Archive for October, 2015

sCMOS runs up the score on CCD

sCMOS scores a touchdown, spikes the ball, and receives a penalty for excessive celebration

The technology in complementary metal-oxide semiconductors (CMOS) sensors has been around for a long time. These sensors are inexpensive to manufacture and have made their way into the many imaging devices found in your pockets (phones, cameras, MP3 players, etc.).

As a technology, CMOS is not merely relegated to funny cat pictures or a video of your buddy’s failed attempt at an American Ninja Warrior course. Instead, CMOS has evolved from a simple sensor design to a niche high-speed camera, all the way to a robust technology that benefits a wide variety of microscopy applications as diverse as time-lapse applications, to cell trafficking, to light sheet microscopy.

The coming of age of CMOS happened a few years ago with the launch of a new Fairchild sensor design incorporated into cameras such as the Hamamatsu Flash 4 v2, pco.edge, and Andor Neo/Zyla. What has been coined scientific CMOS (sCMOS), in many arenas, has overtaken CCD as the gold standard for fluorescence imaging.

Although the new generation of CCD sensors has its place alongside sCMOS, as noted in a previous blog post, sCMOS technology has eroded, if not completely replaced, CCD’s position as the preferred sensor technology for advanced imaging applications.

sCMOS Scores Another Victory in Technological Advancement

Just in time for the 45th meeting of the Society of Neuroscience in Chicago, IL, Hamamatsu, pco, and Andor have announced major technological advancements in their respective sCMOS cameras. Photometrics announced its entry into the sCMOS market with very unique shot noise reduction technology.

For Hamamatsu, pco, and Andor, the release of the new cameras include sensors that employ improved microlenses. The microlens improvement increases the overall QE of the sensor by 5-10% across the visible light spectrum. This offers users the ability to capture more light in less time, increasing signal-to-noise ratio, shortening exposure times, and increasing frame rates.

Although three companies have announced new cameras, only Hamamatsu has access to the new chips now, where other companies will not have access for several more months.

Without a sCMOS camera to offer microscopists until last week, Photometrics has tossed an 80 yard touchdown-scoring bomb into the sCMOS market. Named the Photometrics PRIME, the standard 4.2 megapixel sCMOS sensor used in the vast majority of cameras in this class has been juiced with noise and data-reducing algorithms. These advanced features are unique and stay with the tradition of advanced technology in flagship cameras from Photometrics.

Striving for Superior Signal to Noise Ratio

In low light fluorescence imaging, one of the most important aspects of the detector is signal-to-noise ratio (SNR). Although the equation to calculate signal-to-noise ratio (SNR) is complicated, the concept is simple: How well does the camera sensor read out signal above the level of electronic noise?

Smart engineers from the various companies have been working on improving SNR and appear to have addressed it in force. The new 82% QE sensors offer an incremental improvement. However, with Photometrics Prime Enhance technology, Photometrics reports an improvement of 3X-5X in SNR. The data provided in Photometrics technical notes provide a glimpse at what is possible. And what is possible is amazing!

Higher Frame Rates with Fewer Photons

A characteristic of sCMOS that has always been attractive is high frame rates. If you thought a base, full resolution frame rate of 100FPS was impressive, the Hamamatsu Flash 4.0 v2 can achieve over 25,000 FPS with ROI. These frame rates, however, are exposure-time limited. What is the most common cause of limited exposure time? Photons.

With a higher QE in the Hamamatsu Flash 4.0 v2 PLUS and the Prime Enhance technology from Photometrics, sCMOS cameras are set to challenge the conventional wisdom that EMCCD’s are required for low light, high speed imaging.

As documented in the technical note, Photometrics Prime Enhance technology can generate equivalent data in 100 milliseconds for what would normally be 800 milliseconds in standard operation! Take that, EMCCD!

