Life Science Technologies
Microscopy
Produced by the Science/AAAS Custom Publishing Office
Souping Up Your ‘Scope
The diversity of options for today’s microscopes—objectives and cameras,
advanced stages and software, sources of illumination, and more—challenge
even the experts when designing a new system. The process gets even more
complex when faced with constraints, such as a budget or a specific set of necessary applications. Consequently, life scientists need to know where to spend
and when to save. The experts interviewed here provide some pointers and
give us insight into some of the newest components in the fast-changing field
of microscopy. By Mike May
build
your
microscope
depends
entirely on how
you plan
to use it.
A
Upcoming Features
Big Data—June 13
Digital Lab Management—July 25
Metabolomics—September 19
536
microscope?” So Richardson would put more money in an objective
first. Next, he’d invest in a good detector.
How you build your microscope depends entirely on how
you plan to use it. For example, certain types of cameras work better in different lighting situations than others. Likewise, microscope
stages can address specific needs, such as mobility or environmental
constraints. To get the optimum results, you need to combine the
parts that work well together to meet the specifications that your
experiments demand.
CONNECTING THE RIGHT CAMERA
Many life scientists attach a camera to their microscopes to both document and analyze images. In today’s microscope cameras, an electronic
sensor takes the place of film. One of the oldest sensors is a chargecoupled device (CCD), in which an array of pixels captures light and
turns it into an electrical signal. Many manufacturers continue to use
this type of sensor, which is extremely reliable.
A less expensive technology, complementary metal-oxide
semiconductor (CMOS) sensors, came from the integrated-circuit industry. This kind of sensor also turns light into an electrical signal, but it requires more circuitry on the sensor, which takes
up space for sensing, thereby making this technology less sensitive
to light, on average, than a CCD. Nonetheless, CMOS sensors enable faster frame rates (images per second) than CCD sensors since
pixels are detected in groups rather than individually. A new version
of CMOS, called scientific CMOS (sCMOS), addresses the technology’s limitations by providing increased sensitivity while maintaining
the speed.
Different applications, however, often determine which unique
camera features are necessary. “If you’re building a simple microscope to snap a picture of a sample stained with H&E—hematoxylin and eosin—your camera does not need to be overly extravagant.” If you need higher imaging rates or the ability to detect weak
fluorescent signals, says Richardson, “you could be going from a
$2,000 camera to a $20,000 or $30,000 one.” The former could
be a high-end consumer CCD camera, and the latter might be an
sCMOS camera.
Manufacturers continue to make advances in microscope cameras using a variety of sensors. As an example, Scott Olenych, product
www.sciencemag.org/products
CREDIT: © ANYAIVANOVA/SHUTTERSTOCK.COM
How you
scientist’s ability to visualize the intricate details of their experimental
model depends upon their microscope’s capabilities. “An old adage is that a
microscope is only as good as the sum of its
parts,” says Douglas Richardson, director of
imaging at the Harvard Center for Biological Imaging in Cambridge, Massachusetts. “If one component—lens, detector, or
anything else—is of lower quality than the
rest, it affects the image in the end.” He adds,
“You need all of the parts to function at their
highest levels.”
A scientist can easily dream of their ideal
microscope system, but building one takes
more work. As Richardson says, “It’s a lengthy
process to select which components to put on
your system. It needs to be customized for
your own applications.”
With so many components available
today, every scientist needs to make some
choices, often based on price, about which
ones are must-haves. When asked if any
particular pieces matter more than others,
Richardson says, “With any system, the most
important component is the objective.” He
adds, “When we look at a system, one of the
first things that I consider is: What objectives
are available and which ones will work for
the applications that we have in mind for the
Life Science Technologies
Microscopy
Produced by the Science/AAAS Custom Publishing Office
marketing manager of imaging products at Carl Zeiss Microscopy
To do that, scientists must maintain specific environmental
in Oberkochen, Germany, points out his company’s new Axiocam 506, conditions when viewing live cells. To decide what devices provide
which uses a CCD sensor with a 16 mm diagonal dimension. Com- the best conditions for a specific application, a scientist can ask an
pared to cameras that use a sensor with an 11 mm diagonal or less, expert, such as Jeffrey D. McGinn, president and director of instruthe Axiocam 506 can grab a larger area of view. In addition, this ment sales, McCrone Microscopes & Accessories in Westmont,
camera comes in color and monochromatic versions with software Illinois. He says, “When someone contacts us, we go through some
that controls image acquisition and provides other tools, including needs-assessment questions, like asking about the types of samples
the ability to add labels or scale bars to an image. In describing this the customer wants to look at and if the samples need to be in some
software, Olenych says, “People generally agree that it’s fairly simple particular environment.” As a result, he might suggest outfitting a
to use.” That really matters,
system with a temperaturebecause, as Olenych says,
controlled stage like that
“You interact with the microavailable from Linkam SciEach part matters, as does the interaction between
scope at the computer, and
entific Instruments in
the software determines the
Surrey, United Kingdom.
them, and these must all merge into
overall experience.”
