Archive for August, 2015


Sony announces CCD production to end

Sony announces end of life for CCD production: The CCD zombie apocalypse is upon us!

Just in time for the mid-season restart of AMC’s The Walking Dead, on January 30th 2015 Sony let the cat out of the bag that they would stop production of its existing line of CCD sensors. There is a difference, however, in announcing the end of production, and actually ending production. Current reports suggest that the end will not actually happen for another 10 years.

When the end does finally arrive, unlike the zombies popularized in The Walking Dead, CCD cameras will not die and come back as half dead creatures. Cameras that employ CCD sensors will continue to function happily for years to come – with new cameras built on CCD technology ready to release now, and into the future.


The Future of CCD sensors

While many might assume Sony’s announcement signals the end of life for CCD sensors, there will be plenty of time to prepare. Although the rumor mill is inaccurate with dates, there are reports that Sony targeted final ship dates for 2025. As of today, 2025 is a full 10 earth years away. Or in technology years, approximately 2,200 – that’s a joke people.

Unfortunately there is no public announcement from Sony regarding the end of CCD’s, however there are CCD distributors that made the announcement for them.

Thankfully there are other companies out there that produce CCD sensors who will be happy to pick up the slack provided by Sony – such as Texas Instruments. There are many other, smaller companies that may pick up the slack as well which can be found on an unconfirmed list of CCD manufacturers.


Impact of Sony’s discontinuation of CCD’s for scientific cameras

In the short term, there is little impact. With years to prepare, camera manufacturers can continue using the wildly popular Sony ICX-285 sensor cameras such as the Photometrics HQ2, Leica DFC365, and Qimaging QIClick.

In addition to the continuation of existing camera models, companies such as QImaging are actually preparing the launch of a new camera line which includes Sony sensors. The all new QImaging R1 camera uses the successor to the Sony ICX-285 chip, the new ICX-825 sensor. With and improved quantum efficiency of 75%, 16 bit digitization, higher frame rates, the QImaging R1 is betting on the availability of Sony CCD’s for years to come.

Interestingly, Hamamatsu may also be prepared to pick up the demand for scientific cameras based on CCD technology as it owns the technology in its ER-150 sensor. Since Hamamatsu does not make public the company they use for production, with the many manufacturers of CCD sensors, one must assume the Hamamatsu ER-150 is safe.

Hamamatsu has kept its technology secret for years and with the ER-150 sensor being used in the Hamamatsu Orca R2, Orca D2, and 8484 series of cameras, Hamamatsu is uniquely positioned to meet the demand for scientific cameras built on CCD technology.


Expected lifetime of CCD’s in scientific cameras

If the CCD sensor built into your favorite camera is working well now, chances are it will work well for years to come. Particularly when purchased from an established manufacturer such as the brands represented by W. Nuhsbaum, Inc.

Many of the first generation monochrome CCD sensors are still being used today in many scientific labs and Research and Development facilities around the country. Although the Hamamatsu Orca 100 was discontinued many years ago, there are plenty of 15 year old Hamamatsu Orca 100 cameras that still function well for low light fluorescence imaging.


Buying technology with a defined end date

Although purchasing a camera with a component that is slated for cancellation may be intimidating, consider that hundreds of components go into scientific cameras – any of which could be changed with very little notice. Sony is doing everyone a favor by making the end of life clear to manufacturers so they can prepare.

The current time frame of 10 years is very, very long particularly in the technology sector that develops so quickly. There are numerous cases of technology becoming obsolete after 10 years not because of hardware or electronics inside of a device, but because of software support, driver support, data transfer interface, or a new innovation that supplants the existing technology. Although the device may still work, it is unusable because of something as silly as computer manufacturers making a switch from 5V PCI bus to 3.3V PCI bus.


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. The second step is to disregard rumors and big announcements, and instead focus on the image and application. If your application is best addressed with a CCD sensor, fear not, CCD’s will be around for a long time. Although they will go away some day, it will not resemble a CCD zombie apocalypse where all CCD’s die and come back as half dead creatures.

