Live Debugging

Get a Full list of Supported Drivers

Testing a Specific Driver File

Testing Host and Sub Devices

Live Debugging

Due to the open nature of our driver system, it’s relatively straightforward to write a new driver file for almost any imaginable device in Nebula. However, driver development can be a pain, so I though I’d share some debugging tricks.

For all commands, we will navigate to the command line using windows Command prompt or powershell. Here I show command prompt syntax. Nebula is commonly installed under program files. So in the windows start bar type “cmd” and press enter. This should open the command prompt. The commands below navigate to the install location.

cd\

c:\>cd "Program Files"

c:\Program Files>cd Nebula

c:\Program Files\Nebula>

First, is the use of logging to the command line. When Nebula is launched from the command line, a series of optional switches can be used to report logged output. By opening this alongside the app, it’s very fast to figure out what or where something is going wrong. To see log output, use –printlog.

c:\Program Files\Nebula>Nebula.exe --printlog

This launches the app, but shows a live log output on the console at the same time.

Get a full list of Supported Drivers

This command will return a complete list of drivers found in the Nebula supported list. To print this list, use:

c:\Program Files\Nebula>Nebula.exe --listdrivers

Here is live example of that command and the expected result. A list of drivers is shown, press enter to close the listing.

Testing a specific Driver File

Next, let’s load up one of these drivers in the test utility. Now, this is where things get cool! The test utility can load a host device driver, inspect it’s shown devices, attempt to connect them, and even test every sub-device!

So, let’s assume we have a new camera driver we want to work on. The camera should do things like snap an image, show various properties etc. In my example I use a real UVC (web) camera, and inspect the driver. The driver actually pulls the real camera info and even snaps a test image. The goal of this function is twofold, first to validate a given driver, and second to validate the device itself.

For this command we will use –testdriver *drivername* and optionally –port *portname*, for example, COM5

c:\Program Files\Nebula>Nebula.exe --testdriver --driver UVC_Camera

Here we can watch the expected output in real time. Note a new window is spawned to show us the results for these tests.

Testing Host and Sub Devices

Let’s say we have a controller, for example, an ASI or Marzhauser box, which has a Z motor attached. In Nebula, the controller can be thought of as the Host (the resident controlling brain), and the device can be considered an appendage attached to the host. So all drivers must have a Host, but a Host may only have one device. In this example, we use a demo Focus device. First the host is loaded and analyzed, and then, you ca select to test any device provided by that host. I choose option # 1 (focus) and the test runs, even showing my driver has a small error I missed!

Obviously, if you have a driver which isn’t behaving, please contact us – yet I hope these options provide a first-line of interface for power users, so simple things can be corrected and analyzed quickly!

-Austin

• The newly announced Excelitas Tetrem LED Light source
• Our Starlyte Nebula software (Now supporting the Tetrem)
• The Triggerscope Mini
• The PCO Edge Camera

TL:DR

The Problem

For illustration of the device control, I’ve captured a few real example frames from my oscilloscope, but also added some simulated oscilloscope examples that I hope will better depict the work behind the scenes for these devices.

I think it’s most important to frame this in a question,

When my software controls devices, how fast do they respond?

Assume we click a shutter button in any software. This can be for a laser, an LED, even an old mechanical shutter. In every case a bunch of things must happen for the end device to actually change it’s state. For that state (let’s say, “OFF” to change to “ON”,

• the software must accept the command
• and issue control signals to the device connection.
• That connection talks to the device controller (normally, a microcontroller)
• The microcontroller reads the command and does what it’s told
• The state changes.

Now, along the way, each device has a communication bus, and each bus has a traffic control system to insure multiple communications don’t conflict. As a result, the timing from the start of the button click to the end is variable – sometimes, up to 20 milliseconds!

Now, because the software is typically programmed to “confirm” each thing it does, it doesn’t just shout orders into the dark. Instead, the software waits for a confirmation, or acknowledgment, of the command being received, and oftentimes it will confirm the new state is active.

Consider this means a new set of jobs which must be completed.

• The device controller changes state
• The device controller then replies, or reports state
• The communication connection on the PC receives the reply
• The software reads the reply, and confirms it’s correct
• The software continues to the next job.

And so again, we can see a stack-up of not only delay, but a variable amount of delay.

If we visualize this in a graphic example like the one below, we can see the software code is sent to an LED microcontroller, the command is processed, and the led illuminates. However, we can also see there is a lot of room for improvement!

Another way to look at this control is what happens with a typical complex instrument. Below is a video where I control 3 devices. The PCO Camera, a Zaber filter turret, and the Tetrem. Notice how long it takes just to get 3 time points for 2 channels. The exposure is short, that's not the issue - the issue here is of course, the devices need to move, but also each device must receive + process + move + report positions, and this takes a long time.

TTL Control -> Old + Fast = Awesome

So what is TTL? Basically, nothing more than an on/off switch! Just like a light switch allows an electrical signal to feed a light, TTL uses a voltage to send a signal to another device. You can find a thorough history on this communication method here if interested.

What we care about is that we have an on/off control system. Because it's nothing more than a voltage, no crazy translations need to happen for things to immediately react. If a TTL control line is on, or "High", then the device should be doing whatever ON is. Many devices have this input, but few people even know about it! For example, most Focus controllers, XY Stages, Peristaltic Pumps, Perfusion systems, and many other devices like lasers, shutters, and actuators accept a TTL input for control.

TTL Logic typically comes in 2 flavors. Older systems will use 5V for "on" and 0V for "off". The device being controlled might use thresholds or a midpoint. Some devices, assuming you give them at least 2.5V, will go active. Others want at least 4V or 4.5V before they enable. Newer devices often use 3.3v control. This is the same concept as 5V, just with lower voltages. One word of caution here - before connecting things up, it's good to confirm the logic of your devices. Generally, the amount of power these use is quite low, and signal inputs won't break things - but in the case of an exposed microcontroller pin, you can definitely cook a 3.3v input with a 5V line!

TTL Blanking - A partial Solution

So what can be done about this overhead? First, we can consider that the only time we really need the illumination on, is when the camera is truly collecting data. One way to do this is to use the I/O control cables found on most scientific cameras. In my case I routed the expose signal from the edge camera into the Tetrem on a single channel, and then, told the tetrem in my software to use "TTL Control". With these changes, the LED source now waits for a TTL input to go high, before it enables it's emission.

So now, let's assume that when the camera is exposing, we can enable the LED line. The software can act as a logical "AND" if needed, but the line won't enable unless there is real collection of light by the camera. You can see an example of this below.

For the example, the yellow line is the camera output, or trigger output, the teal line is the LED B brightness result, and the purple line is the software-only reaction. In this example, we can see the delay (latency) of the response, yet in most software, the purple line will illuminate before the camera exposure. What this means for research is your cells are being exposed to excitation energy, and you aren't even collecting that light onto a sensor! So, we are in effect just cooking cells for fun. NOT GOOD!

Using the Triggerscope for State Control of Devices

This is great for a single LED, but how can we control more than 1 channel? For this, we would use a logic controller like the Triggerscope Mini! With the mini, the camera expose line is routed to it's TRIG input, and up to 4 output channels are connected to it's TTL control pins.

With this done, we can assign an active line using the triggerscope. This provides fast easy state switching, and can even be done over multiple devices. Doing this will mean, for this example, we won't be moving our filter cube device. Instead, we would use a multi-band dichroic. However we can switch the LED excitation, and that's how we will gain some speed here.

In the example above, we see a high speed switch. This is simply by telling the Triggerscope Mini to change channels, instead of the LED. But - there is still more to do to improve our speed!

Streaming for the Win

The above example uses the triggerscope via USB, and while we only have 1 device, we are still using a "set + capture + set + capture" type of sequence. What if we just told the camera, "run as FAST as you can." Then, we used the output signal from the camera to count frames, and we told the triggerscope, "every other frame, switch from LED 1 to LED 2, then back to 1". THIS is the power of "Streaming", or also called "Sequencing". With this capability, we pre-load stuff to do into the triggerscope. Then, we arm the triggerscope. With that done, our triggerscope is sitting, locked into a state, simply waiting to receive camera frame TTL inputs. Below is a graphical depiction of this technique.

Now, we tell the camera, "Give us 2 channels * 10 frames = 20 pictures, and do so as fast as possible". In effect, "give us video of 20 frames".

In this method, there is zero control overhead. The camera free-runs, the triggerscope switches, and the LED reacts. Below is a video example of this, note also there is a small in-view live feed from a web camera (for all these video samples), and you can see that, indeed, streaming is crazy fast! For the below example, we've tied the camera "transfer" line into the triggerscope. This way, the triggerscope recieves a brief pulse before capture occurs. We enable on the "low" side of the signal, and illuminate. You can see for the CMOS style readout on the left of the example, once readout is done, the channel switches.

So what does the above look like in real life? Check out this example below! Note that I select in my "lambda" control box the "Stream" checkbox. Then once start capture is pressed, the triggerscope is armed, the camera is readied, and we snag 20 images in < 300ms!

