Archive for July, 2015


Digital microscopes… with eyepieces?

I wanted a digital microscope, but you brought me one with eyepieces!

I had a conversation with a customer recently who asked about “digital microscopes.” To that I said, “Define ‘digital microscope,’” complete with air quotes. Why? Because although the term “digital microscope” is often referring to an optical macroscope and camera combination – without eyepieces, the fundamentals of a “digital microscope” are present in many of Leica’s stereo microscopes and macroscopes.

Precision optics – Check

High resolution camera – Check

Coded microscope components – Check

3D Imaging and measurements – Check

Versatile – Check

Modular – Check and Check

In fact, according to the International Organization for Standardization’s (ISO) definition of a digital microscope (ISO/DIS 18221), the presence or absence of eyepieces is not mentioned:

3.1 – Digital microscopy system: Instrument consisting of an objective, an image sensor and a digital display to make visible minute details that are not seen with unaided eye

Despite this definition, the perception of a digital microscope is one without eyepieces. This mainstream definition is both incorrect and limiting to end users and their respective businesses. The marketplace has become obsessed with adding a digital microscope to their set of available tools when, in many cases, a traditional microscope system with an advanced camera would produce comparable data, provide more flexibility, and most likely come at a lower price. This is, in part, due to digital microscope manufacturers producing a “one size fits all” strategy to their systems. This approach has forced customers to purchase systems with features they will never use – without the ability to accommodate upgrades that address future needs. Sure, users can upgrade a lens or software license but cameras, lighting, automated stages, and computers are unavailable to upgrade. This means that a business will need to purchase a completely new system to address future needs.

There are, of course, exceptions to these limitations. Some manufacturers have opted to offer more modularity or upgradability. Thankfully many companies are moving away from the integrated system computer, opting instead for a separate computer. This limits the risk of a computer failure rendering a digital microscope useless, but it highlights the shortcomings of a “one size fits all” strategy.

One manufacturer that is leading the way in the evolution of the mainstream definition of the digital microscope is Leica Microsystems. The recently introduced DVM6 comes in three different versions with the ability to upgrade lenses, software, and computer as necessary. It currently meets or exceeds the draft standards set forth in ISO 18221. If the DVM6 is not the right fit, there are also the DMS1000 and DMS300 that round out the “digital microscope” offering by Leica Microsystems.

If the digital microscope phrase is opened up to include microscopes with digital components, the options are even greater. Regardless of your experiment or project demands, Leica has a digital microscope that will meet the needs of any project requiring optical observation or analysis. Project needs can be addressed by an upright compound microscope, inverted compound microscope, stereo microscope, or macroscope with hundreds of options.

Leica’s commitment to the modular microscope design also offers businesses the opportunity to upgrade when an unforeseen challenge arises. This is something that Leica has been doing for years. In fact, Leica microscopes that are 20 years old are still in use today because of their quality manufacturing, but also modularity. Leica has made a point to manufacture systems that are compatible across generations of microscopes. This is demonstrated in the cross compatibility in the stereo microscopes of 15 years ago. In fact, I have personally upgraded a 40 year-old microscope with a new trinocular head to accommodate a camera attachment. This commitment is also demonstrated in Leica’s continued support of digital cameras that are over 12 years old – several of which were first generation digital cameras!

Although these systems are not the type that can be purchased “off the shelf,” they do come with highly skilled experts to help advise on critical configuration decisions. The microscope and imaging specialists at W. Nuhsbaum, Inc are prepared to help you identify the microscope system that best fits your projects, regardless of whether the microscope is digital or includes eyepieces.

Get in touch with us and experience the expertise for yourself.

“The Count” on Digital microscopes: You are having too much fun with numbers!

Since the days of Antonie van Leeuwenhoek and his custom microscopes, the most exquisite detector was used for critical observations – the human eye. The human eye was critical for describing the first microorganisms, but also, as the story goes, the quality of the thread he was using for his textile business. The eye was able to open the door to unknown areas of science, albeit with the help of glass lenses.

Today, the eye is still a critical part of every scientific advancement based on an image. Whether the image is generated by a stereo microscope, compound microscope, electron microscope, or the Hubble Space Telescope – the eye is the final destination for the data generated by these systems. So, naturally, the eye is the final calculation for total magnification in a microscope system. For over 160 years, Leica has been calculating total magnification by multiplying the magnification of the objective lens, intermediate lens, and eyepiece lens. For instance, a 20X microscope lens, without an intermediate magnification lens, and 10X eyepieces, would have a total magnification of 200X.