Trust the experience of W. Nuhsbuam, Inc to weather the technology winds of change and advise on the proper technology for your experiments.

sCMOS v CCD – adversaries or partners?

Scientific CMOS cameras: Arch rivals to CCD or partners in crime?

When building a microscope system, there are many important components to consider, such as the microscope stand, objective lenses, and contrast methods. However, increasingly software and cameras are playing a critical role in the design of new microscope systems. At the forefront of this paradigm shift are the many new university faculty members setting up new labs, making decisions that will affect the next several years of their research projects. Therefore, considering the investment required for a quality camera and software package, it is imperative to get the decision right – the first time.

While every point in time has new technology to consider, the decision facing new investigators regarding fluorescence microscope cameras has never had two such strongly opposing forces as it does today. Researchers are accustomed to opposing technology for color and monochrome fluorescence microscope cameras, but now there is a dramatic difference in fluorescence microscope cameras – Charge Coupled Device (CCD) vs. scientific complementary metal oxide semiconductor (sCMOS).

In the past, the difference was cooling, length of exposure, and resolution. Today, the decision includes frame rate, noise, resolution, bit depth, gain, binning, dynamic range, and quantum efficiency. These many variables can make even the most tech-savvy scientist’s head spin. When considering a microscope system, new investigators can feel the tension between the two technologies. Adding to the pressure is the sense that one is buying “old” technology if choosing a CCD microscope camera instead of the newer sCMOS – but are they really opposing forces where sCMOS will replace CCD for monochrome fluorescence cameras? Or are they two independent technologies that work together to cover the wide range for fluorescence microscopy applications?


Applications that determine the sCMOS or CCD microscope camera


High speed

When looking for pure speed, it’s hard to find a camera for fluorescence imaging that beats sCMOS. There are some electron multiplying CCD (EMCCD) cameras that with region of interest (ROI), binning, and unreasonable clock and clearing settings that can challenge the raw speed of the sCMOS camera, but at 100 FPS full frame and 25,000 FPS or higher with ROI, the sCMOS camera is king of speed. By contrast, CCD sensors are slow – most topping out around 100-150 FPS with ROI and binning. So not only is the camera slower, but to get to the base line speed of sCMOS, the user needs to compromise on resolution and field of view.

Winner: sCMOS

High speed in low light fluorescence

Speed is where the clear victory ends, because when considering frame rates, one must also consider exposure times. So unless your exposure times are on the microsecond time scale, stop dreaming about 25,000 FPS. However, at 10 milliseconds, 100 FPS is at least plausible. Although 10 ms is a very short exposure time for fluorescence, the 16 bit register and 65,536 grey levels means the sensor can produce significant grey levels above background noise. Combined with the lower read noise in sCMOS the user gets more of what they need: pixel data.

The CCD sensor, surprisingly, has some virtuous characteristics that benefit users in low light conditions, most notably analog gain and binning – two features that are unavailable on sCMOS cameras. These two functions, although they amplify noise and reduce resolution, provide the user with the ability to amplify signal by multiplying the electrons produced by the sensor and create “super pixels” – effectively increasing the sensitivity of the sensor. These two functions, combined with a 12-, 14-, or 16-bit digitizer with 4,096, 16,384, or 65,536 grey levels respectively, can amplify signal and get what users need most: pixel data.

Winner: Tie

Multi dimensional live cell

Live cell imaging presents its own challenges, although live cell could require both speed and low light sensitivity, a lot of live cell is slow, time lapse imaging. Long exposure times? Who cares! There are 10 minutes between each image.

As far as field of view, sCMOS has a 50% larger field of view over most fluorescence CCD cameras. This means each position can increase sample size and possibly decrease the number of images required to collect the necessary data. When smaller areas are required, the user can simply add an ROI and create a mosaic image with smaller images, albiet larger than the single image FOV.

Another area for importance is sensitivity (QE), noise, and the resulting signal to noise ratio. Whenever real data is required for image analysis, more signal and less noise is always preferred. With readout noise around 1 electron rms, the sCMOS wins the day compared to readout noise of 6 electrons rms found in the most popular CCD sensors.

