2014 Nobel Prize in Chemistry to be Awarded for Superresolution Microscopy

October 8th, 2014 Comments off

The Nobel Prize in Chemistry for 2014 has been announced. It will be awarded to Eric Betzig, Stefan W. Hell, and William E. Moerner for the development of super-resolved fluorescence microscopy.

The award recognizes Stefan Hell’s idea to circumvent the diffraction limit of light microscopy with stimulated emission depletion (STED), W.E. Moerner’s discovery of fluorophore blinking and reactivation, and Eric Betzig’s idea of overcoming the diffraction limit by combining images of sparse sets of fluorophores, which laid the foundation for Photoactivated Localization Microscopy (PALM).

From The Royal Swedish Academy of Sciences, “How the Optical Microscope Became a Nanoscope”:

“The methods developed by Eric Betzig, Stefan Hell and W. E. Moerner have led to several
nanoscopy techniques and are currently used all over the world. The three Laureates are still active
researchers in the large and growing community of scientists spearheading innovation in the field
of nanoscopy. When they direct their powerful nanoscopes toward the tiniest components of life
they also produce cutting-edge knowledge. Stefan Hell has peered inside living nerve cells in order
to better understand brain synapses. W. E. Moerner has studied proteins in relation to Huntington’s
disease. Eric Betzig has tracked cell division inside embryos. These are just a few of many examples.
One thing is certain, the Nobel Laureates in Chemistry 2014 have laid the foundation for the
development of knowledge of the greatest importance to mankind.”

There is also a more detailed summary from the Royal Swedish Academy of Sciences, “Scientific Background on the Nobel Prize in Chemistry 2014, SUPER-RESOLVED FLUORESCENCE MICROSCOPY.”

New molecular motion tracking mode for Nano-Cyte®

August 6th, 2014 Comments off

The Nano-Cyte® Single Molecule Imaging system now features a “tracking” mode capable of surveying a wide sample area. This new feature builds on the existing capabilities of the Nano-Cyte® fluorescence imaging system: nanometer scale stabilization in three dimensions, image acquisition, device control, particle localization analysis, particle position rendering and active positional control.

The tracking mode is capable of surveying a 200 micron by 200 micron area within a sample, representing 16 typical fields-of-view (FOVs), while maintaining 3-dimensional stability at the nanometer scale. Using this feature it is now possible to track the motion of particles at the nanometer level as they move through multiple FOVs for extended periods of time.

New Nano-Cyte® Video

March 7th, 2014 Comments off

Video of Nano-Cyte three dimensional image-based stability for live cell imaging
Video: Nano-Cyte®: 3D Image Stabilization System

The new Nano-Cyte® video highlights some of the particle tracking and rendering capabilities of the standalone Nano-Cyte® program, shown toward the end of the video. We are constantly expanding the capabilities and features of Nano-Cyte®. Call us today to find out what Nano-Cyte® can do for you.

Nano-Cyte® Newsletter: Update on Nano-Cyte® features

February 24th, 2014 Comments off

Nano-Cyte® Advantages
• 3D stabilization up to 3 nanometers
• Active stabilization over days
• Corrects for temperature gradients and drift
• Simultaneous image acquisition and stabilization
• Particle tracking capability
• Integrated hardware and software
• Microscope platform independent

New Nano-Cyte® Features:
• exportable DLL
• ability to load pre-existing calibrations
• 3D localization with output to text files
• 3D rendering with output to AVI video
• support for camera binning

In response to customer enquiry, Mad City Labs has now made available the Nano-Cyte® DLL with all Nano-Cyte® instruments. This DLL allows access to Nano-Cyte® technology from Windows user mode applications and provides access to most of the functionality of the Mad City Labs stand-alone Nano-Cyte® application. The DLL exposes the localization algorithms, find fiducial algorithms, and control loop adjustments. This allows commercial applications such as LabView™, μManager or other proprietary OEM software to control image acquisition and hardware while running Nano-Cyte® stabilization in the background of the application. The use of the DLL is simple, in that the OEM or customer software acquires the image, and passes that image to the Nano-Cyte® DLL for analysis. The Nano-Cyte® DLL returns a feedback signal to stabilize the system with the Nano-Cyte® hardware.

Nano-Cyte® is compatible with
• LabVIEW™ • μManager • ImageJ & ImageJVI • rapidSTORM

In addition, Nano-Cyte® has an exportable DLL to allow wider functionality with 3rd party software platforms.

