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Step 1. Evaluate Your Research Needs
Step 2. Understand the Options
Step 3. Budget Your Purchase
Step 4. Learn About Reputation & Support
Step 5. Know the Terms
Step 6. Make a Confident Decision
Step 1.
Evaluate Your Research NeedsPresent and Future
With such a variety of confocal microscopes on the market, each providing a unique set of
benefits, the first step in the buying process is to evaluate your labs current areas of research,
preferences, and anticipated needs.
Deep
tissue
Super-resolution
Multicolor
Live cell
Time-lapse
Intercellular
Intracellular
Quantitative
imaging
3D imaging
You want your investment to last. Since it is difficult to envision the types of projects youll delve
into over the next five to 10 years, you may want to consider a confocal microscope that will
grow with your future needs.
In addition, consider who will be using the imaging system over its life span. If you are a
small, high-volume imaging group with very specific imaging needs, your search may lead you
to a highly sophisticated, customized instrument. But if youre looking for a confocal microscope
to be used by an entire department of researchers with varying levels of expertise, you will
likely opt for a product that is very simple to use with a broad range of imaging capabilities.
In either situation, it's best to evaluate your imaging needs in terms of the Big Three.
Sensitivity
laser lines needed for excitation, which beam-splitting device is best, the number of detectors
needed, and whether any specialized detectors are necessary.
Speed
of acquisition? These questions will help you determine how fast of a scanner you need.
Resolution
Step 2.
Understand the Options
There are many confocal microscopes available to match your application
requirements and budget needs. Heres a look at the two main classes of
products on the market today:
Basic Research Model: A Powerful Start
The lowest cost models fall into this category. A basic research confocal typically covers routine fluorescence microscopy
applications and requires minimal training. With these ready-to-use platforms, even confocal newcomers can quickly produce
spectacular 3D, multicolor images. A basic confocal will help you easily advance your research from regular fluorescence to
clear, crisp imaging. While these basic research models use the same underlying technology as their big brothers, they have
fixed configurations and limited options for upgrades and accessories for live cell imaging.
TIP: If you go with a basic research model, understand that you will be restricted on laser lines and detectors, with
limited accessories available for live cell imaging. However, this model is perfectly acceptable if you are mainly imaging
fixed, sectioned tissues with two or three fluorescent dyes. If not, you may opt for a system that will move with you as
you advance into new research avenues.
TIP: Uncover any limitations of each vendor's configurable platform. Many people purchase a configurable model
with the plan to add upgrades as needed in the future. But some features are not available as upgrades after the time
of system purchase. When comparing models from different companies, ask specifically if your desired features will be
available in the future, and most importantly, if these features will perform exactly the same whether you add them
now or at a later date.
Tandem scanner. The best of both worlds. A tandem scanner allows you to have two imaging scanners in one system. Based on your
experimental needs, you can switch between a conventional scanner and a resonant scanner. The resonant scanner can be used to image
rapid cellular dynamics, and the conventional scanner can be used to capture a large field with image formats up to 8k x 8k. Each
confocal brand has a slightly different definition of tandem scanning. Be aware of this and ask specifically what the tandem scanner does
and how it works.
DETECTION
When evaluating detection systems, youll find that each one offers a variety of special characteristics. These features are
not exclusive of each other; in fact, many systems offer more than one combination of these features.
Spectral detection. Define your own spectral ranges within the emission spectrum rather than having to use a defined, fixed filter.
The tunability and flexibility of a spectral detector makes your system ready for new dyes and markers today and in the future.
GaAsP photocathode. A favorable option when youre seeking higher sensitivity than whats possible with the standard photomultiplier
tube. A GaAsP photocathode provides greater sensitivity when imaging dim or weakly stained samples, but operates with the sample
noise characteristics of a standard photomultiplier tube due to traditional dynode amplification. For better signal-to-noise ratio, a hybrid
detector uses a GaAsP photocathode but offers a lower noise amplification stage.
Non-descanned detectors. Captures more light to record the faintest structures from deep tissue sections, and allows for elaborate
multicolor, multiphoton experiments. This is the detector choice for multiphoton microscopy.
OTHER CONSIDERATIONS
UPRIGHT OR INVERTED
Most confocal microscopes are available in two configurations: upright or inverted.
Consider your lab's preferences as you begin the buying process.
Upright microscopes allow you to view a specimen from above. There are two types
of upright models. In one, the stage is used to move the sample up or down for focus. In
the other type, the stage is fixed, but the objective nosepiece moves up and down. If
you commonly image larger organisms such as a whole mouse, or any sample that you
do not want to disturb, a fixed stage is best.
Inverted microscopes position the imaging objective below the sample. They are
most commonly used in applications that involve studying cell cultures in liquid, with
the flat part of a vessel serving as the base. You can image most samples on an
inverted microscope that you can on an upright.
superresolution
tandem scanner
optics
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3D imaging
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Widefield
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Resolution
Choosing the best super-resolution system requires an understanding of how each imaging method varies in performance with
various sample types. Remember, lateral resolution isnt always the most important factor. Speed and z-resolution are two other key
considerations when selecting your super-resolution approach. Heres a look at the three super-resolution technologies in use today:
Localization:
A widefield technique that uses thousands of images of stochastically excited fluorophores to
generate a super-resolution reconstruction. Known commercially as PALM, STORM, and GSDIM.
Benefit: High lateral resolution for fixed samples
Structured Illumination:
A widefield technique that calculates a super-resolution result from images taken of a sample illuminated with a series of structured
masks. Known commercially as SIM.
