Buyer's Guide: Confocal Microscopes for Life Science Discovery

Bringing focus to your decision-making process so you make the best choice for you and your lab.

Inside… › 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

Choosing the right confocal microscope is one of the most important decisions you will make for your lab.

Confocal microscopy has come a very long way since its invention more than a half-century

ago. Today, with novel technology driven by the leading imaging companies, it has become the

standard for fluorescence microscopy. Choosing the right confocal microscope for your specific

research requires careful consideration of the appropriate mix of features related to resolution,

sensitivity, and speed.

Confocal microscopy offers life science researchers a crisp, clear view into the inner workings

of cells, structures, and tissues — visualization that goes well beyond what can be seen

through conventional widefield microscopy.

Empowered by this technology, researchers are making unprecedented headway into a wide

range of biomedical specialties including immunology, neuroscience, oncology, and much

more.

This greater potential for meaningful discovery goes hand-in-hand with the ability to be

published in the most respected peer-reviewed journals. Many researchers are switching to

confocal from widefield microscopes or upgrading to a newer confocal technology to get

closer to the research answers they’re seeking. Some labs are finding it beneficial to

purchase their own confocal microscope rather than continuing to share one at a core lab.

In this guide, you’ll learn the key considerations for each step of the purchasing process,

from budgeting to comparing different technologies to asking the right questions of sales

representatives.

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Step 1. Evaluate Your Research Needs — Present 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 lab’s current areas of research,

preferences, and anticipated needs.

You want your investment to last. Since it is difficult to envision the types of projects you’ll 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 you’re 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.”

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Live cellDeep tissue

Time-lapse

Quantitative imaging

3D imaging

Multicolor

Intercellular

Super-resolutionIntracellular

Depending on your research area — be it neuroscience, biochemistry, optogenetics, or emerging

specialties — different factors will be more important to you than others. For example, if you focus

on cell biology, high-speed imaging is a must and you will want to compare products based on their

offerings in this area.

Keeping your primary applications in mind, consider these questions:

What are our detection needs?

What fluorophores will my lab be using, and how many? How will we split excitation from

emission? How many imaging channels will we be working with? Does our research require

simultaneous multicolor imaging? The answers to these questions determine the number of

laser lines needed for excitation, which beam-splitting device is best, the number of detectors

needed, and whether any specialized detectors are necessary.

What are the dynamics of our experiments?

Will our samples be fixed or live? If live, then do we need incubation equipment to keep live

cells thriving during an experiment? How much data will our lab be collecting, and at what rate

of acquisition? These questions will help you determine how fast of a scanner you need.

What structures will we be imaging?

Will our lab be imaging intracellular or intercellular structures? Will we be mapping tissues and

groups of cells, as with tile scanning experiments, or focusing on single cells? If you’re seeking to

capture interactions between proteins, or other structures and events on the very small scale,

super-resolution technology may be required. To ensure that the confocal hardware can accommodate

the resolution your work requires, consider your experimental design, including depth and size of the

tissues or samples you’ll be imaging. Zoom factor is a meaningful parameter, but it’s not the only

parameter that contributes to resolution. Numerical aperture also comes into play.

THE BIG 3: PRIORITIZING SENSITIVITY, SPEED, AND RESOLUTION

Speed

Resolution

Sensitivity

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Step 2. Understand the Options

There are many confocal microscopes available to match your application

requirements and budget needs. Here’s 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.

Configurable Model: “Grows With You”

The key word here is modularity. These advanced models provide greater flexibility for simultaneous multicolor imaging. If you

are performing live cell imaging and require advanced spectral capabilities, a modular confocal system is your preferred

choice. A modular confocal also gives you the option of adding advanced techniques such as multiphoton imaging for deep

tissue, and it opens the door to more sensitive detectors.

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.

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UPGRADES THAT MATTER

Special Accessories for Deep Tissue ImagingWhen you’re regularly imaging thick specimens such as a living organism or brain tissue, multiphoton microscopes are the answer. Multiphoton systems combine advanced infrared lasers and non-descanned detectors for elaborate multicolor multiphoton experiments. Especially if deep tissue imaging is central to your research, it’s a good idea to weigh the benefits of investing in a mutiphoton confocal instrument.

