The techniques mentioned by others (electron microscopy, atomic force microscopy, and STORM) are all great techniques. The budget AFM looks impressive. However none of these techniques is going to give you the resolution to see interactions between a protein and a small molecule. These techniques would be great for looking at the level of a whole bacterial community, like a biofilm, or even at the scale of single bacteria cells or virus particles. You might even be able to see a single protein with some of these techniques, but it will be just a blob, you won't have the detail to look at and characterize the chemical interactions that might be present.
On Thu, Dec 10, 2015 at 4:46 PM coolcash2004 <davidconwayj@gmail.com> wrote:
Thank you all for the incredible information!Bryan, do you think that the methods that Nathan mentioned or the machine simon mentioned would provide a clear enough resolution to be able to design interactions? Or is there really not a suitable DIY machine at this point to get the resolution needed?The bacteria I want to look at is borrelia burgdorferi http://www.rcsb.org/pdb/results/results.do?qrid=930DD4CE&tabtoshow=Current . Many proteins have been identified, but from what I can tell more are being discovered, so I don't think we have a full picture yet. Also, I want to specifically look at persister bacteria with biofilm that has undergone treatment, and that is a new area of study. I do not yet know the target, so I'm hoping to get a full idea of the structure first (and I think its incomplete at this point). All the existing targets of pharmaceutics have just recently been proved to be ineffective against the persister bacteria, so unless we aren't hitting the right known targets in the right way, I think I'l need to find a new target. To find a target I'm hoping to look at changes in the bacteria from non-persister to persister state to see what mechanisms convert the bacteria to persister state and enable its survival. The only research I have found at this point, other than testing specific treatments (none worked fully), was a report about the genes that were expressed differently from non-persister vs persister state and the corresponding proteins - but it includes the known proteins only and its unclear which of the genes or proteins was responsible for the survival of the bacteria.Sorry for the long message / extra info. Any thoughts are appreciated and thank you again for the guidance.David
On Thursday, December 10, 2015 at 5:02:37 PM UTC-5, Simon Field wrote:On Wed, Dec 9, 2015 at 9:35 PM, Mac Cowell <m...@diybio.org> wrote:Regarding 1: generally, no. Most biomolecules are smaller than the resolving power of conventional light microscopes, so you cannot see molecular structures.Atomic Force microscopes could be used to probe molecular structures. And there's even a new DIY kit that is about the price of a fancy laptop - maybe you could raise funds for one with your local network / school / etc (simultaneously becoming the coolest kid on the block): Strømlingo Basic DIY AFM Kit + Strømnest ($3000).Otherwise, if you are on a serious shoestring budget, then perhaps building your own super-pixel resolution lensfree optical tomopraphic microscope (what a mouthfull!) - you won't be able to see individual proteins, but in principle you will be able to resolve microbes in 3D in near-realtime. And the components are dirt-cheap off-the-shelf digital cameras (used would be fine), a computer, some carefully arranged LEDs, and software for deconvoluting the captured signals. See details below.Lastly, you can get a very affordable high-power (binocular 2500x) optical microscope from amscope for $300-500.Add some filters and a wideband (halogen would be good) light source, and you might be able to DIY yourself an epifluorescence microscope, By using fluorescent dyes that only stain particular cellular biomolecules, you'll get pretty close to your goal of imaging molecules directly. Even though you won't be able to see them individually, you may be able to explore their activity and localization in realtime. Base your DIY design off the specs of one of AmScopes Epi models (http://www.amscope.com/special-microscopes/epi-fluorescence-microscopes.html), or just buy one straight up, they start at around $2500. But really you are just getting a $300 2500x microscope they've upgraded with a bright light source and filter set. I'm sure those components could be scrounged up and added to one of their $300 models for less than $2000.So there you have it!I'll leave you with some more info re: lensfree tomography:Multi-angle illumination with pixel super-resolution enables lensfree on-chip tomographyIn conventional imaging, lenses resolve a sharp image of the target onto the sensor by focusing just the light emitted by the target within the lens' "focal plane" (a volume in front of the lens determined by the width of its aperture and the wavelength of the light passing through it) while blurring light from everywhere else into background noise. One can think of this system as amplifying certain information (the light from the focal plane) while filtering out other information (the light from everywhere else) from the total input light.