Of course, we can extend this using the 5V DAC control line on the triggerscope for capture of Z as well as channel, and if using the more capable triggerscope 4B, we can control even more devices.

I hope this article has helped explain not only why I make devices such as the triggerscope, but also how it's important to use software that provides complete control for your light sources and other devices, as well as the ability to do things like this. My sincere thanks to Excelitas for lending me the Edge camera and Tetrem LED, both of which have worked flawlessly on my system. Nebula has drivers for both products fully integrated for streaming control if you have interest in using it.

-Austin

I was able to work with two different variations. One is a complete transmitted illumination setup with camera and objective at 20x. The second is a reflected light device with integrated LED illuminators.

It’s neat to see such a compact setup on the desk. Using the 20x with a typical gut section stained sample was a piece of cake. Field uniformity on the illuminator was great, and color balance was as expected.

The reflected design unit has both a coaxial and ring-style illuminator position – both are provided in Nebula as control LED options.

Use of the camera was straightforward. It supports 8 and 12 bit readout on a color sensor. I can definitely imagine hooking this up to a small XYZ gantry or even a 3d printer for inspection use. Thanks to Opto for the demo unit!

Here is a video review and live demo of hte unit while running on Starlyte Nebula.

These cameras support Sony IMX-174 sensors, use a USB 3.1 (locking!) cable, and are straightforward to hook up and use. A GPIO connection is provided for I/O, and some models also have opto-isolated IO as well.

Binning, ROI and bit depth readout modes are also supported.

The camera I had hands on had a color version of the sensor, and while today has been a lot of rain, I was able to get a gloomy scene outside for my demo video below.

For questions on running this camera in Nebula, please contact us at www.starlyteimaging.com

If you are interested in these cameras, please contact Scientific Imaging.

Here is a brief video demo of the camera in Starlyte Nebula!

• Gain modes
• Color & Bit Depth selection
• Mono / Color Debayering

This addition enables users with existing cameras to easily integrate Nebula on machines used for inspection, materials processing, and other industrial applications.

-Austin

If you’re in a hurry, I’ve also outlined the basic steps below with screen shots.

Connect the triggerscope mini to a USB-2 compliant port, via it’s USB C connection cable (included).

Open micromanager, select the device configuration wizard, and browse to the add device page.

Scroll the device list until you see “Triggerscope-MM” expand that and select the -Hub device.

A window will appear, set the COM port to the port used by the triggerscope mini, and set the baud to 115,200, then click OK.

After a few seconds, a list of components will appear. Note this is for the full triggerscope, so we will only choose the TTL 1-8 Bank, and DAC 1 + DAC 2. Below is a basic chart showing what devices are supported on the mini vs the standard triggerscope.

When prompted for the DAC voltages, only select 0-5V (default), and click OK.

That’s it! Follow the manager to complete the install. Once done, you’ll have the common TTL output control, DAC control, blanking, sequencing and other features of a standard triggerscope!

To test this, I’ll walk through my goal. I need to get a lens made and I need it fast. I also need it to work properly. Lastly, I need it to be manufacturable at scale which means using glass substitutes often found in China.

So – using Claude Chat, I took performance specs from several existing lenses, handed them over and added:

“Make me a lens design that meets these general performance criteria.”

I also asked :

• Use Chinese available glass (e.g. K series)
• Validate the design using a raytrace
• produce estimated performance data
• produce design drawings
• produce a prescription
• produce a ZMX file

Here is the design it produced, it sure doesn’t look bad so far!

After a while it decided to build it’s own python analysis tool using multiple metrics, it did the job and generally things look ok. However – is this actually OK? Let’s drop the lens design into Zemax.

Using a COTS 200mm doublet as a comparison, I pulled a image, plot, ray fan and Huygens spot to inspect. All look quite similar, the spot size can be realized a bit better, but consider the aperture on the reference file was slightly larger, so that means difference in NA.

AI Doublet Left — COTS Design at right

Ray Fan Diagram

Note there is a difference here, this design is not optimized – with an optimized second surface, it’s obvious a lot of improvement can be made. Yet for a commercial type lens, I think this is a good example of a sufficient result – it’s less than the diffraction limit, which is a suitable bar for pass failure.

Huygens spot size is below, again note the difference in the psf due to optimization missed.

Finally, we can compare the simulated image formation – here I don’t think there is a clear winner, which is what we are shooting for in a commercially produced result. Very cool.

Of course, the first thing I made using this engraver was a 3D engraving of the USS Enterprise 1701-D inside of a BK7 Prism. Because why not?!?!

I’m happy to share a new device I’ve been working on after encouragement from numerous clients. This new smaller version of the Triggerscope is intended to bridge the gap between large complex systems and more streamlined setups.

With the Mini you get:

• 4 TTL Outputs at 5V
• 2 DAC Outputs at 5V
• 1 3.3V to 5V input Trigger
• Status LEDs for all connections!
• A single USB-C Connection to the computer
• Full supported Micromanager Triggerscope-Hub Behavior

This was a super fun project for me, as for the first time this board uses a bare MCU, which enabled my team to use any connector we wanted (hence the USB C) as well as full USB-Serial emulation on our own terms.

I’m also quite fond of the baby side-emitting LEDs on the outputs. These LED’s draw almost no current, but having them for all of the outputs makes it so easy to know whether a given control line is on or off. For the DAC LED’s, they change brightness with the output!

I tried to keep this design in-line with the current TG4, as it’s worked well for us over the years. This one is of course thinner, lighter and smaller, but in effect a truly miniaturized version of the larger model.

If you’d like to buy a Mini, we have them ready for sale on the store page here. For more information on the unit please see this flyer or the product page, thanks!

We had a recent customer asking to solve a rather standard but annoying problem. If I have a manual filter turret, and still want to overlay images, how easy is it to do?

To solve this, we added the ability to drag+drop any image from our capture sidebar into our main view. By adding a demo filter turret to the system config, we captured the real ex, di and em filter settings from the image, insuring metadata is transferred with the images collected. The resulting view is a true multi-dim architecture, saved as an OME set or RGB combined view. Our team made the addition, ran bug testing and edge case validation, pushed the changes and compiled the application in 1 business day.

Being able to react to our customers’ needs in a short timeframe is one of the best parts of working with my team. Nice work!

-Austin

Below is a 2 channel illumination sequence using the Excelitas Tetrem, captured via a Hamamatsu Fusion camera. A nice feature of the Excelitas units is a commonality of protocol. By keeping the communication back end similar between different units, it’s a straightforward job to add new models as they release. Thanks to Gary Tokman from Visual Dynamix for allowing us the system access to get this done!

Nebula can now control and support:

• Xylis Series
• Novem Series
• Tetrem Series

Each driver has a unique entry in the driver list. All drivers support the maximum possible device response time and full intensity range profile per device. The Mini will be added soon as well.

Contact us for more info at www.starlyteimaging.com !

-Austin

Recently Tucsen had a presentation on their new Libra CMOS cameras which includes some nice improvements in sensor performance.

Key highlights were:

• Improved QE at 92%
• Large Full Well Capacity (48,000 e)
• Native binning / debin system

In a recent presentation here a series of improvements were listed, among them a smaller pixel size selection option which supports a drop in pixel size to ~ 3.27um. From the explanation I assume the camera runs natively in a binned mode to produce 7.x um pixels, but can be optionally de-binned.

Each of the cameras (3 models announced) appear to have this feature, as shown below.

I’m more interested in the full well, as having a ~50k e full well seems a nice improvement in dynamic range over current options. Once item that was very interesting to me is that the full well capacity does NOT reduce when enabling the higher resolution mode. If this is correct, a 3.x um pixel with a 48ke full well would still be quite a nice bump in range and quite a feat!

For pricing, the presentation noted 0.4x the cost of a “classic CMOS”. I’m curious what that means in an actual dollar amount.

-Austin

The Why

20 years ago I fell into a career in the life science imaging industry, working for an integration and microscope dealer in southern California. One of my first jobs was installing a copy of software application called MetaMorph. This was an interesting thing to me, having a background in building computers from the days of ISA cards and DOS boot menus.

Metamorph could move things like a filter wheel (then a Sutter Instrument 10-2), and capture images from a camera like the then-new Photometrics 1300 YHS.

It fascinated me to watch the camera snap images with it’s mechanical shutter as the filter wheel switched positions, compiling images into a singular display. I was intrigued by this ability and also how it was used in research.

As time went on, I eventually moved from building the basic systems to maintaining them in-field, and then to offering complete hardware and software systems as a direct rep for the company who made this software, Universal Imaging Corporation.

The Need

What made MetaMorph appealing was the wide array of devices it could control at speed – from stages, to microscopes, to illuminators, basically anything. The company had no specific lockout, and would integrate device drivers for any product.