Fore more information, visit the Nano-Cyte® Newsletter in your browser or as a PDF.

The Nano-Cyte Concept: Image Centric Stabilization

March 11th, 2013 Comments off

Mad City Labs understands the importance of taking the image centric view of microscopy. We developed the Nano-Cyte® image stabilization system to eliminate microscope drift in an image centric way by closing the loop between the sample and its image.

Nano-Cyte® image stabilization uses the following:

1. A fiduciary element within the sample. A good fiduciary element has a good signal to noise ratio in every image used for stabilization* and has a point like point spread function that is unobstructed by other objects in the image. For super resolution fluorescence microscopy this might be a nanometer scale fluorescent bead, quantum dot, or other structure. I will discuss fiduciaries in more detail in subsequent posts.

2. 3D localization of the fiducial. Localization can be accomplished many ways with varying precision. There are several ways to localize a point spread function to the precision needed for sub-resolution microscopy, but one of the simplest is with center of mass calculations for XY and cylindrical lens astigmatism for Z. I will discuss the details of this in later posts.

3. A 3D nanopositioning system with high precision, used to adjust the field of view based on the position feedback information. Nano-Cyte® uses differential position calculations to determine how far the fiducial has moved in X, Y, and Z relative to the last image used for stabilization. These differential position calculations are used as position feedback to stabilize the image (closing the loop between the sample and the image) by incrementally moving each axis of the nanopositioner. The easiest way to accomplish this is by putting the sample on an XYZ nanopositioner. Details of this will also be discussed in a later post.

This is a very broad view of what Nano-Cyte® is and what it does.

*Nano-Cyte® does not demand that every image be a stabilization image. This means that it is possible to have a fiducial element OUTSIDE of the field of view of the area of interest on the sample because the nanopositioner can be used to very precisely move between fields of view. This is a very important point that I will discuss further in a future post.

An Image-Centric View of Microscopy

November 16th, 2012 Comments off

It’s clear that microscope drift is a huge problem for super-resolution (SR) microscopy (see Why Microscope Drift is Disastrous for Super-Resolution Microscopy). Before we can talk about eliminating microscope drift, we have to change our perspective. Microscope drift results in the sample moving relative to the detector being used to collect the image. This demands that we establish a reference frame. Historically, the sample has always been the reference frame.  This made sense when the image detector was the eye. But modern experiments typically use a fixed image detector, and now the image is the only reference frame that matters. For example, if the image detector moves, the image would undergo a corresponding positional displacement. What is the reference frame you use to judge drift? It is the relative motion of the sample to the detector. Ultimately it’s the detector that records the data: photons hit the pixels on the camera and these events are recorded and ultimately rendered as an image. In fact, any drift in the imaging pathway will produce drift in the image.

Is it worth expending the time and effort necessary to try to eliminate every possible cause of drift? Historically, people have attempted this, but I argue that it’s not worth the effort because the problem is too complicated and is instrument and experiment specific. How do you measure drift? You measure it with the image detector.  Therefore it makes much more sense to stabilize the image rather than the microscope, and be done with it. This is the reason why an image centric view is critical, as discussed previously in the post Image IS Everything. The question is how to solve the problem of microscope drift? The answer is to displace the sample by whatever amount is necessary to keep the image stable on the image detector. This clearly requires a closed-loop feedback system that includes the image and a sample positioner.

In the future, I will discuss the Nano-Cyte® from Mad City Labs in detail. It is the only complete solution for microscope drift, and it eliminates microscope drift in three dimensions in real time.

Why Microscope Drift is Disastrous for Super-Resolution Microscopy

November 9th, 2012 Comments off

Microscope or sample drift is a significant practical problem for biological microscopy. Z-axis microscope drift is commonly referred to as focal drift. Thus far, several methods are used to correct focal drift; but these offer limited success.

Microscope drift can be most accurately described as a system problem, as it results from small movements in components throughout an entire microscopy system over the course of an experiment. Over long time periods and at small distance scales relevant for super-resolution microscopy methods, this system drift is inevitable. In even the best cases, drift complicates experiments; and at worst it can interrupt an experiment in progress or render it impossible.

For many fluorescence imaging techniques, sample, microscope, and focus drift is tolerable. However, for super-resolution microscopy techniques with resolution limits of 10s of nanometers, system drift is disastrous. In a very real sense, a “stability limit” has now replaced the diffraction limit for the resolving power of fluorescence microscopy. Put simply, if you can’t hold the sample still enough, you can’t tell exactly where the particular molecules or structures of interest are within it.