Where CCD provides a counter punch is with dark current noise which can range but is normally on the order of 0.0002 electrons/pixel. By contrast, the industry leading Hamamatsu Orca Flash 4 comes in at 0.06 electrons/pixel. This difference is orders of magnitude in favor of the CCD, however, the dark current noise doesn’t present itself until exposure times go out into multiple seconds to minutes of exposure. And really, who wants to wait that long for an image particularly if there are multiple channels, Z stacks, and stitching taking place.

Winner: sCMOS

Deconvolution

Deconvolution requires proper digital imaging sampling rates, pioneered by Harry Nyquist, which is the topic of an article unto itself. However, the good news is that both CCD and sCMOS have an excellent pixel size for proper deconvolution with 63X and 100X lenses.

Most CCD sensors have a 6.45 micron pixel size, sCMOS have a 6.5 micron pixel size – which is perfect. However, CCD sensors produce smaller data sets which when working through the calculation heavy deconvolution algorithms dramatically speed the time to result. Although the larger file size of the sCMOS takes time for deconvolution, the end result is completely stunning. The high resolution 4.2 megapixel image with zero light scatter is simply unbeatable.

Winner: Tie

Mosaic Image Stitching

When capturing a large area with a high magnification, the only method for acquisition is mosaic image stitching. In this application field of view equals speed, and the sCMOS sensor has a 50% larger field of view, which means 50% fewer images and 50% more speed. When a CCD is used in combination with Objective Imaging Surveyor software and Turboscan, the CCD can go faster, but not as fast as the sCMOS and its huge field of view.

Winner: sCMOS

Ratiometric Imaging (FURA and FRET)

When comparing the difference between image 1 and image 2, ratiometric data is only as good as the grey level difference and time in between the two images being compared. One might assume higher frame rates are better, however, frame rates are dictated by the biological process they are measuring. In this case, biology is the neutralizer because FRET and FURA measures fluorescent proteins or calcium dyes, not extremely bright quantum dots. So attributes such as a high bit depth, gain, and binning can be very helpful. However, this needs to be balanced with speed and all of that depends on the experimental biology. In this case, there is no clear winner.

Winner: Tie

Quantitative fluorescence

If you were to ask 10 people involved in biological imaging to explain quantitative fluorescence microscopy you would probably get three or four answers that focused on different concepts. Comparing fluorescence intensity, ratiometric imaging, and photon counting would probably arrive at the top of the list. Your journey through the world of quantitative fluorescence microscopy would begin there and you would embark on a long voyage, for which a camera would naturally be required.

Important characteristics for a quantitative fluorescence microscopy camera would be low read noise, good full well capacity, high dynamic range, and a high bit depth. All of which sCMOS cameras have. For example, the pco.edge 4.2 has a read noise of 0.8 electrons, dynamic range of 37,500:1, full well capacity of 30,000 electrons, and a 16 bit digitizer. By contrast, CCD cameras such as the Hamamatsu Orca R2 have a read noise of 6 electrons, dynamic range of 3,000:1, full well capacity of 18,000 electrons, and two digitizers of 12 and 16 bits. If the numbers look intimidating, sCMOS is bigger, and in this case, bigger is better.

In the case of quantitative fluorescence microscopy, sCMOS is truly the better fit. Ratiometric imaging depends on the biology, so there is no clear winner. If one is interested in photon counting, welcome to the land of EMCCD cameras, where signals are low and camera sensitivity is high.

Once the camera is selected, the method for qualifying fluorescence begins. Expanding on this part of the journey is best left to highly experienced scientists. A great resource for learning more about this method is from the book Quantitative Imaging in Cell Biology: Methods in Cell Biology by Waters and Wittmann.

Good luck on your journey!

Winner: sCMOS

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. The second step is to not get caught up in any trend or hype around a specific technology. The differences are clear, but although there is some overlap in technology, both sCMOS and CCD have their place in fluorescence microscopy. So although they oppose each other on occasion, in many situations they compliment each other under the large umbrella of fluorescence microscopy.