Over time, UIC was acquired by Molecular Devices, and after a long run ceased offering product sales in 2023. By that time, the industry was somewhat locked into the major microscope companies selling brand-specific products. Further, these companies would refuse support (understandably) for specific competing devices. Yet, this posed a genuine challenge for users:

Once invested into an automated microscope, I am forced to use proprietary software with restrictions, or use open-source software alternatives like MicroManager.

Now, MicroManager is an exceptional suite of software with device drivers, but for customers who want a simplified approach to control, with minimal hands-on configuration, and someone to call when things go wrong, there really aren’t many commercial applications filling the need. This situation led me to conclude that a new company was needed in the industry to:

• Provide Custom Integration Support for existing microscopes
• Offer hardware driver support for smaller and newer companies excluded by the major brands.
• Wrap device control into a simple, easy to use interface
• Provide exceptional support for products.

With this in mind, I engaged with my friend and colleague Jacob Gewerth, to launch Starlyte Imaging, and build our core product, Nebula. It’s been a long development process, but what Nebula does is something very special:

The What

We imagined, and have released, a software application geared to capture, and capture alone:

• We are dedicated to controlling devices as the priority – it’s all about getting data
• Our drivers are completely developed in Python, and our driver standard is open – meaning YOU or any company who wants to, can write device drivers for our software.
• Our pricing is approachable and fair – no huge budget outlay for software – we offer fixed licenses, or service-based pricing.
• We can support old as well as new devices – many older microscopes are perfectly suitable as a chassis to perform any modern study, they just need to be tied into the capture control scheme.
• We also can support data collection and metadata recording from perfusion systems, peristaltic pumps, or any other environmental control device you want to talk to.
• More than anything else, I wanted to bring the simple, fair and honest product support I’ve enjoyed providing to this industry for my career. Things fail, mistakes happen, but what makes the difference to me, is the dedication to see projects through, supporting your research, and seeing the project through.

In keeping with our vision, we have teamed up with Zaber technologies to showcase our software at this years Society for Neuroscience convention in San Diego. We will be at Booth # 2711 controlling the Zaber Nucleus MVR inverted modular microscope, and are looking forward to seeing you there!

Austin

The Venerable Nikon Ti-E remains in service in research labs all over the world. One of the first autofocus-enabled instruments deployed with the integrated PFS, there isn’t much to ask for when it comes to a modern compound fluorescent stand. For owners of the instruments, it’s important to be aware of a big maintenance item on the scope – it’s motorized epi filter cube turret.

These turrets use a stepper motor to rotate the large carousel into various positions. The motor, control electronics, and positional sensing system are all high quality and should last many years, however, there is an internal gear which connects the belt drive to the carousel, and over time and stress of use, these gear rings have a tendency to crack.

I recently had a colleague ask if we could reverse engineer a solution for these, as Nikon charges a mint for them and in some cases they may be difficult to obtain depending on your region. You can see from the one supplied to me indeed had cracked, and under inspection, stress cracks from aged plastic are apparent.

It’s important to note here that these might “work fine” under manual rotation (where the motor isn’t applying stress to the belt drive). If you are having problems where the filter isn’t moving to position, or hearing any snapping, clicking or other unusual noises, it’s highly recommended to remove the carriage assembly, and to inspect the gear closely for cracks. This is a ~ 15 minute procedure and only requires a flashlight and removal of the top cover of the carriage.

If yours is cracked, we can supply a replacement within 1 day of order placement from our web store.

Here is a video of how to inspect and replace the ring as well.

-Austin

-Austin

One of the toughest aspects of working as a consultant engineer is that the coolest and most interesting projects are the ones I don’t get to talk about or share online! But as time marches on, sometimes I get to share a few of my secrets. One of those secrets is Zaber. Years back, I needed to find a reasonable cost linear motion system, with integrated motor drive, that provided a well documented library and good customer support. I ended up working with Zaber on that system, and came to rely more and more on their linear and rotary motion products for such systems. As I grew more familiar with these devices, Zaber released a few products into the microscopy industry, starting with an XY stage platform a few years back. Recently, Zaber released an entire microscope system. This microscope provides 3 channel LED excitation for fluorescence, a linear motor Z drive, motorized fluorescent filter cube changer, and XY top plate motorized stage.

One of the product engineers was kind enough to ask if I would give this new microscope an eyeball, so I spent some time with one of the younger employees at ARC to see if we could set it on fire or otherwise break it. 🙂 I’m happy to report it survived our abuse, no small feat for a microscope in my shop!

Unboxing & Assembly

Microscopes typically require a knowledgeable sales rep or service expert to build. As a test of difficulty and complexity for system setup, I decided to assign my relatively new employee Garrett to the build. His report from the build experience was insightful:

Overall, the assembly and the initial testing of the microscope was simple and concise. The assembly is pretty easy for a person’s first time building a microscope. The directions were clear and provided great examples of what to look for in terms of parts and what to do. The assembly itself wasn’t too hard as screws were accessible and easy to get to. It was also a little fun seeing the microscope work at the end of the assembly. The whole microscope was packaged in a pelican shipping case, so the parts were well protected. Directions were very concise and provided much detail aligned with picture guidance. The motor locks are very good, they never moved or slid in the slightest during assembly.  Cable management is fairly good, doesn’t get in the way of any moving parts at any time. -Garrett Bunch

We also noted some areas for improvement along the way. For our model, the directions for mounting the microscope body and brackets, (5th hole) do not allow enough room for both the motor controller and the LED controller side-by-side as the picture shows. It would need to state at least the 6th or 7th hole. While used in all zaber systems, the the type of male cable connector for the X-DC02 cables were a little tricky to get connected. We had to fiddle with the inside thread on some of the internal drive connectors for a while to get it to connect properly.

In summary, while we had the regular amount of small questions, this was quite a straightforward system to build. There are far fewer components on this microscope for the user to assemble than on other systems. It results that the build requires simple tools and techniques, and all of the hard jobs are already done at the factory.

First Runs and XYZ performance

For our tests, we used a Hamamatsu Orca Flash 4.0 generously loaned to us from Hamamatsu. Connecting the camera to the microscope did demonstrate that cameras with a larger body would have a tough time fitting into the microscope access location on the sensor connection. We brought this to Zaber’s attention and were informed that they offer a riser kit for larger cameras, and can support cameras with large bodies.

Motion performance on the system was as expected. The XY stage uses a standard setup of lead screws and stepper motors. The focus/Z drive uses a linear motor, which supports fast operation, high accuracy and repeatability, but at a higher cost of components. I think it was a good decision to use a linear motor with the focus drive, and would love to see Zaber add linear XY to this microscope.

The control over the microscope can be performed using an included joystick, the Zaber Console software, or a Micromanager driver. We elected to first configure things in the Zaber Console to confirm proper operation, then to set up MicroManager for automated capture. Micromanager’s driver is a bit confusing to link up between drivers (you have to determine what controller and axis are operating a given function, 3.g. Controller 01 Axis B might == the Filter turret changer). But once things were configured, we had zero communication issues with the driver. Below is a video captured demonstrating the speed on a repeated XY move using MicroManager MDA for control.

We used MM 2.0 Gamma for our testing. A demonstration of the XY motion can be seen in the video below. This example included large Z moves and multiple channel settings to better visualize the speed of channel switching and Z stepping.

Initial samples used were a quite beat up kidney section, to demonstrate typical fluorescence performance. The objective we added to the system was a Zeiss 40x without UV apo correction, so some Z shift in color is apparent. Field illumination gradient was adequate, overall image field to the camera was well covered. This camera is a great tool for such testing as almost the entire 25mm output from the tube lens can be captured.

Collection of Z stacks for PSF analysis was straightforward. For these tests we used typical tetra-speck bead sets from Life technologies. What I was interested to look for here were coma or aberrations due to off-axis construction, none of which were observed. The system appears to be stacked up squarely, which was great to see.

To analyze the repeatability of the XY stage, several locations were saved on the bead slide, and then imaged in XYZ using MDA with timelapse. The resulting locations were then projected to produce a single image. Results showed repeatability in all axes within Zaber’s specification. The GIF below shows total drift over several hours. Note the stage is moving away from and re-visiting this location. Z drift from thermal is also evident, but to be expected without an automated focus tracking mechanism.

Z stacks captured in a similar manner to the prior experiment demonstrated even better Z performance, due to the employment of a linear motor stage on the focus axis by Zaber. Here, a single section is scanned multiple times in Z. The timelapse of these scans is stacked, so that numerous re-scans on a single set of images represents the same location scanned over time. When projecting this scan orthogonally, variations in Z will be visible. While expecting some linear shift due to thermal, there is almost no “wiggle” in the Z axis, due to the linear motor being so precise.

LED Performance

To test the LED performance we wanted to target two areas: field illumination performance and linear intensity regulation. Testing both can be somewhat tricky as almost everything will bleach over time. I used the blue isolation region of my bead slide, and it’s white label, to capture 2 multichannel timelapse series. Overall field analysis of each channel was then graphed to demonstrate intensity regulation. While a very small spike was observed in the UV channel, all channels performed considerably well, and it appears intensity regulation is well handled on the drivers for these LEDs.