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Let’s take a look at the most common upgrades to configurable confocal microscopes. In general, these upgrades can be added to your instrument at the time of sale or added later as your research requires or as your budget allows. Keep in mind that there are always tradeoffs, depending on the samples you’re imaging and the resolution you need. For example, if you need high resolution, you may have to

compromise on speed. That said, the power of today’s confocal technology is quickly making compromise a thing of the past.

EXCITATION SOURCES • UV laser. The 405 nanometer (nm) UV laser is commonly added to image counter

stains such as DAPI. Other common uses for this wavelength include photoactivation or uncaging.

• Infrared (IR) laser. IR lasers are used for deep tissue imaging since there is less scattering with longer wavelengths.

• White light laser. Allows continuous selection of multiple excitation wavelengths from blue to red through a continuous spectrum, plus the additional benefit of pulsed emission.

SCANNERS • Resonant scanner. Need speed? The resonant scanner is an alternative to the

conventional scanner that’s usually included in a confocal microscope. Researchers with high-speed requirements often choose a resonant scanner; it is ideal for live cell imaging with rapid experimental dynamics.

• 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

• 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 you’re seeking higher sensitivity than what’s 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.

When evaluating detection systems, you’ll 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.

OTHER CONSIDERATIONS

UPRIGHT OR INVERTEDMost 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.

DATA MANAGEMENT — WHAT YOU NEED TO KNOW When evaluating confocal options, be sure to consider what software is available for your

application and how easy it is to use. You will be interpreting and managing an immense

amount of data, and the software's features and capabilities are important.

• Most confocal microscopes come with proprietary software, so be sure that you

(and other microscope users) are comfortable with the interface. Look for an

intuitive, user-friendly workflow, and test it out during your product demonstration.

• A smaller imaging group of confocal experts is more likely to benefit from

sophisticated and customized data analysis features. On the other hand, a core lab

with dozens or more users might desire a static interface that’s extremely easy to

use – even for beginners.

• To get an accurate pricing picture, learn what software features are part of the

core package versus what’s considered an add-on.

Resonant Scanning for Fast Live Cell Imaging It used to be assumed that spinning disk technology was the only option for fast live cell imaging. Spinning disk confocal microscopes take a parallel approach to point scanning by using rotating Nipkow disks in the excitation and emission paths, each with tens of thousands of pinholes. An EM-CCD or sCMOS camera is used for detection. Spinning disk systems have been used historically to image highly dynamic processes such as cell division, protein trafficking or interaction, and vesicle movement in live cells.

Now there’s another option to consider: Resonant scanners. Resonant scanners offer the speed advantage of spinning disk technology with the added benefit of simultaneous multicolor acquisition. So if fast live cell imaging is your priority, test a resonant scanner to see whether it will help you accomplish your research goals.

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super- resolution

UV Laserinfrared laser

3D imagingoptics

resonant scanner

tandem scanner

IS SUPER-RESOLUTION RIGHT FOR YOU?

Choosing the best super-resolution system requires an understanding of how each imaging method varies in performance with

various sample types. Remember, lateral resolution isn’t always the most important factor. Speed and z-resolution are two other key

considerations when selecting your super-resolution approach. Here’s 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

For many researchers today, the answer is yes. With optical resolution

down to 20 nanometers, super-resolution microscopy has dramatically

improved the understanding of cellular dynamics at the molecular level.

WidefieldMicroscope

Super Resolution

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100

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100

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Confoc

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Electr

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Human

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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 lab’s 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 don’t 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.

Don’t 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: Rank Based on Today’s Needs

So you’ve reviewed all of the options and

compiled your wish list and... you’re over budget.

To prioritize your needs, rank features relative to

the experiments you have planned right now.

This will allow you to more easily see what you

can postpone for a later upgrade versus the

features to include at time of sale.

Get Creative About FundingLook beyond the National Institutes of Health and the National Science Foundation, both of which have seen serious budget cuts. Consider other grant-writing organizations that would support the discoveries you’re pursuing.

