Oliver Kim puts out a nice magazine called Microbehunter. It’s a great resource for microscope nerds. I’ve listed a few blog articles below, but actually, the full PDF issues are where most of the meat is. Lots of in depth, well-written articles on the history of microscope technology, and, of course, microbes. Highly recommended.
Connecting a camera to a microscope (very thorough)
Cover glass thickness and resolution
Setting up a home lab for microscopy
Springer is starting a new open access journal called “Scio Cell Biology”. It’s intended to be the first of what they hope will be a whole family of “Scio”-branded journals. They’re a gold open access/libre publisher with a new business model that allows them to offer two excellent features:
1. No author fees.
No authors pay to publish. This includes big rich labs. No color charges, no publication fees, no author fees whatsoever. No institutional fees, society dues, or subscription fees either.
2. Reviewers get paid.
This is the first time I’ve seen anything like this. The amount they’ll get paid hasn’t been announced yet, but they’re hoping that this will let the editors go back repeatedly to the best, most constructive reviewers. They also hope that it will raise the prestige of the journal. The idea is that the reviewers are financially motivated to be as constructive as possible and so authors will want to have their papers reviewed by them.
So what’s their funding model? As a hint, here are some excerpts from their upcoming inaugural issue:
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Sebastian Seung has a new book out, and to promote it he’s having a debate with Tony Movshon. The event sold out in less than two hours. So they added a simulcast in a nearby room, and are streaming it on the web at radiolab.org. It’s Monday, April 2, at 7pm New York time.
Seung’s position (as summarized on the event website): “… research would be radically accelerated by finding and deciphering ‘connectomes,’ maps of connections between neurons. […] connectomics will be as fundamental to neuroscience as genomics is to molecular biology.”
Movshon’s position (as summarized on the event website): “…maps of the brain by themselves cannot offer much insight into how this remarkable organ does its job. Just as a genome by itself is only a blueprint with little power to explain how an organism works, a connectome is at best a framework with little power to explain brain function.”
I doubt anyone would argue that mapping connections isn’t useful. But just how useful? It really comes down to funding priorities: should we spend a billion dollars on connectomics right now? Would that data be really transformative? Or would we get more bang-for-our-buck if we spread that money over several neuroscience research efforts, including connectomics, neurophysiology, genetics, molecular biology, and so on?
There’s an inherent appeal to large, simply-stated projects. And there are great success stories. The advocates of spending big money on connectomics love to draw parallels to the human genome project. The space program is another large, expensive, worthwhile project. But not all big projects are worth the price tag. For example, although it’s a clear, attractive goal to have a permanent manned outpost on the Moon, the price probably isn’t worth it right now. In a similar case of wrong price, wrong time, the Superconducting Super Collider project was abandoned in 1993 after $2 billion spent, although the science was certainly worthwhile. Perhaps spreading those research dollars to other efforts was the right decision at that time.
Numberfactory is a very useful, clean reference site. Unit conversions, formulas, bolts, nuts, screws, and more.
More…
Bolt sizes
To recap the previous post on axial resolution and numerical aperture in two-photon microscopy:
For excitation deep in scattering tissue, higher NA can actually be detrimental because the light cone at the periphery has to travel a longer distance through the scattering tissue compared to moderate NAs. In addition, spherical aberration is more of a problem at higher NAs.
To increase axial resolution, first ensure that you’re overfilling the back aperture of the objective before trying a higher NA objective. A 0.8 NA objective’s axial resolution is only about 50% broader than a 1.0 NA objective. By contrast, underfilling the back aperture significantly makes the axial resolution broader by 200% or more. So before buying a higher NA objective, ensure that you’re actually using all of the NA in your current objective.
For collection, high NA is good, but so is low magnification. For example, a 16x 0.8NA will collect more scattered fluorescence signal than a 63x 1.0NA. A rough image brightness factor can be computed to compare among objectives: average transmittance of visible light * (NA^2/mag)^2
The figure at the top of this post summarizes the brightness factor for a range of different NAs and magnifications*. Several objectives are noted on it as well. At the bottom is the relationship between NA and axial resolution (theoretical best, ref).
Optimal: So what has been recommended for years is to use a high NA objective and underfill it a bit.
In two-photon population calcium imaging, the neuropil response can contaminate neuron responses. This happens when the axial resolution is poor, such that the excitation volume extends out of the soma. This often occurs when the back aperture of the objective is underfilled, resulting in a lower effective NA.
Here’s the relationship between numerical aperture and neuropil contamination.
The influence of neuropil contamination is partially dependent on the signal-to-noise (S:N) of the somatic spike-associated calcium transients. If S:N is high, then a small amount of neuropil contamination can be negligible.
