Posts archived in Hardware

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February 17, 2012

Fast z-galvo for 3D two-photon imaging

Perhaps you’re familiar with the various AOD and piezo-based methods for two-photon imaging in 3D quickly. Here’s another way (Botcherby et al. 2012, PNAS, open access)

The used all galvos, rather than AODs or piezo z-steppers, as used in other implementations (e.g. 1, 2, 3, 4). That is, stock Cambridge Technology mirrors for x and y, and their custom galvo system for z.

The “aberration-free” part of the title is perhaps a bit misleading. They aren’t correcting for aberrations with a deformable mirror. What they’re referring to is that they do the z-scanning in a clever way to decrease the aberrations that would occur if they simply diverged and converged the beam. (figure below)

They used a second objective L1, which was matched for magnification but not necessarily NA, and air immersion rather than water immersion like the main objective, L2. The scan beam was projected through this objective onto the z-galvo mirror.

This approach is much simpler, from the engineering side, than multiple AODs. It’s also easier to get wide fields of view than with AODs, and there are little to no dispersion issues (again, unlike AODs). Although it is slower than AOD-based scanning, it should be faster than piezo z objective movers.

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February 16, 2012

Intrinsic imaging on the cheap

Intrinsic signal optical imaging is a functional imaging modality where the reflectance of red light indicates active portions of cortex. It is used for many applications, including imaging individual barrels in rodent somatosensory cortex, maps in visual cortex, and the tonotopic organization in auditory cortex.

Here is a prior Labrigger post with tips for intrinsic signal optical imaging. One of the key things is to use a high quality scientific camera.

Recently, a friend pointed out that newer digital SLRs have absolutely fantastic specs and could maybe be substituted for a scientific camera. The advantages would be that digital SLRs can be cheaper (especially second-hand) and can be used with off-the-shelf software.

So why not use a newer digital SLR instead of a scientific camera?

Short answer: For paradigms that rely on a lot of averaging, it could probably work. But for Fourier analysis-based paradigms, it probably won’t work.

Long answer: Here are the key issues to overcome:

1. Can the camera see the light?

700nm light is often used for intrinsic imaging. This report says that there is massive roll off around 700nm. The Bayer filter may contribute to this, but often there is a separate IR filter. Astrophotographers remove these filters to get results like this:

There are tutorials all over the web. For example, this is the source for the above image and it’s a good place to start. Also, plenty of people use wavelengths below 700nm for intrinsic imaging, so this is not a limiting factor for everyone.

2. Getting the raw data

Digital SLRs do all kinds of tricks to make the photos look nice. All of which will screw up your data. Fortunately, many cameras offer the option of reading the raw data off of the camera, in a format helpfully called RAW.

Special considerations for Fourier analysis

The amplitude of intrinsic signals are typically about 1 part in 10,000. Since most cameras, even the latest scientific cameras, top out around 12 bits (i.e., values range from 0 to 4095), it’s almost impossible to detect the signal without some amount of averaging.

There are two main paradigms for intrinsic imaging:
1. Averaging like hell in the temporal domain. This is what most people do. Just average a whole bunch of frames at rest, and then a whole bunch of frames during stimulation. Subtract the two images. Declare victory.
2. Averaging like hell in the frequency domain. This is a trick from fMRI. Kalatsky & Stryker implemented it for intrinsic signal optical imaging. It’s harder, but is typically much faster and can yield much more information.

For paradigm 1, any decent camera will work. But paradigm 2 has some special requirements:
1. Digital SLRs still typically have rolling shutters rather than global shutters. This means that the top of an image is captured over a different time than the bottom of an image. This can distort the phase of signals, which is important for this paradigm.

2. Image quality. Let’s start with pixel size.
The Nikon D3 and D4 sensors are about 3x the size and pixel count of a Dalsa 1M30, the classic choice for Fourier analysis intrinsic imaging. The Nikon FX chip’s pixels are smaller than those of the 1M30.. it’s also a CMOS chip rather than a CCD. (Though that distinction means less these days.)
pixel size:
8.45 x 8.45 µm (Nikon)
12 x 12 µm (Dalsa)

Now let’s talk about dynamic range:
I’m guessing the 66dB dynamic range of the Dalsa 1M30 is still better than most digital SLRs.
For example, the Sony Alpha 900 and Canon EOS 5D both top out at less than 40dB. (ref 1, ref 2) Consumers typically don’t need to pick a 1/10,000 signal out of their images.

3. Output
Can you commonly get 30fps of uncompressed, RAW data at 1 megapixel resolution out of consumer dSLRs? I have the impression that you can get RAW stills, but video is still typically compressed. “Unfortunately there are no HDSLR cameras on the market that will give you a clean (non-overlay), uncompressed 1080p HDMI output.”

If you decide to go the scientific camera route, sCMOS and CCD are solid choices. If you are operating in a light-limited regime (e.g., flavoprotein fluorescence imaging), EMCCDs are the way to go.