Today’s tougher funding climate means you have to prepare a compelling grant application. Seek help from the companies who make and sell confocal microscopes; ask for tips and resources that you can use in your applications. You’ll want to provide the committee with evidence that the instrument will play a central role in solving an important research question.

Likewise, if you’re seeking funding approval from within an institution or company, you’ll need to show and tell a compelling story about how your new confocal microscope will help your organization accomplish key goals.

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Step 4. Learn About Reputation & Support

Just as with most significant purchases, you’ll want to feel confident in the brand

you select. This is where a company’s strong reputation, established community of

users, and responsive service support is an advantage.

Investigate the vendors you’re 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, it’s 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 you’re 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 MicroscopyAs 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 TERMSAiry 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.

FEATURES & HARDWAREAOBS. Acousto Optical Beam Splitter technology is used in confocal microscopy as a replacement for dichroic beam splitters. Unlike fixed dichroic splitters, AOBS is capable of dynamically tuning reflection or suppression of multiple, simultaneous laser lines, making it a highly customizable beam splitter. Also, the extremely narrow reflection band around each selected laser line increases the transmission of emission light.

Dichroic beam splitters. Dichroic mirrors, or beam splitters, spectrally separate light by transmitting or reflecting light as a function of wavelength. In fluorescence microscopy, they are used to reflect the excitation light into the sample and transmit emitted fluorescence to the detector. Dichroic splitters used in confocal microscopes can be designed to reflect fixed laser lines (typically one to four lines) and transmit emission spectra to the detectors.

HyD. Hybrid Detection (HyD) employs characteristics of both photomultiplier tubes (GaAsP-PMTs) and avalanche photodiodes (APDs) to produce a two-step photodetector capable of vacuum acceleration followed by electron bombardment and avalanche gain. HyDs are capable of photon counting for quantitative imaging applications.

Resonant scanner. Point-scanning confocal systems typically use galvanometric mirrors to raster scan the laser across the sample. A resonant scanner is a special variant of such galvo scanners, which can be added to the confocal microscope to provide the speed necessary for real-time imaging of live cells. The resonant scanner can allow line frequencies up to 24 kHz (as compared to the 3 kHz that non-resonant scanners can achieve). The reduced pixel dwell time during resonant scanning reduces photo-stress on fluorochoromes.

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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.

METHODS & PLATFORMSCARS. Coherent Anti-Stokes Raman Scattering (CARS) is a label-free imaging method. CARS allows the analysis of samples based on intrinsic vibrational contrast with specificity to molecular bonds within the sample. Since no fluorophores are required, labeling steps can be bypassed to image the sample in a minimally invasive manner.

GSD. Ground State Depletion (GSD) technology produces 2D and 3D super-resolution images with a precise localization microscopy method. Based on the localization microscopy technique GSDIM (Ground State Depletion microscopy followed by Individual Molecule return), GSD works directly on standard fluorophores to achieve super-resolution images with a resolution down to 20 nm.

Multiphoton. Multiphoton microscopy enables researchers to image deep into thick samples. This technique uses laser wavelengths in the infrared to reduce scattering and eliminate out-of-focus fluorophore excitation. Applications range from whole brain imaging to imaging real-time processes within organs.

PALM. A localization-based super-resolution method that uses photoactivatable fluorophore constructs to stochastically achieve super-resolution images with a resolution down to 20 nm.

SIM. Structured Illumination Microscopy (SIM) is a method of super-resolution imaging that uses a modulated illumination pattern and computational restoration to generate a super-resolution image.

Spinning disk. True confocal laser scanning microscopes focus a single beam on the specimen plane to sequentially point-scan a region of interest with spatial filtration of the emission light through a single pinhole, which rejects light originating from regions that are out of focus. Spinning disk systems use many pinholes arranged on rotating disks (or equivalent arrangements), compromising the sectioning performance in comparison to true confocal scanning (single point scanning) to increase frame rate.