I will be summarizing a few key points about microscope drift in my next few posts, but I also invite you to follow this link to read about the only real time, 3D solution for microscope drift.

The Building Blocks of Super-Resolution Microscopy, Part 4: ImageJVI

November 2nd, 2012 Comments off

As I mentioned in my previous post, Making Super-Resolution Imaging Straightforward and Accessible, assembling an SR capable-system from its component parts is a significant challenge. Not only must the hardware components be compatible, but the software components must also be compatible in order to build an integrated system.

Many researchers are already familiar with LabVIEW, a proprietary visual programming environment from National Instruments used for data acquisition, automation, and instrument control. Because of its wide adoption by researchers, all Mad City Labs products are compatible with LabVIEW. Many researchers with biological interests are also familiar with ImageJ, a public domain, Java-based image processing program developed by NIH that is used to analyze microscopy and metrology imagery. Until now, there has been no easy way to merge the hardware control capabilities of LabVIEW with the image processing capabilities of ImageJ. This was the driving force behind Mad City Labs’ development of ImageJVI.

ImageJVI is an ImageJ – LabVIEW interface that allows users to easily pass data from LabVIEW Virtual Instruments (VIs) to ImageJ and vice versa.

The Building Blocks of Super-Resolution Microscopy, Part 3: A Multi-Color Laser Source

October 26th, 2012 Comments off

If you intend to build your own super-resolution microscopy system, you will need a light source. In order to maximize the number of fluorophores that can be used in a particular super-resolution technique, it is useful to have a variety of laser wavelengths available.  A number of vendors provide laser light sources. Some of them integrate multiple wavelengths. Here again, Mad City Labs is thinking ahead. The Cyto-Lite™ is a relatively inexpensive laser source that can switch between three wavelengths on a single fiber output. The default wavelengths are 405nm, 532nm, and 640nm. Other wavelengths are available. Users can configure amplitude, wavelength choice, and timing in software.

Cyto-Lite™ is compatible with National Instruments’ LabVIEW. Synchronizing laser pulse timing with other events in the experiment can be difficult. Mad City Labs’ Nano-Drive® controllers have the ISS (integrated scan synchronization) option that can be configured to send out TTL pulses to synchronize stage movements or sensor readings with external hardware. The Cyto-Lite™ can be synchronized with Mad City Labs nanopositioners and micropositioners in software, making it easy to design experiments that minimize photobleaching and maximize results.

The Building Blocks of Super-Resolution Microscopy, Part 2: A Flexible Imaging Platform

October 15th, 2012 Comments off

One challenge that researchers face when adapting a legacy microscope to new microscopy techniques is flexibility. Most legacy inverted microscopes have an enclosure with a limited number of access ports in fixed locations. This can be a problem; for example, if the experimental technique requires an astigmatic lens, it must be located in a precise place relative to the rest of the optics of the microscope. When a new technique is required, it can be difficult or impossible to adapt the legacy microscope to a new technique without destroying the ability to go back to the old technique. Because of these issues, some researchers have abandoned legacy microscopes in favor of building their own microscopes directly on top of an optical table. This can be challenging because of optical alignment issues and lack of support for interfacing hardware components such as dichroic mirrors, TIRF modules, and cameras or any other optical component.

Mad City Labs has solved many of these issues with the RM21™ imaging platform. The RM21™ is precision aligned to extend an optical table’s geometric grid in three dimensions, making optical alignment and construction easier. The RM21™ is supported by three to six legs with an open frame that makes it easy to insert or remove components. It includes a 2 in. travel [50 mm] stepper motor Z axis with 95nm step size for objective positioning. Accessory mounting bars with ports for optical components like EMCCDs are provided. It is possible to configure the optical paths to exit three of the four sides of the microscope. Furthermore, because the RM21™ is manufactured by Mad City Labs, it can interface with all other Mad City Labs products like low drift XY micropositioning stages and the nanopositioning systems I discussed in the last post: “The Building Blocks of Super-Resolution Microscopy, Part 1: Nanopositioners”.

RM21™ offers significant advantages for users that do not require a legacy microscope platform. With its unparalleled flexibility and affordability, it is no longer necessary to spend time or money on components that are not required for advanced microscopy applications.

RM21™, coupled with Cyto-Lite™ and ImageJVI™, form a complete development platform for advanced microscopy. I will be discussing the Cyto-Lite™ excitation source and ImageJVI™ software in future blog posts.