Although the traditional “total magnification calculation” still applies in optical systems that include eyepieces, the math changes when a camera is used for total magnification. If I had a dollar for every engineer that asked me, “What is the total magnification of the camera,” I would probably have about 15 or 20 bucks. Don’t laugh, in the microscope world, 15-20 are a lot of people asking the same question! The answer every time is “I don’t know.” There are too many sensors available with different pixel sizes, length, and width measurements. Pair these variables with objective lenses, intermediate lenses, and the lenses in the camera coupler and it’s enough to befuddle even the most talented engineer.

When a camera is included in the total magnification equation, it is the monitor, not the camera that dictates the magnification. Even with a camera, the monitor, and ultimately the eye is the final calculation for total magnification. In systems where the monitor has a variable size, the magnification is calculated based on the viewable area of the live or captured image. For example, the magnification is less on a 17-inch monitor compared to a 30-inch monitor. With this in mind, a digital microscope, without eyepieces, and a 30-inch monitor has a greater magnification than the same digital microscope with a 17-inch monitor.

Commence “fun with numbers”.

This numbers game goes a step further when digital zoom is incorporated into the equation. When a 10-megapixel sensor is “zoomed” to only include a center region of 2.5 megapixels, the magnification is tripled. What if the digital zoom goes all the way to a region of 16 pixels squared, is that really magnification? Most reasonable people would answer “no.” However, on a spec sheet, that digital microscope has magnification that exceeds an electron microscope! Put it on a 60-inch 4K television and the numbers become even more outlandish.

Is the scenario described above actually happening in the marketplace? The simple answer is no, but it could, which is why an ISO standard for digital microscopes is being developed to standardize digital microscopes. ISO/DIS 18221 is being developed by an international committee to put a stop to the digital microscope hijinks.

In reference to ISO 18221: This International Standard specifies the minimum information to be provided to the user by manufacturers of microscopes with digital displays, regarding imaging performance. It further specifies terms and definitions for describing the optical performance of the digital imaging path of microscopy systems including the observation of the image on digital displays.

NOTE: Terms and definitions for the direct visual observation with eyepieces are specified in ISO 8039 and ISO 10934-1. More information on the new digital microscope ISO standard can be reviewed at the ISO website:

https://www.iso.org/obp/ui/#iso:std:iso:18221:dis:ed-1:v1:en

http://www.iso.org/iso/catalogue_detail.htm?csnumber=61810

In short, the draft standard is designed to account for more than magnification, but also consider field of view and resolution (line pairs per millimeter). The flagship digital microscope from Leica Microsystems, the DVM6, was designed with these draft standards in mind. Although these standards are only in draft status, the Leica DVM6 already meets these standards.

Those in the market for a digital microscope should seriously consider these standards when evaluating digital microscopes. Will your digital microscope stack up well to the new ISO 18221 standards? The data from the Leica DVM6 is ready for the future – will yours?

My microscope stinks! The misunderstood 40X correction collar lens.

When consulting on an inverted microscope purchase, one question I always ask is whether the lab intends to use coverslip bottom dishes or plastic vessels. In approximately 9 of the 10 conversations the investigator says, “both.” How silly of me to ask.

Many companies would simply provide a long working distance lens that was designed for plastic, but worked just fine for glass as well. However, many years ago, Leica designed a lens with a correction collar that would allow the user to account for the different properties of glass or plastic – particularly thickness. The user simply needs to twist the lens housing to adjust internal lenses up and down. Whether the lenses are “up” or “down” will align the lenses to provide the sharpest image possible for the vessel being used. Thankfully there are even handy little markings on the lens that give the user reference points for lens adjustment.

Inevitably I will visit the lab months, or in many cases years, later and users complain that the 40X lens is bad. Other users say, “We don’t use the Leica because the 40X lens doesn’t produce the images we need.” Comments like this leave the 40X microscope lens feeling sad and forlorn. Fear not, the 40X lens has great resolve-ing power. If you listen closely, you can hear it whispering, “What you perceive as my biggest weakness is actually my greatest strength.”