STED. STimulated Emission Depletion Microscopy (STED) is a method that resolves structures below optical resolution and is therefore attributed to super-resolution. Employing two different diffraction patterns, one that excites and the other that de-excites fluorochromes, this technique enables virtually unlimited resolution.

STORM. A super-resolution method that uses combined activator/reporter dye pairs to stochastically achieve super-resolution images with a resolution down to 20 nm.

Tile scanning. An approach for imaging a large specimen by assembling many single images to form one large composite image. The specimen is generally moved using motorized stages with high precision.

Widefield. A widefield fluorescence microscope is a conventional type of microscope in which the full field is illuminated and imaged. It differs from a confocal, which laser-scans and images the specimen point by point. A drawback of widefield microscopy is that fluorescence emitted by the specimen above and below the focal plane interferes with the resolution.

Z-stack. A data set of spatially ordered optical sections that represent a 3D volume.

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Step 6. Make a Confident Decision Now that you understand your needs and have evaluated the options, it’s 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 don’t 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:

TIP: Be specific or be sorry. The goal of

a demo is to determine whether you can fully

complete your experiments to your satisfaction.

Tell the sales rep exactly what you need for the

experiment you’d like to perform using the

confocal microscope. Talk about your sample

material, desired frame rate, desire for 3D

images, how many colors you’ll use, how many

channels, etc. The clearer you can state those

goals, the better the rep can provide you with the

right solution. Ask each company to fulfill the

same request so that you can compare options

with the greatest ease.

• Reach out to a company salesperson. Formally express

your interest in the brand and set an appointment to ask

questions and learn more.

• Request a demo. Before making a confocal purchase,

perform working demonstrations with a variety of

technologies. Contact the sales representative for the

instruments you’re considering, and explain your specific

needs. The rep should be able to configure a system and tell

you a preliminary price before the demo. Don’t forget to try

out the software; the science is really what you do with the

image after it’s been acquired.

• Make your decision! After you’ve struck a deal, become

the expert on your new system. Contact the manufacturer’s

support team to answer any questions that you have, and

get active in the community of users.

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Confocal Microscope Buyer's Guide Checklist: Images captured with today’s confocal microscopes go well beyond what can be seen through a conventional microscope, allowing incredible new discoveries. Use this checklist to evaluate your options and guide your purchasing decision.

Evaluate your current research needs, as well as those in years to come. Consider the following:

• Sensitivity: What are my detection needs now and in the future?

• Speed: What are the dynamics of my experiments?

• Resolution: What structures will I be imaging, and on what scale?

• Basic models. The lowest cost models have limited upgrade flexibility, but can still produce spectacular images.

• Configurable models. These models allow greater flexibility for simultaneous multicolor imaging. If you will be performing live cell imaging and require advanced spectral capabilities, this is the right choice.

• Upgrades. The most common upgrades are excitation sources (UV lasers, IR lasers), scanners (resonant and tandem), and detection systems, which offer any combination of options such as spectral, GaAsP photocathode, and non-descanned detection.

Do your research and make an informed decision.

• Talk to colleagues. What do they love about their confocal microscope? What do they wish it could do?

• Reach out to a company salesperson. Schedule an appointment to ask questions and learn more.

• Request a demo. Get your hands on the systems before buying and test your own samples.

Step 1 Evaluate Your Research Needs—Present and Future

Step 2 Understand the Options

Step 6 Make a Confident Decision

Prioritize features based on your most common application needs.

• Pay attention to maintenance, workflow improvements, and sample prep costs.

• Understand the benefits of future upgrades to see how the microscope can grow with your research needs.

• Explore the service options and types of contracts.

Step 3 Budget Your Purchase: A Holistic View

As with any significant purchase, you’ll want to feel confident in the brand you select.

• What happens after the purchase? You will want help to get up and running.

• Is field support available? Find out how long it may take to get support when needed, both virtual and on-site.

• Are the in-house service experts qualified? Seek accessible and knowledgeable application support personnel.

Step 4 Learn About Reputation & Support

Make sure that you are up to speed on common microscopy terms and how they relate to your purchase. (See full glossary in Buyer’s Guide for more.)

Step 5 Know the Terms

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