It is worth noting, however, that although Prime Enhance generates clean and beautiful data, the same cannot be said for the visual image. The Prime Enhance algorithms reduce noise in the pixel gray values but, because noise is reduced by factoring in neighboring pixels, the final result is an image with a Photoshop Palette Knife appearance. This is most noticeable as signal decreases to “near noise” levels, but incredibly, the grey level histogram still looks good. If beautiful images are what you are after, fear not, Prime Enhance can be turned off, exposure time extended, and a beautiful image will result. However, if you want to go fast in low light, Prime Enhance makes it possible!

More Information with Less Data

At 4.2 megapixels, 65,536 gray levels (16 bit depth), and 100 FPS, the current generation of sCMOS cameras generates a lot of data! Localization-based super resolution systems are already using sCOMS cameras, which is why the Photometrics PRIME has two more tricks up its sleeve: Prime Locate and Multi-ROI.

Prime Locate allows the data transfer of only the pixels which register a grey value in localization-based super resolution systems. Considering many of these systems generate 60,000 – 100,000 images before building the journal cover-worthy super resolution image, the data savings will be tremendous. This technology also increases frame rates, lowers file size, and reduces storage concerns.

The Multi ROI function in the Photometrics Prime also allows users to capture multiple regions of interest (ROI) in a single field of view. So if the user has two small features in one huge field of view, leave the empty data on the microscope and only acquire the ROIs. Reducing file size and collecting more data, what could be better?

The sCMOS landscape

With the introduction of new sCMOS sensors and sCMOS sensor technology, the market is rapidly changing. The new 82% QE sCMOS sensors have brought more than just high performance. There are now several variants that differ on price, cooling, triggering, resolution, shutter, interface, and now quantum efficiency.

While the new Flash 4 V2 Plus is sure to be priced at a premium, companies such as pco have released USB3 cameras at a much lower price, which are based on the current and popular sensor. Whatever your priority, there is a sCMOS camera to step up to the challenge.

With sCMOS Leading the way, is CCD Obsolete?

Although sCMOS has firmly positioned itself as the premier technology for high-end fluorescence imaging, it does not cover the entire range of scientific imaging – particularly when price and application are considered. Many investigators are not going to be able to justify the price premium on sCMOS cameras for features that will be rarely used on, for example, a routine fluorescent stereo microscope.

Although CCD has fallen out of favor for high-end widefield acquisition, it still has its place on microscopes. Recently QImaging released the Retiga R1, R3, and R6 cameras at shockingly low prices. With the Retiga EXi, what used to cost $12,000 three months ago has bottomed-out at less than $5,000 with the introduction of the new Retiga R1. In addition, the new R1 has deeper cooling, higher QE, and a higher frame rate for live cell imaging!

Interestingly, this opens the door to simultaneous, multi-channel, imaging applications that require several cameras and employ the Multi-Cam from Cairn Research. What used to be a $40,000-$60,000 investment now costs a fraction of the price because of QImaging’s new CCD cameras!

Trust W. Nuhsbaum, Inc.

Choosing a camera can be intimidating, but identifying the needs for the application is the first step in making a smart decision. When evaluating technology for your lab, which will be used for many years into the future, it’s important to consider the latest products and technology advancements.

The Imaging Specialists at W. Nuhsbaum, Inc have seen cameras evolve over the years and can provide perspective on the latest technology to arrive on the market. Trust the experience of W. Nuhsbuam, Inc to weather the technology winds of change and advise on the proper technology for your experiments.

Color infidelity: Why using a light source incorrectly is cheating on your data

Knowledge is power when minimizing error in imaging

Maybe you are one of the people who engaged in arguments about whether the dress is white and gold or blue and black. Or maybe you are just a regular person trying to take a picture of a dress to share it with your friends. Either way, color is important!

With microscopes, most users of digital cameras are familiar with the simple process of going through a white balance procedure to get a very nice white background in images. However, many users often notice that although the background is white, the color of their image is incorrect. This leads to arguments, although less viral than “the dress,” about the true color of a sample! Through the microscope’s eyepieces, the way the sample is supposed to look is clear, but on the monitor the colors appear incorrect!