I certainly hear the whisper when I walk into the lab, which is why I stand up in support for the 40X correction collar lens: “You Sir, were designed for a greater purpose!” Once the new users are educated on the virtues of adjusting the 40X lens, they see the difference in a properly adjusted lens in all lighting techniques – even low-resolution techniques such as phase contrast. Among the many benefits the user will notice are increased resolution, improved flatness correction, and increased brightness. In fact, once the lens is correctly adjusted, many people can’t help but say, “WOW!” This is uplifting for the lens and motivates it to perform at its best!

The difference is critical for techniques such as Differential Interference Contrast (DIC) and fluorescence. DIC depends on correct optical adjustment because of the light sheer that takes place in the prisms and polarizers, which are required for the technique. If the lenses are out of alignment, the angle of the light sheer is not optimal; the image looks fuzzy, and lacks contrast.

For fluorescence microscopy, light is at a premium and lenses that are out of alignment do not transfer light efficiently. They also take the single points of light that are generated by fluorescent labels and turn them into giant fuzz balls – great for stuffed animals, not so great for your eight hour experiment.

Making the correction collar adjustment is simple: adjust the correction collar clockwise, focus the microscope on your sample, adjust the correction collar counter clockwise slightly and refocus the microscope. Repeat this process until you are satisfied with the image. There is no “focus indicator” with this process, simply trust your eyes. They are one of nature’s best detectors! If you use the same vessels for your experiments you may consider making a small mark on the lens housing to make adjusting the lens faster. It will also help you show your less technically inclined colleagues how to adjust the lens properly.

So before you blame your Leica microscope for behaving badly, double check the correction collar on your 40X lens for proper adjustment. And if it’s the 20X lens that you are mad at, check that lens too. Leica makes both 20X and 40X long working distance lenses with correction collars. So don’t get mad, just get smart and look under your microscope stage to see if you have a correction collar on your rig.

Before starting your next experiment or snapping your next picture, do not forget to adjust your lens, it could be the difference you were looking for in your experimental data. Only then will thank Leica for making a lens that gives you the flexibility you need in your lab.

Yokogawa confocal scanning unit (CSU) – Has the competition caught up?

Since the first spinning disk confocal head was released over twenty years ago, there has been a flood of new and different products. Each product has its merits, such as exceptional light transmission, enhanced resolution, flatness of field, speed, and/or versatility to accommodate multiple techniques (FRAP, for instance).

Yokogawa established itself as the gold standard for spinning disk confocal in large part due to its brightness and image quality. The spinning disks themselves have a unique strategy for enhancing laser light to the sample while still providing confocality for the emission light – a dual disk design with microlenses on the excitation disk. The microlenses are used to increase laser light to the sample, but they are not utilized in the emission light path. This strategy ensures that the excitation light is bright, but the emission light passes through a clean pinhole. With an optimized Nipkow disk pinhole pattern, pinhole size, and microlenses, the CSU-X1 is the gold standard for spinning disk confocal applications when imaging with the 100X lens. The system requires a sensitive EMCCD camera, but no computer processing is required to generate a stunning image – at up to 2000FPS with a 10,000 RPM disk motor.

With increasing laser power from laser manufacturers such as Coherent and increasing computing power to leverage super resolution confocal imaging, several manufacturers have started developing, and bringing to market, confocals that could supplant the Yokogawa CSU series as the de facto gold standard for spinning disk confocal imaging.

One system in particular was developed by an upstart company called CrestOptics. With a small team of scientists and engineers the X-Light burst onto the scene in 2012. Their latest generation called the X-Light V2 spinning disk confocal comes with an improved light path and an optional upgrade to high speed structured illumination. With options like these, the CrestOptics X-Light V2 has the potential to unseat the Yokogawa as the go-to spinning disk confocal system for biologists.

The design of the CrestOptics X-Light V2 is unique, it does not use the expensive microlenses and dual disk strategy implemented in the Yokogawa CSU-X1 or CSU-W1. Instead the X-Light V2 uses a simple and less expensive single disk with a proprietary pinhole pattern and superior 15,000 RPM motor. It has the option for a manual emission filter wheel to reduce cost and can work with one of the many multi-channel LED light sources when lasers prove to be too expensive.