Benefit: Simplest transition from traditional light microscopy, compatible with conventional fluorophores
Stimulated Emission Depletion:
A point-scanning confocal method that selectively depletes the peripheral region of the diffraction limited scanning spot while
leaving a center focal point active to emit fluorescence. Known commercially as STED.
Benefit: Live cell and video rate capabilities (28 frames/second), no post-processing required
Step 3.
Budget Your Purchase: A Holistic View
When shopping for a confocal microscope, work within your budget and prioritize
features based on your most common application needs.
Look at the big picture. Pay attention to factors such as maintenance, workflow improvements, and sample preparation
costs. Will your new confocal overcome bottlenecks in imaging that currently hinder your labs productivity? Are you investing
in capabilities you will rarely use? Factor in the productivity improvements, as well as the financial impact of being able to
obtain images to help achieve your research goals and become published in peer-reviewed journals.
Understand the benefits of modularity. Researchers who must adhere to a limited budget dont have to sacrifice quality.
Seek out the best possible instrument you can afford, with an eye for systems that can grow with your research by adding new
modules catered to your applications. But beware: If a system claims to be modular, find out what that means. To what level is
it upgradable? Will it truly grow to meet your needs? Ask to try out an upgraded system so that you can personally attest to
the fact it will be suitable for your anticipated research.
Dont forget to factor in the service contract. Explore the service options and different ways you can buy it. Among
questions to ask: What is the warranty that comes with the system? How is the service contract structured after the warranty
expires? Is there any special pricing offered if you buy multiple years of service contract upfront at the time of sale rather than
waiting until the warranty expires? If you stay with a given vendor, can you obtain a special discount? And what specials are
offered for core lab managers who own a lot of equipment from the same vendor?
TIP:
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Step 4.
Learn About Reputation & Support
Just as with most significant purchases, youll want to feel confident in the brand
you select. This is where a companys strong reputation, established community of
users, and responsive service support is an advantage.
Investigate the vendors youre considering: What is the company's history of
innovation? And perhaps most importantly, what level of service will the company
offer to its customers in the days, weeks, months, and years after the purchase? Here
are some questions to ask:
What happens after the purchase? You want to do business with a company that will help you get up and running, not just
drop off an instrument at your lab. Look for a well defined, personalized onboarding process, as well as ongoing follow-up to make
sure that everything is working as planned. Ask the sales representative whether the company offers extra training or webinars to
help keep your knowledge fresh.
Does the company offer field support? Does the company have field representatives who will make a visit to your lab if you
need help resolving an equipment problem? What kind of lead time is typical for a visit?
Are the company's in-house service experts qualified? Imaging core facilities are often staffed by experts who can help
with new user training, trouble shooting, and routine maintenance. If your lab lacks this internal support, its especially important
to have a reliable tech support system to call for even simple questions. Explore the level of scientific expertise the vendor offers
in-house, and ask how accessible these experts will be to you if you have a question (and you will likely have lots of questions as
you get to know your new system).
What are the company's high-tech support capabilities? Remote access is an absolute must today. You want to make sure
that your vendor support team can easily access your instrument at any time of the day to help resolve urgent problems, or
even non-urgent ones.
Is there an established community? Look for a group of users who specialize in your key applications. Engage with this
community to learn about others experiences using the systems youre considering. Scientists are always inventing new ways
of doing things; an active community offers the benefit of deep knowledge and innovative ideas.
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Step 5.
Know the Terms: Glossary for Confocal Microscopy
As you and your team compare your options, make sure that everyone is up to speed
on the common terminology and how these terms relate to your purchase.
MICROSCOPY TERMS
Airy pattern. Airy pattern represents the ideal focused spot of light that a perfect lens with a circular aperture can make, limited by the
diffraction of light.
Diffraction. Due to the wave nature of light, a propagating light beam acts as infinite point sources, which interfere with each other.
Due to this process, a beam of light passing through an optical element such as a lens spreads out as it propagates.
Diffraction limit. Due to the process of diffraction, the resolution of an optical system is limited. This fundamental limitation is known as the
diffraction limit. In microscopy, this translates into the minimum lateral spacing that can be resolved by an optical system. This is dependent on
the wavelength of light used, as well as the numerical aperture of the objective used.
Lateral resolution. The minimum distance at which two airy patterns of sufficient contrast can be distinguished as distinct objects.
Numerical aperture. The numerical aperture (NA) of an optical system characterizes the range of angles over which the system can collect
or focus light. This dimensionless number directly relates to the resolving power of a lens. It is the product of the sin of the half angle of the
cone of light emerging from the objective, and the index of refraction of medium between the lens and sample.
Square pinhole. The geometry of the confocal pinhole determines the diffraction pattern in the intermediate image plane. The square pinhole is
beneficial because it improves spectral separation by reducing the overlap of colors along a linear detection axis.
Supercontinuum light source (white light laser). Supercontinuum lasers, also know as White Light Lasers (WLLs), are an alternative to
conventional lasers used in confocal microscopy. WLLs emit a continuous spectrum of light between the range of 470 nm to 670 nm. Multiple
wavelengths can be freely selected to optimize the simultaneous excitation of dye combinations with reduced cross-excitation. A tunable beam
splitter, such as an AOBS, complements the tunable output of a WLL.
Step 6.
Make a Confident Decision
Now that you understand your needs and have evaluated the options, its time to find
the perfect match. Talk to colleagues who use different types of instruments to learn
about their experiences: What do they love about their confocal microscope? What do
they wish they had considered before their purchase? If you dont know anyone
personally, do some digging to find another researcher who works with similar
applications, and use your pending purchase as an excuse to make a new
connection in your field. Your next steps:
Reach out to a company salesperson. Formally express
your interest in the brand and set an appointment to ask
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