Field uniformity was great for a built-in LED system. This is always something tough to work on in a short-pathway LED excited system. Performance on multiple surfaces looked quite good. Some clipping can be seen at the upper left, due to camera placement. All channels showed this as well as a simple flashlight illuminated above, indicating this was due to my mounting and adjusting of the camera, rather than an excitation-side issue.

Further Observations

Zaber added a drop/refocus feature to the microscope, similar to an escape function on a traditional scope. Using the linear motor’s speed, it is quite a fast shift to go from escaped to “in focus”. While this demonstrates the speed of a linear motor system, I would take care as an owner to know if I were “escaped” or just assuming so, for if a sample were placed over the objective, and the system to “return” at such a high rate through the sample, I imagine damage would occur to the sample or objective, or both. At the same time, this demonstrates the capability of such a motor, and slowing motion down in firmware/software is easy, while speeding things up is hard!

While Zaber did a great job with the included LED illumination, I can imagine customers would also benefit from using a LLG-coupled or fiber coupled light source. 4 channels is adequate, but many clients need greater diversity in channel selection, and stuffing more than 3 LEDs into an excitation module on-scope can be tricky, so I hope Zaber offers this as an option in the future.

The use of a linear motor for Z shows how great this motion method is for fast, reliable positioning. If Zaber were to add an autofocusing system to this axis, it would provide a nice improvement in market applicability for timelapse observations and high speed high magnification scanning. Similarly, the option to purchase a linear-motion XY top plate would be great, for those clients needing high speed scanning of a large region.

Summary

I really like this microscope. It has the feel of an industrial motion system adapted to an optical platform, which makes sense considering its pedigree. I can imagine that this would make an excellent choice for a plate scanning system, would do well coupled to an incubator, or would be a great fit for commercial clients working on automated analysis of a product, reagent or similar project. The addition of an integrated autofocusing system using reflection would greatly expand the use of this scope into timelapse live-cell work, where it’s shape and design would be a great fit for an incubation enclosure.

Customers who would do well with this microscope are those with some technical chops, who also don’t want to hand-build a microscope due to time constraints, or commercial customers who need a no-frills motorized microscope for scanning. As a company, Zaber has always provided great technical support and has a history of building precision motion systems at fair prices, so I would definitely recommend considering this system if you are in the market for an automated microscope.

-Austin Blanco & Garrett Bunch

(Disclosure) - I was not compensated for writing this review, and my observations and commentary are my own. - AB

EDIT – Fixed Video link!

-Austin

Needing to address this problem, we used a standard level shifting circuit, and stuffed the wires into a small enclosure. After thinking about this, I figured others may have the same problem (not only with PTG or FLIR cameras, but others also), and so decided to roll out a dedicated PCB to address this need. So – it’s posted to the store website. The circuit diagram can be seen below for those interested. You can place orders for these on the storefront here, with shipping as early as the week of 10/12/20.

This can be used with any device that outputs a 3.3v signal. If you have questions on how to connect things or if it will work for your system, please don’t hesitate to contact us! – Austin

New Features

• At the heart of Triggerscope 4 is a New high speed MCU running @ 600 Mhz. This is an 83x improvement over the Triggerscope 3B.
• On board 1024K MCU RAM provides more than enough memory for arrays.
• A new features provides access to a 16GB External SD Card, installed inside. Memory may be used for storing data for sequence access, recording of external parameters and timing events, or saving of advanced settings.
• A new Real Time Clock is included for better timing over long duration experiments and delays.
• DAC update rates can be overclocked, to a maximum frequency of 270kHz. 
• Integrated and simplified external controls for easier operation.

New Software

In addition to the release of Triggerscope 4, ARC is introducing a python-based, multi-platform compatible standalone control application. Our application can be installed on Windows, OS-X, or Linux systems. Source is also available from the GitHub Repo here. Users have two options for use – EXE and APP executables provide a small containerized version of python, so the entire application runs without any external installs required. Or, for customers who already have python installed, simply downloading and running the native python file is an option.

For debugging, all communication between the software and the triggerscope are printed for the user to view, to make diagnostics and custom driver development as simple as possible.

Light and Dark themes are provided to keep monitor glare down during imaging. This software is available for all Triggerscope 3 and up devices, and can be found on our GitHub Repo here.

New Micromanager Firmware Options

Nico Stuurman recently undertook a huge endeavor, and implemented a new approach for using MicroManager and the Triggerscope, with faster syntax, greater memory capacity, and tight integration into the Micromanager sequencing system for fast device control. ARC can install this firmware option before your triggerscope ships, just let us know which version you’d like to use!

• If you already have a triggerscope, and would like to use the new TriggerscopeMM Firmware, please refer to the MicroManager device adaptor variant here.

For Information on setting up the Triggerscope 4, please refer to this guide.

Purchase your Triggerscope 4 at the ARC Online Store here.

-Austin

Thanks to some generous industry friends, I finally managed to get my hands on a seemingly unobtanium RPI “High Quality” camera. I’ll have more info on the camera itself regarding performance, but my first question was – “can it be effectively cooled? ” Fortunately, having run similar experiments on a Point Grey / FLIR camera, I had most of the components and a basic test method ready to go to figure out the answer.

To start, we simply piled up thermal tape on the backside of the camera. This provided a poor but useable thermal coupling to the back of the camera PCB. Ideally, we would wrap the entire camera system in aluminum, but for now, this works. Next, we used a off the shelf TEC, and a CPU radiator, for the heat exchange.

To test performance, we obtained 40 images with an exposure time of 5 seconds. We only ran the TEC at 1V / 125mA. While running the experiment we recorded an IR video to document the reaction. What is quite evident at about the halfway mark, when the cooler is enabled, is that the TEC Cold side goes “dark”, and shortly thereafter the aluminum C mount housing of the camera as well as the exposed PCB begins to cool down. Images obtained showed corresponding drops in background intensity of ~ 10% for this test. Not amazing, but the drop in mean image intensity directly matches the enable and disable of the cooler. Further tests will include a tightly wrapped enclosure to better thermally sink the complete camera sensor cell and PCB.

Of additional interest is the noise in the image from the readout taps. Of the images obtained, a clear pattern can be seen, again with the lens cover on and exposures of 5 seconds. A bloom can be seen on one side. I’m curious how this may change with a better cooling system. Below is a pseudo-colored maximum intensity projection of the 40 frames obtained during the experiment.

A basic example of read noise on the taps can be seen by looking at the standard deviation over the frames. This isn’t perfect but it does show a general amount of error in readout. Performance here was not bad at 3 counts maximum change over the sensor field.

As with all such tests – further study is needed :-). We plan on taking this camera through it’s paces in a lot more detail – so – more to come soon!

-Austin

All over the world people are being asked to work from home… 

Yet how many of us have had to do so for extended periods of time? As a case in point, are you reading this now while sitting at the office? Or, worse – while sitting at home, when you should be working? If so, this guide is for you! I also have experienced this challenge, but have had the opportunity to refine my work procedures while conducting business at home for the last 20 years. So – if you are working from home today, or plan to do so in the future, I hope these tools will help you maximize your enjoyment and impact while away from the office. The following sections I hope will give you clarity of purpose, the proper mindset to work alone, a set of practical tools to work alone, and finally, a method to evaluate how well you are doing at coping with this change. 

Preparing the heart and mind to work alone

To be free to operate independently is a privilege. For most people over the course of history, employers or overseers would not or could not trust those they watched to “work hard”. As a result, work was “managed”, typically under brutal forms of motivation. Of course, there were notable exceptions to this. In the New Testament we find Jesus using a story of three servants who are given large sums of money, “Talents”. All are given rather large, but different sums of money (roughly, 1-3 years’ wages), and told to use the money for the masters’ benefit. Two of them do so, realizing a gain, while the third, buries the money. When the master returns, the two who have profited are rewarded with further authority. The third returns the original sum, saying, “Master I know you are a hard man, taking what you did not earn, reaping what you did not sow, and so I was afraid and hid the money, so here is your Talent back.” But the master replies, “You know I was a hard man? At least you could have put the money in the bank and earned interest!”. The servant is then fired. This to me is a fascinating story. It tells us so much about how life was 2,000 years ago, and what we can basically see is that not much has changed today. Leaders of companies may be tough people, oftentimes we will find ourselves questioning the morality or fairness of the decisions they make. Some wrestle with a love of money so intense that they will allow others to be hurt, so that they can gain further wealth. At the same time, we can see that those of us who use resources wisely are given further trust – and can we not see this principle applies today? If we at our work prove ourselves capable and responsible with the freedom and authority we are granted, most of the time, we will be offered greater freedom and authority. Now of course this is not 100% of the time – when it isn’t, I’d suggest the management is not doing well – as this principle is a simple, effective and fair one for any organization. So – whether by promotion, a change in work policy, a change in life situation, or even a global pandemic, here we find ourselves with this new authority – the authority to do what we want, when we want, and with no one watching over us. So what will you do with this new authority over yourself? The question itself is defined by one word: Integrity 