There are many possible sources of error in the scenario described above. Anything from lighting, optics, detector, or even the monitor being used can all affect how the sample color “appears” in a digital image. Every aspect of the optical system must be considered when working toward an excellent image.

Lighting is Everything

One of the most influential components to accurate color is lighting. When working on customer samples with a stereo microscope, I often tell them that lighting is everything. Proper light is required to eliminate reflection, provide contrast, and highlight features of interest. In compound microscopes lighting is also critical for proper color reproduction.

Regardless of the light source being used, there are multiple factors to consider. Until recently, the most common light source in microscopy was a burning tungsten filament – or, in other words, halogen bulbs. Regardless of the voltage or wattage, all halogen bulbs had the same shortcomings, with low voltage the bulb would produce a yellow color and with high voltage the bulb would produce white color.

For years, microscope users would insert gray neutral density filters into the light path to decrease overall brightness when voltage to the bulb was increased. This technique produces the desirable white light and balances brightess for both camera and eyes. This procedure is an excellent technique, however the vast majority of microscope users do not even know what a neutral density filter is, let alone when to use it.

Ideal Lighting for Accurate Color

With the CRI standard for color accuracy being daylight, in a perfect world we would harness the power of sunlight and pump it through our microscopes. Unfortunately, even daylight isn’t perfect! Daylight can vary depending on time, cloud coverage, and location on the globe! Yikes!

Characteristics of theoretically perfect daylight include a color temperature of 5600K and light intensity that is evenly distributed across the light spectrum. In other words, the intensity of light at 450nm is identical to the intensity at 650nm.

Color Temperature and Color Accuracy, Where Can We Find Both?

When manufacturers attempt to harness the perfection of daylight in a handy and portable light bulb, the process often falls short. This leaves microscope users with a choice to make: Which light will provide the best color reproduction?

Halogen bulbs: Inexpensive and durable, halogen is good, not great, for microscopy. Color is inaccurate because halogen produces a different color temperature depending on brightness. The benefit of halogen is that the light produced is even across the light spectrum, meaning the intensity between red, green, and blue are equivalent. Therefore, color accuracy at peak brightness is excellent.

Light Emitting Diode (LED): LED lights can be made to produce light at virtually any color temperature. Developing an LED to produce light at 5600K is easy. Producing an LED that produces even light intensity across the light spectrum? Not so easy. LED lights often have peaks and valleys in the light spectrum. For instance, the light intensity at 450nm is not the same as light intensity at 650nm. Depending on the quality of the LED providing the light, the color bias can be minimized. However, LED is an excellent option because the color temperature is maintained regardless of brightness.

Arc lamps: Often used in fluorescence microscopy, arc lamps can also be used in brightfield imaging for the full spectrum white light. Similar to LED, the light spectrum of arc lamps have peaks and valleys – which are more dramatic than LED lighting options. Although the light is very bright and the correct color temperature, the peaks and valleys lead to inaccurate color because, for instance, green wavelengths have more brightness than yellow.

Without a Perfect Lighting Solution, What is One To Do?

Without perfect light, the colors of a stained tissue section will be slightly off. The user is left with two choices, correct the color using a post processing program such as Photoshop or Datacolor ChromaCal – or pick a light source that is good enough to represent the sample as accurately as possible.

Considering the advantages of brightness independent color temperature and a fairly flat spectrum for quality LED’s, LED illumination is the best choice for most microscope users interested in faithfully reproducing colors in their samples. However, if a knowledgeable microscopist is ready to run a 100W halogen bulb at full brightness, employing neutral density filters to adjust brightness for their eyes and the camera, halogen would be preferred for its color accuracy across the light spectrum.

Trust W. Nuhsbaum, Inc.

Whether choosing a light source for H&E photography, polished materials for manufacturing, or petrography, trust W. Nuhsbaum, Inc to provide the expertise necessary to independently suggest the correct light source for your application.

Get in touch with one of our microscope or imaging specialists to learn more about the latest in microscope lighting technology. It could be the difference between an argument about whether your sample has white and gold or blue and black colors!