The Imaging Specialists at W. Nuhsbaum, Inc have been around long enough to know about where each technology fits and can advise on what is best for your set of applications. Trust the experience of W. Nuhsbuam, Inc to weather the technology winds of change and advise on the proper technology for your experiments.

Using Köhler every day

Using Köhler every day: Tips you will not want to flush down the toilet

Although most people are familiar with the word “Köhler” because of the brands world class bathroom fixtures, the global brand does not define the people with the last name Köhler. That is certainly the case for August Köhler who changed microscopy in 1893 by developing a method of transmitted light illumination that produced even illumination across the field of view. Prior to August Köhler’s innovation, the predominant method for aligning the lenses required for optimal light was critical illumination. The most significant shortcoming of critical illumination was that the bulb filament is visible in the resulting image. Insert August Köhler and the bulb filament disappears in the final image.

What is Köhler Illumination?

Köhler Illumination is a perfectly aligned optical path relative to the sample and objective focal plane. Put simply, it’s when every lens is in the correct position to provide perfectly even light across the field of view with optimal brightness, color, contrast, and resolution.


Why Köhler Illumination is Important for everyone

Many upright microscope users overlook the bottom half of their microscope, or top half for inverted users. This is the unexciting portion of the microscope because it’s lacking eyepieces, camera mount, and the exciting objective lenses. However, the ignored half of the microscope is critical because it contains optics that determine the success or failure of your imaging work.

When a microscope is aligned with proper Köhler Illumination, the condenser is at the correct height relative to the sample, the field diaphragm is adjusted to eliminate reflections and glare, and the aperture is moved to an optimal position to provide contrast without producing shading artifacts. This benefits the user in many ways with a bright and clean image – but also a dirt free image! When a microscope is in Köhler, the hidden dust and smudges in an optical system disappear! This is the genius of August Köhler, lens surfaces, which are home to dust and debris, are placed in an intermediate focal plane and invisible to the microscope detector (your eyeballs or the camera).

Without proper microscope alignment, the image will look washed out, dirty, fuzzy, or it could look dim, too much contrast and produce halos in the image.


Only upside to Köhler Illumination

Aligning your microscope is easy and it will only make your images, and the resulting data, better and more convincing. So how do you accomplish this task? Excellent question, I’m glad you asked.


Step 1: Focus the microscope to the sample.

Step 2: Adjust the field diaphragm until you see an octagon in the field. No octagon? No problem, move to step 3.

Step 3: Adjust the condenser height until the edges of the octagon are in focus. This is the smaller focus knob directly below the stage. You know, the one you always grab to focus the microscope before you realize you are not focusing the microscope? That one. If all you see is a shadow in the background, or a general darkening of the image, your condenser is too far away from the sample. Move it closer to the surface of the slide and keep an eye out for the octagon!

Step 4: Using the knurled knobs that stick out of the front of the condenser base, those knobs that you always grab when you want to “fiddle” with the image, align the octagon until it is in the center of the field of view. When complete, adjust the field diaphragm just outside of the field of view.

Step 5: Perhaps the most critical and most often overlooked step, adjust the aperture. This is often the slider that is built into the condenser, you know, the one you slide back and forth in the hopes of creating a better image? Well, this time you will remove an eyepiece and look through the optical tube without an eyepiece. Now, slide the aperture back and forth, just like old times. Then, once you see the octagon position it so that it covers 1/3 of the field of view. Put the eyepiece back in and you have a perfectly aligned microscope!

Impress your friends!

If you want to impress your friends or pretend to be a microscope snob, teach everyone in the lab your new found knowledge. You can also visit Leica Science Lab for an interactive tutorial.

If you are tired of constantly correcting the lighting, field diaphragm, aperture, and other optical components, check out the fully automated DM4, DM6, and DMi8 to simplify routine microscope tasks through intelligent automation. If you would like to learn more about intelligent automation, you can contact us anytime to see what an automated Leica microscope can do for you.