These stages can maintain
When outfitting a microa sample at temperatures of
a system that meets the individual scientist’s needs.
scope with a camera, scientists
-196°C to 1,500°C. As an
can choose from other options
example of an application that
with a 16 mm diagonal CCD.
requires heating, McGinn
An example is the Retiga 6000, which is made by QImaging in Sur- points to the pharmaceutical industry. He says, “When you heat
rey, British Columbia, Canada. This camera, says Chris Ryan, product a drug product, it may go through polymorphic transformations.
manager at QImaging, “is for customers who want to scan a whole Some of these may react differently.” So a pharmaceutical scientist
slide or simultaneously monitor large populations of cells, say for high- might expose a sample to a range of temperatures to find the transcontent screening.”
formation points. In other cases, a researcher may simply want
In fluorescent imaging, for example, scientists look for sensitiv- to keep a cellular sample at body temperature during an imaging
ity and resolution, says Ryan. Sensitivity determines the weakest fluo- experiment.
rescent signal that can be detected, and resolution reveals the smallest
Some applications require even greater environmental control,
features that can be distinguished in an image. “Those features are a which scientists can obtain with an on-stage incubator from the
combination of the camera and the objective,” says Ryan.
Advanced Microscopy Group (recently acquired by Life Technologies,
In some cases, a life scientist might prefer a camera with a range which has in turn been acquired by Thermo Scientific), which
of features. For that, they might select an sCMOS camera. As an includes an environmentally controlled chamber. “The device is
example, Orla Hanrahan, a life science application specialist at Andor essentially a plug-in for the EVOS fluorescent microscope,” adds
Technology in Belfast, Ireland, mentions Andor’s Zyla 4.2. “This Hans Beernink, product management leader at Life Technologies.
can provide sensitivity, speed, and a large field of view, sort of an “It integrates into the microscope and is controlled by software.” It
everything-in-one camera,” says Hanrahan.
can be controlled for temperature, humidity, and even gases, such
In low-light imaging, though, a researcher might prefer an as oxygen. continued>
electron-multiplying charge-coupled device (EMCCD) camera. An
EMCCD includes an electronic device that multiplies the output from
Featured Participants
a conventional CCD. As a result, an EMCCD can detect weaker signals.
For example, the Evolve 512 Delta from Photometrics in Tucson,
Andor
McCrone Microscopes
Arizona, uses an EMCCD sensor. Rachit Mohindra, product manager
www.andor.com
& Accessories
www.mccronemicroscopes.
at Photometrics, points out a downside, however: “To work at such
Carl Zeiss
com
low-light levels, EMCCD cameras use sensors with much larger pixels,
www.zeiss.com/microscopy
Nikon Instruments
which collect more light in a given amount of area, so you can’t see the
Femtolasers Produktions
www.nikoninstruments.com
www.femtolasers.com
finer details.”
KEEPING SAMPLES ALIVE
Today’s life scientists often image live cells. Traditionally, scientists
mostly looked at dead cells, which were prepared using a process
that makes the samples robust and able to survive storage for years
without any special care. Live cells, by contrast, must be nurtured—
kept in the right surroundings and temperature—to keep them alive.
As Michael O’Grady, senior R&D manager at Life Technologies in
Carlsbad, California, says, “To get crisp images, you need to keep the
cells healthy.”
Life Technologies
www.lifetechnologies.com
Linkam Scientific Instruments
www.linkam.co.uk
Harvard Center for
Biological Imaging
labs.mcb.harvard.edu/hcbi/
Olympus Canada
www.olympuscanada.com
Photometrics
www.photometrics.com
QImaging
www.qimaging.com
MBF Bioscience
www.mbfbioscience.com
www.sciencemag.org/products
537
Life Science Technologies
Microscopy
Sensitivity determines the weakest
fluorescent signal that can be detected, and
resolution reveals the smallest features that
can be distinguished in an image.