In the Marine Corps, one of the principles drilled into our heads was that integrity is what you do when your drill instructor isn’t there. Similarly for us, integrity is what we will do at home when our management, more importantly our team isn’t there to motivate us and keep an eye on us. So these stories convey operational principles and the question of our own personal morality. We all must ask ourselves if what we are doing is the Right Thing. So, here are some thoughts to keep handy while working, or in case you find yourself browsing facebook during work time:

• YOUR COMPETITOR IS WORKING HARDER THAN YOU RIGHT NOW If you don’t work hard, keep in mInd the people who you compete with. They are likely also at home – and if they are working harder than you, at some point this energy difference will result in your company losing it’s position, resulting in you losing your employment. This is harsh, but I think we can all agree rather obvious. 
• DISCIPLINE PROVIDES OPPORTUNITY AND POWER If you do work hard, you can realize the personal joy and pride of knowing you did well for yourself, your team, all on your own – further, you can grasp the power of your discipline, knowing that you can work on your own in any other job! This opens up a massive new world of employment opportunities – as there are quite a few people who simply cannot discipline themeselves! So you are more capable, a better team member, more employable, and ultimately, have greater freedom as a result of your discipline and efforts. 
• YOU ARE NOT A THIEF – SO DON’T STEAL. If you are being paid to work, and are not working, you are stealing. Not only from your employer, but from those members of your group who ARE working hard while you don’t. You may not consider the owners or management worthy of your integrity, but we should all consider our peers as deserving of our best. Get after it.

Practical tools for working at home

Prepare yourself

For the next few months – keep your regular work routine as much as you possibly can. Get up and dress in your work clothes. Do whatever you need to be presentable and prepared for your job. Eat breakfast as you normally would at the same time. All of these activities will help mentally set the stage to understand that “We are now working”. I know it seems wasteful – but the mind needs this routine, especially when so many changes are all around us. Bear in mind, your work commute now takes 3 seconds, and traffic is always light! 

Prepare your “office”

My first office was a piece of junk computer stand 4 feet from our kitchen in a one bedroom apartment (pictured above). From that little cove I covered a sales territory encompassing all of northern California for 2 years. In that time I attained a salesperson of the year award from Molecular Devices. But I MADE THE SPACE for my office, and when I was sitting there, even when the wife was watching tv 5 feet away, I was “at work” during business hours. Because so many of us find ourselves without preparation today, you might make your office be the kitchen table or even a folding table for now. But – wherever it is it should be away from the living room if possible, and in a place where there are the least possible distractions. In front of a window? Close it if it’s distracting. You can open it after you rebuild the routine. In a noisy location? Amazon prime a headset. But make the space, and remember that while you are there, you are “away at work”, away from the responsibilities of the home, away from the XBOX, away from the kitchen. Those things don’t exist, because you aren’t there – you are at the office! 

Prepare your schedule

As you are now your own boss at home, you will probably find it hard to stay motivated, as until there is a deadline, do I really have to do this now? Yes you do! Using our calendar, block out time for each project as you need. Make sure to include a 15 minute break every so often, as well as your lunch break. This is helpful as when the desire to check Instagram hits – it’s better to have an alternative – e.g. “Well I only have 2 hours to finish this data entry project, so if I hit instagram right now I won’t have time for the job”, instead of “Hey let’s go see what’s up on instagram, there isn’t anything pressing at work right now…”. The difference in mental position is amazing. 

Prepare your family

Kids and the spouse may find it tough to understand why they can’t pester you while at home. This is very important to draw a line while working. If they are interrupting you while at work in the house, they are literally taking food from their own mouths (is your competitor allowing such distraction? No? Then you are losing right now!!!). Your work is important, and they must honor your endeavour. This simply takes training and repetition to realize for them, but give it a few days and they will see your clothes, you sitting at the office, and soon realize, “Mom is working right now, I can’t bother her”. Now – I should note that one of the greatest joys for me is allowing my boys into my work life. I’ve had them help with many work projects, learning along the way.  Over the years we have developed a system where they will visit the office to tell me stories about school, ask questions on things etc. But at first – the barrier must be established for your benefiet as well as theirs. If you allow distractions now, they won’t respect your requests later for peace and quiet. Further, what are you telling them about the value of your work? So have the family meetings as many times as are needed, and keep up the barrier for the short term. In a few months you can experiment with opening that up – but this is a tough change for you – so minimize the number of fronts you need to be fighting on at the same time! 

Prepare your management

Any time we are suddenly gone from the office, management can have trouble. People in leadership positions can have greater fear, greater uncertainty, than those in operational positions. So as bad as things may be for you with this change, it’s worse for them! Further, while you are struggling with how to cope with the change, they are doing the same, but also struggling with how to handle the managerial/directive changes needed to keep things going. In this time of chaos, you can be the stable anchor. This requires some creative thinking, good communication and diligent follow through. 

• Get the tools you need. This is critical. If you do assembly work, check out or record the tools needed from the office and take them home. If you need a soldering iron, buy one. If you need to have supplies shipped to you or you need to have a monday pickup / friday drop-off, talk with management and set it up. Ultimately there is a way to do your work anywhere – it might not be optimal, but it is possible. Be creative and solve these problems for your management, so they can worry about others and not you. . 
• During online meetings, write down the tasks you are supposed to accomplish, and then review them with the team or leader at the end of the meeting. 
• Share your online “self-schedule” with your management – this can be a great way to help them allocate resources. It’s much better to have a conversation where you say, “Hey I am trying to maximize my work in this situation, so I’ve cooked up a work schedule for myself. Please take a second to look at it and let me know if you want anything changed.”. If they see this, and have half a brain, the reaction will be “Wow, his employee is good to go – and I’m lucky they are so self reliant!”
• If you are running out of things to do at home, spend some time thinking up new things you can do to help the team. Brainstorm with management – This is quite important because ultimately, if you do run out of things to do, why should the company keep paying you? With enough creative thinking and hard work, you should never end up with nothing to do.

Conclusion

I have set a rather strong tone on this subject. I have done so because initially, it takes a heap of effort to think differently about your work. But what also needs to be understood is the amazing freedom, efficiency and joy of working in isolation. How long is your commute? 30 minutes? You now have 5 hours back in your life, 5 hours. That’s almost a work day! What can you do with 5 new hours in your life? Learn a new skill? Spend more time with your family? This new time is a gift, and the only loss is the auto company and gas station! Learning to work this way makes you exponentially more powerful and employable. If an employer can see that you are capable of working like this – will they really question you when you ask for greater autonomy? Further, when you do meet in person, your meeting time will be more valued, and you will find greater joy in sharing time with colleagues. The richness of doing work together will be realized as now, it will be rare.

As with all things in life – today can be a chance for us to shrink from what we are called to do, or to rise to the challenge, and grow as a result. But having tools helps in our growth. I hope some of these tools help you in learning to adapt to this new work environment you may find yourself in. Good luck! – Austin

For complicated systems which may use reflection or episcopic combined paths, additional pieces of extrusion can be cut and aligned to a core axis quite easily, and even attached in 3 rotations (left, right and “up” from the primary optical axis). Extrusion pieces can be ordered pre-cut, or easily cut to length using a hacksaw. Right angle clamps avoid any angular errors caused by bad cuts. Personally I use a mill to face off the ends, but most people don’t have access to a mill.

To make things even easier, it’s nice to be able to mark off critical dimensions on the extrusion with a felt tip pen, which is easily later removed from the anodized surface with IPA.

I’ve added all of the designs I frequently start from, in STEP format. If someone needs another format please let me know and I’ll export one.

The files are hosted on my CAD share on Github Here.

-Austin

Some additional screenshots of the tool.

This approach gives any user of the triggerscope far greater control over how sequences are run, and offers users the ability to customize external device commands if needed.

I’ve cooked up a brief video overview here, with some example screenshots of the results on an oscilloscope below.

• If you’d like to use or check the script out, it can be found on my GitHub site here.
• Please note this requires updated firmware (V 600 or up)

A basic example of a Z stack in a single channel is shown below. In this configuration, 1 channel is selected in the MDA window, and a single Z stack is collected. The Yellow line indicates intensity of a laser or LED on Ch1, the pink line indicates the Z voltage output, and the blue line indicates a TTL input from the camera.

In the next example, output from a “channel first” Z capture is shown. Here Channel is prioritized, so the system runs Ch1 , then captures all Z frames, thens witches to Ch2, and captures all Z frames.

Next, is an example of a capture previously impossible in MM. In this capture we stream 3 time loops back to back, with no delay.

Next up is an example of a “Z First” capture, where all channels are acquired for a single Z position.

Finally, a multi channel example can be seen in this image, with 2 channels running in a time series.

Those are the highlights – I hope others find this useful!

-Austin

But – the SM1 thread from Thor is another matter. I had to add one to a part, so I found this nifty trick in fusion which supports adding custom threads to your install.