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February 15, 2012

Clearing tissue for imaging: index matching is not new

The main factor that limits how deep we can image into tissue is the scattering of light. Multiphoton imaging partially mitigates the problem by using infrared light, which scatter less, and by using an excitation process that drops off nonlinearly with intensity. However, it only partially mitigates the problem. Light scattering is still the main factor limiting how deep we can image.

If scattering is such a problem, why not address it directly? Scattering is due to mismatches in the index of refraction at the borders of structures. In biology, this is typically between lipid membranes and aqueous intracellular and extracellular fluids. If the aqueous solution is replaced by something with the same index of refraction as the lipid membranes (or the lipid membranes are replaced), then there should be less scattering and we should be able to image much deeper.

Well, this idea has been around for quite some time. Dating back to the 1950s.

Recently, there has been somewhat of a rediscovery of the technique. Starting with a paper from Dodt in 2007, a paper from Miyawaki’s lab in 2011 (the Scale paper), and then another paper from Dodt this year. There has been some criticism that the earlier work didn’t get cited much by the recent papers.

Importantly, microscope manufacturers have started releasing objectives specifically for cleared tissue. These objectives offer a unique combination of low magnification, high numeric aperature, very long working distances, and are designed for the refractive index of the clearing agents. (e.g., Olympus, Zeiss)

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February 5, 2012

3D Sketch-up models for Newport parts

Thorlabs and Newport have offered 3D models of their products for a long time. However, they’re typically in formats for expensive programs like SolidWorks and AutoCAD. In the past year or two, Newport has been slowly adding to their library of Google SketchUp models.

I still prefer SolidWorks, but I’m optimistic that I’ll eventually switch to SketchUp. Regardless, it’s nice to see a company supporting free tools.

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December 30, 2011

Optics Planet and Braintree Scientific and New Era syringe pumps: Inexpensive scientific equipment

Optics Planet has a nice selection of inexpensive microscopes and other lab equipment. Such as these chubby, potential Cute Overload stars from Nikon (above, the blue one that is taking a bow is $380).

Braintree Scientific also has a really nice selection of reasonably priced equipment. Tons of very interesting, unique products. Get the catalog and flip through it– the website isn’t so nice to browse. They do custom work too, in case you have something specific in mind. One of their new products is a netbook+syringe pump package, pictured below:

I recognize the syringe pump as one of New Era’s OEM pumps. New Era sells all kinds of syringe pumps, from barebones OEM devices ($500, controlled via RS-232), to digital ($750) and multi-syringe units ($1500). You can use one of the OEM units for things like delivering water rewards in behavior rigs.

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November 27, 2011

Thorlabs’ B-scope

Thorlabs’ scope pieces and kits have been mentioned in these pages before. At SfN, they had their new B-scope on display. This is like the Sutter MOM and the UCLA scope, in that the microscope rotates in one plane in addition to x-y-z movement. A few differences with the Thorlabs scope:

1. The objective rotates around the focal plane, and the rotation is motorized.
With the Sutter/UCLA style scopes, the objective rotates about an axis along the scan path, so the focus point changes a ton when rotating. The rotation can really only be changed before there is a prep on there, because the objective swings a big arc whenever the rotation changes and it ends up pointing at a completely different point in space.

By contrast, the Thorlabs scope is set up to rotate about an axis that is in the plane of focus. So you can be looking at a cell and then, while imaging, rotate the scope (since it’s motorized) and still keep looking at the same thing, just from a different angle.

This is why they have the crazy periscope you can see to the right in the photo below.

I remember seeing a scope with this same feature (rotation around an axis in the image plane) at a conference at least 2 years ago. I think it was a group based out of Switzerland. Can anyone fill in the details for me?

2. No conventional scanners, just the Thorlabs conventional scanners.
This might not be true for long. Thorlabs has their own conventional scanners, but they’re not as fast as Cambridge Technologies (CTI) scanners. This is probably why they opted to put their resonant scanners in the system.

I’m guessing that they’ll help out buyers if they want to fit the scope with a set of conventional scanners from CTI. I say this because Thorlabs told more than one person at SfN that they would help them fit the Thorlabs resonant scanner kit to their Sutter MOM scope. This was news to Sutter.

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November 11, 2011

Building a 2p scope, 2011 edition

What’s new this year?

The original post on building a 2p scope still gets a lot of hits. Let’s revisit the topic.

Scientifica

Scientifica has been working hard on developing a kit for multiphoton imaging. It’s all based off of their minimalist, yet versatile SliceScope platform.

I’ve had the opportunity to check out their collection module and it’s really well done. The components are available for individual purchase, so if you just want to buy part of it and tack it onto something else you’ve got, they offer that flexibility. Since they sell a wide range of electrophysiology products, they can offer customized package deals to suit your needs.

The collection module shown above is a nice tight package with the PMTs, filter cube, and preamps all integrated. They really like the R9880U series. I can see why: they’re very small, with an 8 mm diameter active area, and are constructed such that incident light can approach from a wide angle. However, they are bialkali and the QE at 520nm is less than 30%. They have a GaAsP version in the works.