I recently had the opportunity to talk with Andrea Latini, Ph.D., President of CrestOptics , about the X-Light V2 and its improvements over the previous generation.

Q: Brightness has always been an advantage for Yokogawa spinning disk confocal units, how does the CrestOptics system address this traditional Yokogawa advantage?

A: The ‘brightness’ could be defined as overall Confocal System throughput in terms of collection efficiency for sample fluorescence. With our X-Light Confocals (V1 & V2), we’ve been extensively working on both excitation efficiency end fluorescence collection efficiency optimizations.

We decided not to use microlenses in our approach since, due to the microlenses diameter limitation, it is not possible to accommodate an optimum number of pinholes per unit area onto the spinning disk surface. A second reason for eliminating microlenses is that they cannot improve collection efficiency. Fluorescence from the sample, or emission light, doesn’t pass through microlenses – only excitation light. Furthermore, microlenses f/# (f-number) doesn’t match well with the numerical aperture of all microscope objectives. Nor does it match the tube lenses focal lengths of different microscope brands.

Main optimization on our proprietary spinning disk spiral pattern is both on pinholes diameter (available with 70um, 60um, 50um, and 40um diameter), on total number of spirals on the disk and spiral geometrical parameters.

Final results allow for both higher throughput through the disk (excitation + fluorescence photons), and excellent confocality through z-planes with the largest choice of off-the-shelf microscope objectives magnification and microscope tube lenses.

Q: Have you compared a Yokogawa spinning disk confocal in brightness and image quality to a CrestOptics X-Light V2 unit? How did the two stack up?

A: Nikon Amsterdam & Switzerland Test Labs (Dr. Fabian Anderegg, Dr. Daniel Ciepielewski and Dr. Herman Fennema), conducted a complete quantitative comparison among CrestOptics X-Light V1&V2 systems and other confocal systems (Visitech & Yokogawa), following a well-known official methodology for Overall Brightness Measurement and Confocality performances (details are available).

When comparing Yokogawa CSU, Visitek, and CrestOptics, the PSF performance is the same on all three. In addition, the throughput confocal performance ratio (CF/WF %), taking into account both excitation and collection efficiency, results are as follows:

  • Visitech 1% efficiency
  • Yoko CSU (> W1 due to W1 lower pinholes density) 2.8% efficiency
  • CrestOptics X-Light 5.5%-6.3% efficiency
Note: using 200nm beads, 100X 1.49 NA objective and Lumencor SpectraX LED system (not even lasers), Evolve EMCCD. Visitech and Yokogawa tests have been carried out with laser sources

The X-Light performs significantly better the two other confocal brands.

Q: The CrestOptics X-Light V1 was revolutionary for its ability to accommodate large format sensors such as the 1K EMCCD cameras and sCMOS, what changes does the second version have in store?

A: X-Light V2 is mainly a refinement to the V1 version to get top performance for image quality with no aberration on large format sensors. Additionally, The V2 adds the capability to hot-swap the spinning disk box with no alignment needed for maximum pinhole pattern flexibility on the same confocal system. End users could purchase more than one disk box at any time.

Q: The first generation of the CrestOptics X-Light was able to combine spinning disk confocal with FRAP in a single unit, will FRAP support be continued?

A: the X-Light V2 is not intended to be a V1 replacement. The optical solutions adopted to the V2 do not allow for a FRAP system to be combined with it. The V1 will continue to be the only system capable of doing it.The V2 has been mechanically and optically engineered to perfectly match with the new VCS SuperResolution Add-On to get a confocal + super resolved system at once.

Q: High speed Structured Illumination Microscopy has been around for years, what does the CrestOptics system bring to the table that other manufacturers do not?

A: the CrestOptics VCS Super Resolution module allows for nearly 100nm lateral resolution (XY – 115nm measured), with only 1s-3s needed to get the super resolved acquisition (depending on CCD-sCMOS Region of Interest size – a 1920×1080 image requires 5s acquisition-deconvolution time; smaller ROI require faster acquisition time).

This has been possible by combining a full CUDA proprietary DLL that makes use of NVidia graphic card computational power to get to the fastest super resolution algorithms application on the structured illumination acquisitions (normally 36 frames per one super resolved image).

We do offer several deconvolution algorithms which are all proprietary (documentation and literatures are available).