It turns out Thor provides the required information to form the threads, so using this page I was able to pull in the thread settings, and generate my own thread selection.

For anyone who just wants to use the threads in their copy of fusion, please find the file located on my Github page here.

-Austin

Yet for many, the financial or temporal obligations of higher education aren’t possible to accept. Sure, there is night school, online correspondence etc, and these frameworks are excellent sources of gaining credentials, and of accessing new opportunities in a career. But what if I simply want to learn a bit about making parts? What if I want to build a little trinket of electronics, or to learn a little bit about optics? Today, this concise/iterative form of learning is available to everyone, yet only a few I interact with seem to exploit it’s full potential. It’s the way I’ve learned almost everything I know and use today. It does have downsides, but these can be filled with proper guidance. I would call this form of learning “Project-based” in nature. So, how does one attend such a lesson?

Step 1: Take on a project in the field of interest

Let’s say that, like me you have an interest or a curiosity on how machines move. You’ve seen a 3-D printer, or maybe a robot, or a CNC machine, and wondered, “how does the table top move, what drives it? How does it “know” where it is?” All good questions of course! Now, I had these questions, and I never truly was able to answer them until I embarked on my first CNC construction project. This began as a kick-starter investment, and probably had a total cost of ~ $350 by the time I was done. It took 2 months or so of work to finish it completely. Spending a few hours here and there to construct it. From there, I tinkered and modified a bit, and finally, after several months of use, I ended up stripping it down to use for other projects. Now, numerous issues and limitations popped up during this process, and I also realized I was left with several questions. Which brings us to the next level of learning.

Step 2: Learn from the Experts

Some of the questions which gnawed on my brain at the end of this build were:

• Was this machine actually capable of milling aluminum?
• Why did the speed seem so slow?
• Did I need liquid cutting fluid all the time?
• How come these tiny dremel end-mills kept breaking?
• What was the best GCODE generator to use?

These are questions which come not from the top level of a neophyte, but from a marginally deeper question of the enthusiast with some minimal experience. I moved from the neophyte to the enthusiast when I took on the project. I now had a surface level understanding of what questions to even ask! Not only that, but my ears itched for the answer. I HAD TO UNDERSTAND these answers, because my lack of knowledge was killing me, and affecting my understanding as to what this little machine could do! So I turned to people I know in the microscopy automation industry, gents like Chris Ballard from Sutter, John Zemek from ASI, and during the course of other work, would ask things like, “Hey so what exactly do you do with your machines to compensate for backlash?” Now here’s the fun part – no-one EVER denies an opportunity to share their expertise when prompted! So while I absorbed everything they had to teach, they were happy to share and to receive an opportunity to deliver lessons and experiences gained from years of effort. These small interactions may have taken 10, or 15 minutes, but over time I accumulated a series of data points. Some things like, “well, we always use fluid because we are cutting so fast” allowed me to understand that speed impacted heat to a greater degree than I had assumed, prompting me to search down google eggs like this speed & feed calculator. Other times, I’d learn objective known facts like Chris stating, “It’s hard to beat mass, when it comes to CNC, and microscopes for that matter, the heavier the better. ” Now such a statement seems simple, but because I had the background of watching my tiny machine struggle to cut, and had the framework of knowledge for that statement to fit, I quickly was able to understand the why behind the fact, which allowed me to cement a better understanding of what this meant for such machines. Vibration, strength, heat, inertia, it was all making sense.

Step 3: Take the next class

How to proceed? Simple: Build a machine that supported my next level of learning! In this case, it was a used RF-45 geared head mill. Armed with my understanding of how motors drove my current CNC, I snagged a used RF45, and designed my own CNC conversion for it. My design was additive, meaning that even if it completely failed, I could revert back to owning a manual mill – and who doesn’t want a milling machine? 🙂 So the next level of learning, the next class, had begun. Through it I learned my motors weren’t strong enough, my motor drivers were not high enough current and too slow, I had to account for additional backlash on the bigger machine, and so the journey continued. I still use the results from that machine for small projects today. The experience gained during the project has been too enormous to measure as I’ve applied it to things like, “hey, you can’t make a box with square inside corners, because all end mills are round, so how will it be cut?”

Step 4: The finals Exam

How do I know I’ve gained requisite knowledge in a field? The answer is, I never have. At each level of learning I’ve discovered new and more interesting aspects of a field. Liner encoding, DRO’s, automated tool changing heads, variable speed control GCODE, a litany of CAM generators, new 3 phase motors and controller options. So when is learning a field enough? I don’t think there is an upper limit. And this is the most important principle in my humble opinion. Those of us given this gift of curiosity have an obligation to leverage it. In an era in which Khan Academy can teach you trig for free, there is no excuse to stay stagnant. More importantly, I have watched several colleagues declare an end of learning, and subsequently become irrelevant. From, “I don’t do email” to “I can’t understand programming”, these people made the choice to stop.

And the world kept going.

And they were left behind, no longer useful to the community they lived or worked in. So whether it’s art, philosophy, religion, sports, outdoors, history, science, or just learning about the lives of others, we must never accept a point at which we believe, “I have arrived”. And so what is the final exam? Well, it’s likely comfort. Are you comfortable in your work? In your technical experience? Then get uncomfortable. Engage in that arena you were scared to try. Try out Jiu-Jitsu even if you’re scared. Order that 3-D printer kit, even if you don’t have time to build it. Download Python and follow some online tutorials, even if you never use it again. Stay off balance. If you are comfortable today, you failed the exam. The good news is that you have another opportunity to take the test, and learn something new, staring today.

-Austin

For a new Triggerscope 3B shipment, remove from packaging and account for the following:

• 12V power supply
• Mini-USB cable
• SMA-BNC connector cables, if purchased
• ARC certificate of compliance paperwork
• Triggerscope 3B Controller

Hardware Connections

Once unpackaged and all components are accounted for, plug the 12V power supply into the slot located on the left side of the unit labeled “12V,” and plug the other end of the power supply into an available power outlet.

Next, plug the mini-USB cable into the left side of the Triggerscope unit in the slot labeled “USB,” and plug the other end into the computer you are going to be using.

Driver Install

Next, locate the switch on the right side of the Triggerscope 3B, flip the switch either way, both ways turn the Triggerscope on. Open a new tab on your web browser, and download the Arduino IDE Software that matches the Operating System you are currently using. Follow the install prompts to install the software

With install of Arduino IDE complete, open the newly installed Arduino IDE application on your computer.

On the top ribbon, select the “Tools” tab. Then hover over the section that says “board” and then click on the “Board Manager” option.

Next, in the “Board Manager” window, click on the “filter your search” box and type “DUE,” and the following should pop up on the screen, “Arduino SAM Boards (32-bits ARM Cortex -M3) by Arduino.” Click install on this option.

The Arduino application will proceed to download and install the driver package needed to run the Triggerscope 3B unit.

MicroManager Setup

Most end-users will already have MicroManager installed, but if not, make sure to use the nightly builds found here.

In MicroManager, select the “Hardware Configuration Wizard”, Click Next until reaching step 2. In the lower list, browse to the “Triggerscope” section, and add the Triggerscope Hub Device.

For the Communication setup, select the COM # assigned to your triggerscope under the Windows Device manager, read as “Arduino Due Programming Port”.

Configure the Com # found int he device manager, make sure to set the BAUD rate to 115200.

When prompted, select the TTL and DAC lines needed for your application. Also note DAC 16 is tied by default to the FOCUS device. If running sequencing for Z stacks you’ll want to add this.

Continue through the dialog options, if connecting to a Z stage, be sure to specify the maximum and minimum height of your stage so that Z data is properly calibrated. Contact ARC if using a range other than 0-10V.

Next, confirm proper operation by opening up the Device Property Browser. You should have discrete control over the DAC lines added, and on/off state control over TTL lines. Note that the Triggerscope 3B will illuminate an output LED when DAC control is enabled, or when TTL control is enabled. This can be very useful to confirm proper communication with the computer.

This concludes the basic setup for Triggerscope 3B. Please contact ARC with any questions if you are having difficulty!

Daymian joined the team as Technical Systems Engineer Jan 15th, and brings to the company experience in MSCA, fabrication, additive manufacturing and firmware development. His primary role will be hardware & technical systems , as well as supporting work in the software and optical areas we work in. Please join me in welcoming Daymian to the team!

– Austin

• DAC Software Range Selection
  • Range selection is now software controllable for each channel. This means you can get a full 16 Bit resolution delivered to your device in any range from -2.5/+2.5V, to -10V/+10V, as well as -5/+5V, 0-5V, and 0-10V. A demo video of this feature is available below.
• LED Status Indication
  • When integrating or first using any TTL controlled system, it’s difficult to determine whether the controller is properly firing trigger signals, and whether the device is properly outputting changes on the TTL and DAC lines. New LED’s on the Triggerscope 3B indicate an active sequence, TTL status, DAC output status, and input trigger status, all from the face of the device. In addition, these signals can be disabled or enabled for compatibility with ultra low light acquisition environments.
• High Current TTL Drivers
  • When driving Lasers, the 50 Ohm impedance used can cause a tough voltage drop problem. This has been addressed by now sourcing up to 500mA per line on TTL lines 1-4. In addition, lines 5-8 now source 200mA per channel. While most systems should work great at 200mA, the higher current channels may also optionally be used to drive low intensity LEDs if needed, for applications such as transmitted illumination, powered directly from the Triggerscope.
• An External DC Power switch is now included for simple restarts.