STAYING IN FOCUS
Beyond keeping cells in good health, live-cell imaging creates other
unique challenges. Some of the obstacles scientists must overcome are
optical, and others are more mechanical.
Distinctive optical factors come into play with live-cell imaging.
This creates a need for new microscopy solutions since objectives were
originally designed for fixed samples, which have different optical
properties than live cells. As Andrew Millar, marketing manager for
the scientific equipment group at Olympus Canada in Richmond
Hill, Ontario, says, “Our silicone immersion objectives were designed
for imaging live cells.” He adds, “Images collected from live cells are
brighter and more highly resolved when using silicone immersion
optics.” These objectives come in 30x, 40x, and 60x, and work on any
Olympus microscope. They improve the quality of an image because
the refractive index of the silicone immersion media is very similar to
that of live cells.
Live-cell imaging technologies cover a wide range of applications, and some can be extremely specific. In electrophysiology, for
example, once you insert a glass electrode into the desired neuron,
you don’t want anything bumping it, which can abruptly end your recording. But what if you want to look at something nearby, maybe to
even place a second electrode? You can’t move the microscope stage,
but some technologies allow you to reposition the objective. That’s just
what you can do with the Radially Moving Objective, or RMO, from
MBF Bioscience in Williston, Vermont. “It moves the objective lens
in the x and y direction,” says Jack Glaser, the company’s president. “The
specimen stays stationary, and the objective lens moves to scan different points.” Glaser adds that the RMO works with most microscopes
and objective lenses. “You just remove the objective turret, slide on
the RMO, tighten a setscrew, and you’re ready to go,” Glaser explains.
The RMO comes with a control box that allows a computer to move
the objective.
538
FEATURES FOR FLUORESCENCE
Whether a scientist is imaging live cells or fixed ones, controlling the
illumination is an important tool for microscopy experiments. This is
particularly true for today’s imaging techniques that involve multiple
lasers to light up different fluorescent labels in a sample. “People have
their own preferences and needs for their experiments,” says Stephen T.
Ross, general manager of products and marketing for North and South
America at Nikon Instruments, which is headquartered in Melville,
New York.
To help with that, Nikon developed some new components for
its Ti inverted microscopes. For instance, the Nikon LU-N laser unit
can be equipped with up to eight lasers, ranging from 356–756 nm.
“This laser unit also provides up to seven different outputs that can
connect to various devices, like a photo-activation system for fluorescence imaging,” Ross says. “Plus, it’s a solid-state laser system,
and there are no mirrors or adjustments needed when it comes out of
the box.”
Released in conjunction with the LU-N, Nikon developed its
L-Apps system components. “These application components and the
LU-N work synergistically through our software,” says Ross. “This can
take complicated experiments and make them accessible to people at
all different levels of expertise.” When illuminating live cells, laser light
can damage the sample. So having more control over the illumination
comes in handy.
To really lessen the potential damage from laser illumination in
fluorescence imaging, a scientist can use two-photon microscopy. It
decreases the impact of light on a sample by exciting the fluorescent
molecule with two photons at once, with each carrying only about half
the energy required with just one photon. To further prevent tissue
damage, a scientist can use a laser that creates very short pulses, which
also applies less energy to the sample. Today’s fastest lasers for this
kind of imaging produce pulses in the range of femtoseconds—just
millionths of a billionth of a second.
When asked about the key challenges to making femtosecond lasers that can be used in most life science laboratories, Andreas Stingl,
president of Femtolasers Produktions in Vienna, Austria, points
out three things: ease of use, a lifetime of 10,000 hours, and robust
construction. He says, “Currently, tunable titanium-sapphire–based
oscillators operating at repetition rates of 80 megahertz and delivering pulses in the 100 femtosecond range are standard sources used in
two-photon microscopy.” Beyond causing less damage, a femtosecond
pulse can penetrate deeper into tissues than longer pulses. For instance,
Stingl says, “Sub-20 femtosecond pulses show 160% increased penetration depth compared to 120 femtosecond pulses at the same average
power.”
The power to transform images with such minute—femtosecond—changes reveals the precision involved in today’s microscopes. But focusing on such tiny parameters should not overshadow the need to blend together the right microscope components
overall. Each part matters, as does the interaction between them,
and these must all merge into a system that meets the individual
scientist’s needs.
Mike May is a publishing consultant for science and technology.
DOI: 10.1126/science.opms.p1400085
www.sciencemag.org/products
CREDIT: IIMAGE COURTESY OF QIMAGING.
Produced by the Science/AAAS Custom Publishing Office