In other words, the images formed by lenses are lossy by design. This isn't practically a problem at macroscales, but at microscales the focal plane becomes extremely thin (micron-scale), oftentimes 1/100th as thick as the sample being imaged. To image large cells, for instance, a microscopist might be forced to capture tens or hundreds of images of the sample from different focal lengths (a "z-stack"), then deconvolve them to generate an image in which most/all of the sample is in focus.
These new techniques are interesting because they fundamentally attempt to capture more of the "information" present in the incident light. The lens does not capture an "image" that can be directly visualized, but rather a more full representation of the wave-field of all the incident light. Generating a conventional image from the raw data requires sophisticated additional processing.
The Ozcan lab has published some really neat (and eminently DIYable) work in this area:
"Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution", Serhan O. Isikman, Waheb Bishara, and Aydogan Ozcan. J Vis Exp. 2012; (66): 4161. doi: 10.3791/4161. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3487288/
"Existing 3D optical imagers generally have relatively bulky and complex architectures, limiting the availability of these equipments to advanced laboratories, and impeding their integration with lab-on-a-chip platforms and microfluidic chips. To provide an alternative tomographic microscope, we recently developed lensfree optical tomography (LOT) as a high-throughput, compact and cost-effective optical tomography modality. 7 LOT discards the use of lenses and bulky optical components, and instead relies on multi-angle illumination and digital computation to achieve depth-resolved imaging of micro-objects over a large imaging volume. LOT can image biological specimen at a spatial resolution of <1 μm x <1 μm x <3 μm in the x, y and z dimensions, respectively, over a large imaging volume of 15-100 mm3, and can be particularly useful for lab-on-a-chip platforms."
Also see http://spie.org/newsroom/technical-articles-archive/3979-mul...
On Wed, Dec 9, 2015 at 8:59 PM coolcash2004 <davidc...@gmail.com> wrote:--Hi - I am new to biotech (though not to entrepreneurship in general). I have a whole bunch of DIY questions that I am saving for later - for now I've just been doing a lot of research on my own trying to determine how I want to start and planning everything out. Any help with the questions below would be greatly appreciated.1 - For a DIY lab, is it possible to view bacteria on the molecular level? Separately, is it possible to view them while changing (pre treatment, during, and post) on a molecular level? Is the equipment needed for this reasonably priced? Is it ever possible to rent or borrow equipment? (The microscopes I saw online seemed to cost $25,000+ but I can't imagine thats the only way - or I'm looking at the wrong machines).2 - If I were to take the findings from part 1 (and have a detailed view of the bacteria in various states) is it possible to then design a new compound that will accomplish something specific with the bacteria WITHOUT taking existing compounds and without actually doing the lab testing for now? And what would be the best way to approach this? Is molecular modeling the best way to go? Is it reasonable to learn as much as I can about various compounds, etc and then model a new compound from a problem solving approach rather than looking at existing compounds? (Assume to date all existing compounds tested have failed). Where should I look to begin learning the basics of how to engineer compounds? Any links are appreciated. School is not an option at this point. I understand the process in general of looking at bacteria, finding targets, then design compound that will achieve goals within parameters - but I'm struggling to find info on how you actually design a compound (without just testing existing compounds for potential matches).3 - Is the NYC group I saw here still active? It would be great to meet with other people from NYC at some point later on.Thank you so much. Any other feedback / resources are appreciated as well.-- You received this message because you are subscribed to the Google Groups DIYbio group. To post to this group, send email to diy...@googlegroups.com. To unsubscribe from this group, send email to diybio+un...@googlegroups.com. For more options, visit this group at https://groups.google.com/d/forum/diybio?hl=en
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