More specs on the datasheet found here

Here’s a quick demo of the DAC Range function as shown on my Oscilloscope.

List price is $1,500 USD. Current lead times are 2 weeks ARO. Please contact ARC for purchase.

-Austin

Dr. Kuan required an upright confocal microscope, which could perform photoactivation, widefield and confocal fluorescent excitation, all while running on an extended focus throw of 2″ range. The system required several custom pieces of hardware and software, which made things extra *fun*.

Team Contributors

Visual Dynamix LLC, headed by Gary Tockman, was the lead system integrator on the project as well as the primary service provider for the system. Gary led the team on the build and did an excellent job defining input specifications and performance criteria, as well as managing the crazy logistics of building a system from so many different vendors.

Using the same device for slides and for live specimens required some work on the focuser throw. Sutter Instruments provided a great solution via the BOB microscope. Chris Ballard spent a great deal of effort working out a long travel Z motor relying on the MPC control system for XYZ axis.

89 North provided the photostimulation system, confocal, and LDI laser excitation. Scott Phillips and Simona Stelea both spent several days on site working on the system to make sure everything was dialed in properly.

All of the devices on the system needed control via Metamorph. I worked with David Biggs over at KB Imaging to handle this part, and was very glad I chose his work. David has extensive experience with driver development, and due to the proprietary nature of the firmware on this instrument, his experience saved the day on dialing in the driver for speed and reliability.

Finally, I took on the system I/O side for lasers and triggering, as well as building the imaging PC and configuring Metamorph. Because the latest Triggerscope 3 model needed to drive the lasers,I also managed to work with David to create a Metamorph driver, so if you happen to run MetaMorph and want to control the triggerscope, please get in touch.

Systems like this usually require a day or two for the install, but in our case, most of the install team spent a week on site, to be certain that the details were ironed out and to get some training on the system in for the end users. In our situation this was a good call, as we found the inevitable last minute changes which needed to be addressed. Having the team together made addressing these things straightforward.

Results

A bit after leaving, the client sent over a beautiful video showing experimental data which includes the visualization of vasculature, with a clot stimulated by the laser exciter, and a resulting timelapse of that clot forming.

It’s great to be able to participate with clients, manufacturers and engineers who can pull builds like this together, and better still to watch this important and exciting research move forward. I’m even happier I get to share this build!

-Austin

One additional trick I use for mods like this, is a router speed control, available form Harbor freight or Amazon. It allows the motor speed to be adjusted. The little Ryobi is super fast, so I ended up suing the speed control on it’s lowest setting. These controls work well for most motors – some using “soft start” motors may not work, but it did great in this case!

If you would like to print your own (assuming the hole spacing is compatible), the STL file and STEP for modification are available on my Github page here.

• Austin

Basically each camera can capture 4 polarization states in one image. I can imagine these may be used for several microscopy applications, chiefly in polarization light microscopes, but also in DIC, and stereo applications where specular reflection is prevalent.

-Austin

I am aware that the vast majority of end users rely on Windows for control of devices, so if you read this and have an interest in a compiled x64 version, I’d be interested to know.
Over time I plan on expanding this application to include full configuration over delay timing, sequencing, and other functions.

Here’s a brief video showing the device, you can purchase it from the online store here.

The FAA classifies certification for commercial use of drones as an “sUAS” cert. Or, a small Unmanned Aerial System, Remote Pilot certification. This certification allows us to operate a drone up to 55lb, for profit, in much of the US airspace. Below are links and resources, as well as questions and answers that come up during classes. Feel free to post here with more questions if you have them!

This is for part 107 operations, or sUAS operations.

• FAA Advisory Circular 107-2 – many common questions answered here 
• FAA’s B4UFLY App, this is a great too to confirm you are free to operate in an area.
• Waiver for operation in controlled airspace (request for from FAA)
• Interactive test questions
• Excellent summary of numbers & facts
• FAA Test Supplement PDF (2016)
• Drone Logbook References
• Free Online Charts 
• Excellent online test with chart images

If you are having trouble with reading of charts, especially the Lat/Long stuff, here’s a great video that walks through the steps.

—-UPDATE—-

Some answers to the questions which came up during the class:

Question: “Can a rPIC certificate holder operate within 5 miles of a non-towered airport?”

Yes – refer to FAA AC 107-2, section 5.8.1. “…Unless the flight is conducted within controlled airspace, no notification or authorization is necessary to operate at or near an airport. ” (Applies to certificated sUAS pilots)

Question: “Are sUAS operations permitted in MOA’s?”

Yes – There si a great study table on airspace from this website, search for “MOA”.

-Austin

This research continues to advance the work of attempting identification of Alzheimer’s via the retina. If we can continue to refine this approach, someday, people will be able to have frequent checkups to determine whether they are approaching a risk of Alzheimers – early enough for intervention to occur.

For the human trials, images were obtained of the retina, using a custom modified confocal ophthalmoscope. The retinal imager obtains images from the patient’s eye, and the images are then analyzed for a number of potential plaque sites (those sites which may contain AB plaques). If enough sites, and or site density is found, the patient indication shows higher, whereas a control would show less grouping and/or less sites. This is a simplified explanation of course, and a ton of work went into figuring out how to best identify these sites. Here’s an image of such a patient. Int his image the heat map indicates the grouping and intensity of the AB plaque locations.

Hats off to Maya & Yosef Koronyo, the team at Neurovision Imaging, and the folks at Cedars for this awesome work!

(Ref) https://insight.jci.org/articles/view/93621

-Austin

I made a short video showing the results – I hope someone can make use of it!

-Austin

-Austin

Not sure the money pans out considering how much time it would take to build, but if one wanted to learn mechanical control, this would be a good way to dive in!

https://www.drdflo.com/syringe

• no cooling whatsoever
• cooling to + or – some delta from ambient
• fixed-temperature cooling to some set point.

Of these methods, the fixed temp cooling is the most difficult to achieve, for a number of reasons, and I don’t believe these types of cameras would benefit greatly from such an attempt, so my idea was to simply answer the question, “Will cooling improve the performance of these cameras in a measurable way?”

Normally, the cooling of a sensor takes place as close as possible to the back side of the sensor itself. In this case, I started with an already-encased camera, the Pt Grey Chameleon 3. Because I didn’t have access to the sensor back directly, I figured making contact with the case, at as much surface as possible, would suffice. In order to do that I simply used 2 l channel strips of aluminum, and placed thermal tape on all 3 sides touching the camera. The graphic below shows what was added after the aluminum wrapper. First, a peltier (thermo-electric heat exchanger) was coupled to the aluminum. From there, a large CPU heat sink/cooler was attached to the hot side of the TEC. The general idea here is that the aluminum should absorb the heat from the camera. The peltier moves a lot of that heat to the CPU cooler, which radiates the heat into the air. This is a rather rudimentary setup, but it works for the proof of concept.

Below is an image of the assembled system. To provide the required 5V and 12V power to the cooler and peltier, I used my old reliable ATX Power supply.  This assembly low cost components from Amazon and the like. I think the total cost was $27 or so. Maybe more including thermal tape.

So how did the system perform?

First I allowed the stack to heat up for ~ 30 minutes. I wanted the camera to heat-soak the wrapper, so that when cooling was applied any measurable change could be collected. I think the act of adding the non-powered stack aided in the camera performance a bit, as simply having the radiator attached means that the hot camera can cool more readily from greater surface area exposure. In any case, a thermal camera was used in timelapse mode to collect the change in temperature over time. The recording was started after 30 minutes of warm-up. Images from the thermal camera were captured during a 1 hour period, while the camera was run in timelapse mode using Micro-Manager 1.4. First, here’s a GIF showing a time-compressed thermal image.

Several things to note here. One is the large drop in camera body temp during the experiment. Another is the subsequent increase in radiator temperature, as it works to shunt the heat from the peltier. Also note the wings on each side of the camera body. They start at a similar temp as the camera body, then drop in temperature significantly (40-50°F) over the duration of the timelapse. It’s important to also note that the emissivity of the plastic camera body differs from that of the aluminum. As a result, even if the temperature is exactly the same, a slight difference in the IR image will be visible.

Below are the results of the image captures. First is a graph showing maximum measured intensity of any pixel in-frame, over the duration of the timelapse (120 frames @ 30Seconds interval). In the case of “hot pixels”, as the camera temperature decreases, the collected signal from those pixels drops.