More info:
Part I of axial resolution and numerical aperture
High NA, low mag objectives
* I’ve omitted the transmission characteristic in these calculations. Although IR transmittance varies considerably among manufacturers, in the visible range transmission is consistently around 85% for water dipping, low mag, high NA objectives. Thus the relative measures are unaltered.
Recently, microscope manufacturers have been releasing ever higher NA objectives for multiphoton imaging. Although higher NA objectives should give better axial resolution, they might not be ideal for imaging deep into the brain compared to more moderate NAs.
I think the perception that higher NAs always improve images arises when people try out new, high NA objectives that have smaller back apertures than their old objectives (e.g., an Olympus 20x/0.95 NA or a Nikon 16x/0.8 NA). If the back aperature on the 25x, 1.0+ NA objective they’re trying is smaller, then suddenly they’re overfilling more than before and their axial resolution and S:N are improved. They chalk it up to the NA and swear never to go back to 0.8 NA objectives. However, their old objective might actually be better, and what they really need to work on is their scanning optics.
The key issue is this: high NA objectives bring a large portion of their light in at a high angle. This high angle results in longer paths for the excitation light to take, and this results in more scattering events. The end result is that excitation intensity decreases. This has been shown theoretically and empirically. So if you’ll be imaging deep, consider moderate NA objectives.
By contrast, underfilling the back aperture is a great way to destroy one’s axial resolution. Since the lateral resolution is relatively unaffected, this problem often goes unnoticed (see figure below, its link, and this review). If the excitation beam is less than half of the diameter of the back aperture of a 20x/0.95 NA, then the axial FWHM could be 3x what it should be, or roughly the equivilant of a 0.60 NA objective (theoretical FWHM 5.6 microns), or worse.
Even many commercially available scopes fail to overfill the large back apertures of today’s low magnification/high NA objectives. The major microscope manufacturers need their objectives to fit onto their existing microscope bodies and systems, and this is a major engineering constraint in their design for new imaging systems.
As previously noted, homemade cables are to be avoided. However, stock cables are not always up to spec. For example, if one wants to drive stepper motors using a 9-pin serial cable, are the individual conductors able to carry the 1 A of current required? What about high frequencies over repurposed speaker cables?
The chart above (larger version) should help you decide what gauge of wire to look for in a particular application. Note that these are relatively conservative engineering specs, so in practice you can get away with underspec’ing a bit.
The maximum current is the current that, if sustained, won’t result in too much heating. The maximum frequency is the signal frequency at which there is 100% skin depth– i.e., the entire cross section of the wire is carrying the signal. At frequencies higher than this, the effective resistance of the wire increases.
To answer to the above questions: Yes, a 9-pin serial cable will work fine for driving steppers if the duty cycle is low (i.e., the stepper motors are typically not moving). Get a heavier guage if you can (e.g., 20 or 22 AWG), but since the currents are fairly brief the wires won’t heat up much even if you underspec them. However, with high frequencies through speaker cables, there might be problems. Even with 1 MHz signals through 18 AWG wire, there will be significant signal degredation.
Recently I needed a 3.5 mm TRS (tip-ring-sleeve, aka 3-conductor) phono cable that would carry fairly high sustained currents. Typically these types of cables are used to plug iPods and similar devices into the Aux inputs of car and home stereos. In that application, fairly light gauge wire is ideal since the currents are small. However, I was able to find a heavier gauge cable assembly from an audiophile shop.
Data source file (tab-delimited text, note that gauges 00, 000, 0000 are recorded as -1, -2, -3, to get the chart to plot properly)
via
I thought I needed a $30 multimeter. But then I saw that for just a few bucks more I could get some more features. This process repeated itself until I ended up buying a $98 multimeter. But that’s still relatively inexpensive.
One feature I’ve found myself using a lot is the IR temperature measurement. It works like a laser pointer and is accurate enough for most of my needs. This way I can check the temperature of a homeothermic blanket, a set of galvos, or anything else. And I don’t have to awkwardly place a temperature probe on the surface (although the meter has that feature too, of course).
I highly recommend buying a meter with this functionality.
Thanks go to commenter ybot for this one. They pointed out this handy iPhone-to-Microscope mount iPhone case.
From the pictures, it looks like the prototype was printed on a Makerbot, which isn’t terribly high resolution as 3D printing goes. Hopefully the files can be printed as-is by Quickparts, Shapeworks, or Ponoko and still fit correctly. I double check the measurements, particularly for the eye pieces, before I sent this out. Regardless, this is a great idea.
By the way, there’s also this similar device. It’s not as elegant as the above iphone case, but it a bit more flexible and should work with an array of different eye piece sizes.
Perhaps the best-looking one right now is the SkyLight, which was funded via Kickstarter in January. It looks thoughtfully engineered, and can accomodate and array of eye piece sizes and many different smartphones.