Their platform can be configured for slices (below, left) or in vivo (below, right).

Thorlabs

Thorlabs has made some improvements in their own software for their 2p kit, and Vijay Iyer’s ScanImage 4 will interface with the resonant scanners. I think this is an interesting starting place for custom rigs. Thorlabs has add-ons like deformable mirrors that you can purchase at a later time. There’s no conventional galvo scanning option, and since resonant galvos are not good for arbitrary line scanning, you’re pretty much locked into (fast) raster scanning. If that’s not an issue, it’s a good option. And I’m guessing they’ll have a conventional galvo scanning option at some point– they do actually sell them, all they have to do is integrate it into the software. Neither their software nor ScanImage 4 supports regular galvo scanning at this time, but at least the latter intends to add that functionality.

Till Photonics

Till Photonics has a couple of systems on offer too. First up is the 2p version of their iMIC platform. These octagonal monoliths look like they should be launched into space.

Till Photonics’ modules are popular, particularly their Yanus scan head. The heart of it is a set of Cambridge Technologies 6210s, but they have them packaged up nicely in top quality optics and an easy-to-implement module. They have modelled the nonlinearities and can squeeze a bit more scan speed out of the mirrors if you use their systems.

Next up from Till is their Intravital 2p that came out this spring.

It boasts a fairly large scan field (about 15.5 mm to a side in the focus plane, divide by your objective’s magnification to get the field of view) and a voice-coil driven z-axis with 7.5 mm of travel.

By the way, Till Photonics runs Colibri, an open source, LabVIEW-based laser scanning microscope software package. It runs off of the NI PCI-6110 board that most people use. It’s modular, uses 4 MHz sampling, and has support for cameras, motor controllers, and beam control. So you don’t have to have a Till system to try out the software. The author, Christian Seebacher, has some interesting information about the software on his website.

I’m sure there’ll be more new stuff at SfN this year… let me know if you see anything interesting.

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November 9, 2011

Sensapex piezo manipulators

Sensapex is the new kid on the block for micromanipulators, and theirs have an ultra small footprint with 20mm of travel on 3 axes. Here are some pictures of one of the first production runs:

To change pipettes, the manipulators have a tilt-back action.

The tilt-back action should help conserve space in crowded setups, but the arc might not be clear. Some sort of sliding back and/or twisting motion might be needed.

They’re very small. Check out the Axon headstage next to them.

It’s really built to be a pipette holder-type manipulator rather than a larger, headstage holder-type manipulator. They have magnetic and bolt-on headstage mounting options for Axon, Heka, and npi.

They have a “high load” version that should handle 200g (the MultiClamp headstage is about 90g). So it should be possible to mount about any headstage directly on the manipulator. Having the headstage too far away from the pipette can cause noise problems, so this might be what people want to look for.

Here’s the controller:

They’re also considering releasing the user interface as open source. This is from Mikko, the CEO:

We are using PC-software in the R&D and testing, but we don’t yet have computer interface for the customers. We have had some requests for it though so it is in our R&D plan. However, we are happy to provide drivers, function calls etc. if someone wants to implement control to their existing software (Matlab, C or Labview based). I’ve been thinking of going for the open-source approach for the user-interface software.

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November 1, 2011

Thorlabs’ SM2 fits Nikon lenses

Fun fact for the day: ThorLabs’ SM2 lens tube standard screws right onto the front end of Nikon’s SLR lenses. Other manufacturers probably use the same threading, I just haven’t tried them.

I don’t know if this is by design or not, but it makes coupling 35mm SLR lenses into optical setups fairly straightforward. I’m using it for a tandem lens macroscope. In the picture above I used ThorLabs part SM3A2. BTW, they also sell some F-mount adapters for connecting to the other side of the lens.

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October 31, 2011

No homemade cables

One of Josh Trachtenberg’s rules that I have adopted is “No Homemade Cables”. It’s so natural to think that this is the best solution to hook two things up: “Wire and bare connectors are so cheap, let’s just make our own cables.” However, homemade cables, even when made with great care, are usually of poor quality compared to commercial cables. As a result, you’re just giving yourself something else to repair in the future.

Option 1

Adapter boxes, aka breakout boxes, are more reliable than homemade cables. If you take this route, put it in a transparent, easy to open box so that you can quickly see (a) how things are wired up, and (b) whether there are any loose connections.

Option 2

Another option is to have custom cables made. I started doing this in London for a rig where I needed two fairly long 48-conductor cables. Custom cables are actually not that expensive and are of very high quality. It’s practical because you don’t have to buy an entire spool of 48-conductor wire (or whatever the job requires); nor do you have to source obscure connectors, strain reliefs, jackets, and whatnot. Not to mention time saved soldering.

Here are a couple of places that you can get quotes from if you’re interested. Any of them will do one-off jobs, you don’t have to bring them volume jobs.
Circuit Assembly
Technical Cable Concepts
Custom Cable Assemblies

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