme of things (big ones are quick to test; little ones must first be optimized = money+time).
But maybe I was just being incompetent. Happens.
phx.
----------
From: Jürgen Bosch
I once tested such a commercial system in Seattle about 4 years ago. It did not impress me. In particular the discrimination between salt and protein did not work for about 10 different proteins from which we already had collected data. sure those were small between 10 and 100 micrometer. Excuse was to few tryptophans
So in theory it is nice but a cheaper variant might be to add Gfp to your protein and screen for something green.
Jürgen
......................
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
----------
From: Andrew Purkiss-Trew
Sorry, I don't know, I think that a new PI at our institute has ordered one from his new equipment budget. But I don't have a price to hand, I can ask though.
--
Andrew Purkiss-Trew
X-ray Laboratory Manager
London Research Institute
Cancer Research UK
----------
From: Bosch, Juergen
I'm replying here to myself :-)
So in an off-board discussion it turns out that the "microscope" in question was a special emitted light and not a UV microscope. So real UV microscopes might be better for the purpose of detecting real crystals.
Sorry for the confusion - had too much sun today :-)
Jürgen
......................
Jürgen Bosch
Johns Hopkins University
----------
From: Edward A. Berry <BerryE@upstate.edu>
Date: 16 September 2011 03:07
To: CCP4BB@jiscmail.ac.uk
A "real" UV microscope requires quartz optics, right?
Probably conventional microscopes use glass.
And you can't see 280 nm (and its not good for your eyes)
so you need some kind of phosphor screen to view the image?
----------
From: Nagarajan V <tatoosh@gmail.com>
Date: 16 September 2011 04:57
To: CCP4BB@jiscmail.ac.uk
Typically, what you image is Trp fluorescence by exciting at around 280 nm and observing at around 350 nm. Standard silicon based detectors do fine at the detection wavelength, although, as you can imagine, increased sensitivity in the UV means increase in the price of the detector. If your excitation and emission light paths do not overlap, you also can get by with standard glass (crown, flint, etc.) optics since they do allow some of the 350-nm light to get through. Therefore, yes, it is possible to build an inexpensive UV imager based on inexpensive excitation light source (Douglas Instruments offers a pen light), and standard lab microscope. Of course, for increased sensitivity and contrast you need a very good light source, optics made of quartz and calcium fluoride that let almost all the UV light through, highly discriminating filters and a sensitive detector.
V. Nagarajan
JANSi
----------
From: Klaus Fütterer <k.futterer@bham.ac.uk>
Date: 16 September 2011 09:19
To: CCP4BB@jiscmail.ac.uk
From the experience when our (commercial) UV imaging system was set up, I can confirm that signal-to-noise is a non-trivial parameter for imaging in the UV range.
I find the additional info gained from the UV capability very useful, not just to distinguish salt from protein crystals, but also to tell protein from buffer precipitate, buffer phase separation from protein phase separation, etc.
Klaus
=======================================================================
Klaus Fütterer, Ph.D.
Reader in Structural Biology
Undergraduate Admissions
School of Biosciences P: +44-(0)-121-414 5895
University of Birmingham F: +44-(0)-121-414 5925
Edgbaston E: k.futterer@bham.ac.uk
Birmingham, B15 2TT, UK W: http://tinyurl.com/futterer-lab
=======================================================================
----------
From: Shiva Bhowmik <gene1267@gmail.com>
Date: 16 September 2011 18:13
To: CCP4BB@jiscmail.ac.uk
Would be curious to know the current limitations on UV microscopy employed for screening protein crystals - such as content of aromatic amino acids, protein size etc.
Cheers,
Shiva
----------
From: Patrick Shaw Stewart <patrick@douglas.co.uk>
Date: 19 September 2011 12:35
To: CCP4BB@jiscmail.ac.uk
As Nagarajan mentioned, we do have a "UV Pen" that we offer at a reasonably modest price.
However we have not been promoting it because we've been struggling to find a camera that we can recommend to use with it.
We use it with an old Russian microscope (normal glass optics) and a cheap consumer Panasonic camera on a "starlight" setting, which works very well. We developed it for our own crystallization project and we have found it very useful, although we occasionally get both false positives and false negatives.
One problem is that the cheap camera we use is now obsolete, and we are investigating our own solution, which will involve writing our own software for an off-the-shelf CCD and lens. (CCD seems better than CMOS.) If anyone knows of a suitable more expensive SLR-type consumer camera, please let me know. We can't find one.
We are looking at just the tail end of the fluorescence, the part that is in the visible or near-UV spectrum, so you need a sensitive camera. We have found that you need to push everything, taking e.g. 30 exposures and combining them, subtracting the "dark" image etc. Presumably the Panasonic point-and-click camera does this.
One solution is to buy a camera from one of the microscope suppliers, but they use very old technology, and charge you a fortune for e.g. the controller (which is essentially a laptop that was designed 15 years ago). The results are no better than the Panasonic camera, and we hesitate to sell you a UV source for around 1000 pounds then recommend a camera system for 6,000. However the pens are available to anyone who wants to order one!
We're not claiming that our approach is as good as the more expensive commercial systems, some of which have quartz optics. Each image takes 30s, you have to work in a dark room, and you can't use it in transmission mode (allowing you to see absorbance) for which you need quartz optics.
Another way to go is to use the approach that we published recently, where you covalently (therefore unambiguously) label your crystals after they have grown. It's pretty much 100% accurate. See the methods section of the ref below.
Hope this helps
Best wishes
Patrick
Patrick D. Shaw Stewart, Stefan A. Kolek, Richard A. Briggs, Naomi E. Chayen and Peter F.M. Baldock. "Random Microseeding: A Theoretical and Practical Exploration of Seed Stability and Seeding Techniques for Successful Protein Crystallization"
Crystal Growth and Design, 2011, 11 (8), p3432.
On-line at http://pubs.acs.org/doi/abs/10.1021/cg2001442
--
patrick@douglas.co.uk Douglas Instruments Ltd.
Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK
Directors: Peter Baldock, Patrick Shaw Stewart
http://www.douglas.co.uk
Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034
Regd. England 2177994, VAT Reg. GB 480 7371 36
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