Next, I wanted to measure the total # of hot pixels. Now, it’s somewhat nebulous to define a hot pixel, but I used anything 1.5x greater in intensity than the minimum background, or offset, of the image. On this experiment, the average background was ~ 14, resulting in a threshold of 21. I was really surprised by this one. There is a rather large spike in the data, and I’m not certain what that is from. However, this again shows that the cooling is indeed reducing the hot pixel count, as would be expected. Additionally, the count can be seen to normalize at around 1500 pixels, which I expect is where the system started to thermal stabilize.

For a final set of interesting images. Here’s a shot of the stack after the experiment finished. Note the low temp of the wings and the back wrapper block.

Another interesting shot, this one of the sensor face. Using the cooling system it was at 104°F.

Turning the system off produced a temp of ~ 130°F.

My conclusion is that it is possible to cool even encased cameras using external cooling methods, to improve the performance of the camera(s). I do not think such adaptation will yield comparable results to a camera designed with cooling from the start, but this is an interesting option to achieve better results from a camera with a low price point.

-Austin

• PWM duty cycle should be controlled form presets
• PWM should be called to “off” from a TTL or other “shutter” style command
• Once off, a similar “ttl on” type command should re-enable PWM at the alst used duty cycle
• multiple output lines should be supported at different duty cycles.

Version 407 of the Triggerscope firmware now supports this, and uses the existing driver available for these devices. I’ve made a short demo video showing how this works on an oscilloscope output. I’ll place an option for this on the store. Please get in touch with any questions or post here – thanks!

Scope Selection

What we were looking to build on this system was a low cost, yet fully automated imaging scope that would be easy to modify, simple to operate, and compatible with Micro-manager. In the short term, only Z control is required, but as the work grows, we wanted provision for XY automation, as well as autofocus tracking. For fluorescence excitation we an LED illuminator. After looking at several options, we selected the ASI RAMM microscope, with the inclusion of the Tiger unified controller, and the ASI LED excitation module. This system offers a complete microscope from one control box, on a scalable build platform, at a competitive price. I reviewed the RAMM scope a few years back, and happy to see that the quality and options have only increased over time. When I reviewed the system, it was required that I build the frame and align things correctly. The new systems ship completely assembled. I literally pulled it out of the box, plugged it in and things were operational. This out-of-box experience isn’t normally seen even in the “big 4” microscopes.

Another feature of this scope is the inclusion of all needed driver components in one box, ASI’s Tiger controller. This is a modular control box which can accept riser cards as-needed to drive various components. The controller on this setup was actually the largest electronic box in the system! From left to right are installed driver cards for joystick/control input, Z stage, LED output, and filter wheel control. Another great feature of this box is that when installing in Micro-manager, only a single USB connection, and single hub device are required.

LED Illumination

ASI offers a 4 channel LED illuminator, with option for almost any available LED module. This is a simple illumination setup using a common ladder-dichroic design. The open nature of the hardware allows for changes to the dichroics, or the LED modules, as may be needed by the customer as things change in the future. Each LED is driven using a simple audio style TRS cable, so connecting or disconnecting the LEDs from the driver is straightforward. On the output side of the module, an aperture diapragm is provided to avoid overfilling the back aperture of the objective.

Camera Selection

When selecting a camera, we wanted good QE, decent resolution, but didn’t require any advanced cooling or long exposures. Of the available options, the Pt Grey (FLIR) BlackFly camera fit the bill. Recently, Nico Stuurman @ UCSF wrote up a driver to support this camera in Micro-manager, and after working with it for w while I’m quite impressed. I was able to configure some tricky stuff, I tested various binning modes, subarrays, a wide range of exposure times, and things seemed stable. One suggestion if using this camera is to always snap an image after making changes, vs. simply clicking “live”.

The camera is unbelievably small, roughly the size of a 1″ cube. While cool at first glance, there are some drawbacks to such a design which I note below. I tested the camera at no-light conditions for 2 things – dark current over exposure time, and read noise. Read noise shows a 16-bit count standard deviation of 214.78. Value ranges on the noise floor ran 3264 counts max-min. This works out to a noise level of 4%. Calculation for this measurement was from 2 images in a sequence.

(Image1+2000) -image2 = 200 count centered noise level. (ranging around the 2k level).

Not the best performance from a typical sCMOS camera, but if we consider the next lowest-cost camera runs ~ $5,000, this is quite good in my opinion. There is some pattern noise apparent in the camera, below is an average of 20 exposures, all using 10ms exposure time. The image has been contrast enhanced and pseudo-colored for presentation.

So it appears the taps are drifting a bit. Just for grins I tossed one of these images into an FFT and redisplayed the spectrum for view. Looking at the output, my gut says the image below results from temperature bias on the output taps, and this bias is distributed across the back side of the sensor, or at least wherever the taps are located on the readout layer of the PCB. The higher frequency events at the center look like the typical clock timing jitter seen on any ADC. But this is only my non-expert observation – I’d love for any uber-camera experts to weigh in!

I also wanted to look at how bad the hot pixel effects would be using longer exposures. For this test I ran a set of exposure times from 1ms to 3000ms.

Increase in dark current over time was about as expected.

I think the bottom line on this camera is that it runs as hot as any other camera, but has neither the active cooling nor the inherent surface area to radiate heat well. I took a pic of it using my Flir One camera, and it runs at about 95°F while idle.

Does this mean the camera isn’t suitable for microscopy? Not at all? This is an amazing piece of tech for a very low price. I can imagine buying 3 of these, one for each of 3 emission channels on a single scope! My goal in presenting this information is to share what the current limitations are for such a product. I think the raw QE and ~2.3MP resolution of the camera make it a perfect fit for most microscopy jobs. Of course, there will always be need for higher end cameras, but for many run of the mill operations, this is a nice solution.

NUC Computer

When searching for an “imaging computer” on this project, I had initially considered using a Mac Mini, dual booted with windows 10. I’ve used this for a number of other projects, and it has always run well in windows. John suggested looking at the NUC as an option, which provided later opportunity to upgrade the RAM and disk space. After building it up (which only required installing memory and an SSD), this little guy kicked butt!I ran Windows 10-Professional x64 for this build, and used the latest version of Micro-manager. Funny that a $500 computer and a $500 camera can be had for what used to run ~ $15k!. I successfully ran this on timelapse for 10 hours, and captured streams in MM of 100 frames. I didn’t push the buffer too much or analyze the USB-3 performance, but overall this is a great little machine for simple capture of images.

Building this system was a lot of fun, and I really enjoyed using non-traditional components for it. I think this type of system will become commonplace in the future, as more and more customers realize that traditional components, at a traditional high cost,  aren’t always the best fit for their needs. If you are interested in this type of solution for your lab or company, please get in touch, as I’d love to build one just for you!

-Austin

I’d imagine that memory handling issues related to image capture would be the biggest problem for the rpi, but it wouldn’t be surprising to see that addressed in the future. It looks like accessing cameras such as the point grey chamelion has been accomplished, however over 1394, not USB.

-Austin

It’s interesting to consider what techniques fit into the field of computational imaging. Optical microscopy has pushed into an ever smaller domain of resolution, to the point that Abbe’s laws need to be avoided in order to pull resolution out of an image. Consider the initial techniques used for this – phase, DIC, confocal, TIRF, and the like are contrast enhancement techniques of enhancing an image’s contrast, by either increasing the signal intensity, or by reducing the surrounding noise/background intensity.  Beginning with the Apotome, and maybe earlier, with deconvolution, a combination of using multiple image samples with post-capture image processing, to gather more information than can be obtained by a single image, started to emerge.

Building on these first techniques, we can see further sampling methods in the optical domain, like STORM/PALM, where optical control over emission is coupled with high sampling rates, and back-end processing, to statistically avoid Abbe’s laws. I’d imagine FLIM falls into this category as well, using time and multiple images, again to extract more data than may be obtained by a single sample. Non-microscopy applications for yet another method in this domain, the employment of coded apertures, similar to this work from MIT,  are growing in a number of imaging fields.

This combination, of computing power + controlled image fields, can be employed to do all sorts of as-yet untouched things with a conventional microscope. I can imagine that very soon, we will see a few of the following products available for use:

• phase contrast, and fluorescent, multi-capture resolution enhancement systems, all built into an “off the shelf” microscope
• angular changes to fluorescence excitation, used to improve resolution
• wavelength-based restriction inside an image field, to eliminate the potential for crosstalk in emission
• multiple excitation power sampling, to pull greater dynamic range from images
• multiple cameras used with restricting apertures, used to pull focus information, or multiple focal fields, from a single position in a sample

These are only a few ideas where this can go, but the general path I can see in the future is lower cost components, which are leveraged to do better and better work, which gives more imaging power to a greater number of researchers. At least – that’s a future I’d like to see 🙂

-Austin

Here’s an example of one of my breakout board shields. Overall this makes review of a gerber output MUCH easier than other programs I’ve tried. An app store DL is available here – It’s $14,99 on the app store.

-Austin