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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.

Evaluate Your Research Needs ›› Step 2. Know the Terms ›› Step 6.Inside… ›› Step 1. Make a Confident Decision . Budget Your Purchase ›› Step 4. Understand the Options ›› Step 3. Learn About Reputation & Support ›› Step 5.

Empowered by this technology. and tissues — visualization that goes well beyond what can be seen through conventional widefield microscopy. from budgeting to comparing different technologies to asking the right questions of sales representatives. 3 . Choosing the right confocal microscope for your specific research requires careful consideration of the appropriate mix of features related to resolution. Confocal microscopy has come a very long way since its invention more than a half-century ago. In this guide.Choosing the right confocal microscope is one of the most important decisions you will make for your lab. 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. sensitivity. researchers are making unprecedented headway into a wide range of biomedical specialties including immunology. neuroscience. and speed. This greater potential for meaningful discovery goes hand-in-hand with the ability to be published in the most respected peer-reviewed journals. Some labs are finding it beneficial to purchase their own confocal microscope rather than continuing to share one at a core lab. clear view into the inner workings of cells. you’ll learn the key considerations for each step of the purchasing process. with novel technology driven by the leading imaging companies. oncology. Confocal microscopy offers life science researchers a crisp. it has become the standard for fluorescence microscopy. and much more. structures. Today.

high-volume imaging group with very specific imaging needs. In either situation. and anticipated needs. your search may lead you to a highly sophisticated. In addition.Step 1. Since it is difficult to envision the types of projects you’ll delve into over the next five to 10 years. the first step in the buying process is to evaluate your lab’s current areas of research.” 4 . you will likely opt for a product that is very simple to use with a broad range of imaging capabilities. Deep tissue Super-resolution Multicolor Live cell Time-lapse Intercellular Intracellular Quantitative imaging 3D imaging You want your investment to last. preferences. you may want to consider a confocal microscope that will grow with your future needs. If you are a small. But if you’re looking for a confocal microscope to be used by an entire department of researchers with varying levels of expertise. customized instrument. each providing a unique set of benefits. Evaluate Your Research Needs—Present and Future With such a variety of confocal microscopes on the market. it's best to evaluate your imaging needs in terms of the “Big Three. consider who will be using the imaging system over its life span.

and whether any specialized detectors are necessary. and at what rate Speed of acquisition? These questions will help you determine how fast of a scanner you need. which beam-splitting device is best. if you focus on cell biology. optogenetics. 5 Resolution . biochemistry. Numerical aperture also comes into play. as with tile scanning experiments. For example. SPEED. including depth and size of the tissues or samples you’ll be imaging. To ensure that the confocal hardware can accommodate the resolution your work requires. 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 Sensitivity laser lines needed for excitation.THE BIG 3: PRIORITIZING SENSITIVITY. or emerging specialties — different factors will be more important to you than others. Zoom factor is a meaningful parameter. What are the dynamics of our experiments? Will our samples be fixed or live? If live. super-resolution technology may be required. What structures will we be imaging? Will our lab be imaging intracellular or intercellular structures? Will we be mapping tissues and groups of cells. then do we need incubation equipment to keep live cells thriving during an experiment? How much data will our lab be collecting. but it’s not the only parameter that contributes to resolution. AND RESOLUTION Depending on your research area — be it neuroscience. or other structures and events on the very small scale. consider your experimental design. or focusing on single cells? If you’re seeking to capture interactions between proteins. high-speed imaging is a must and you will want to compare products based on their offerings in this area. consider these questions: What are our detection needs? What fluorophores will my lab be using. Keeping your primary applications in mind. the number of detectors needed.

A basic research confocal typically covers routine fluorescence microscopy applications and requires minimal training. These advanced models provide greater flexibility for simultaneous multicolor imaging. A basic confocal will help you easily advance your research from regular fluorescence to clear. Many people purchase a configurable model with the plan to add upgrades as needed in the future. and most importantly. But some features are not available as upgrades after the time of system purchase. you may opt for a system that will move with you as you advance into new research avenues. crisp imaging. When comparing models from different companies. While these basic research models use the same underlying technology as their “big brothers. 6 . A modular confocal also gives you the option of adding advanced techniques such as multiphoton imaging for deep tissue. If you are performing live cell imaging and require advanced spectral capabilities. even confocal newcomers can quickly produce spectacular 3D. TIP: Uncover any limitations of each vendor's configurable platform. TIP: If you go with a basic research model. 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. Configurable Model: “Grows With You” The key word here is modularity. this model is perfectly acceptable if you are mainly imaging fixed. However.” they have fixed configurations and limited options for upgrades and accessories for live cell imaging. Understand the Options There are many confocal microscopes available to match your application requirements and budget needs. and it opens the door to more sensitive detectors.Step 2. multicolor images. if these features will perform exactly the same whether you add them now or at a later date. a modular confocal system is your preferred choice. sectioned tissues with two or three fluorescent dyes. with limited accessories available for live cell imaging. understand that you will be restricted on laser lines and detectors. ask specifically if your desired features will be available in the future. If not. With these ready-to-use platforms.

it is ideal for live cell imaging with rapid experimental dynamics. These features are not exclusive of each other. A GaAsP photocathode provides greater sensitivity when imaging dim or weakly stained samples. you’ll find that each one offers a variety of special characteristics. Define your own spectral ranges within the emission spectrum rather than having to use a defined. That said. Allows continuous selection of multiple excitation wavelengths from blue to red through a continuous spectrum. depending on the samples you’re imaging and the resolution you need. a hybrid detector uses a GaAsP photocathode but offers a lower noise amplification stage. The resonant scanner can be used to image rapid cellular dynamics. Be aware of this and ask specifically what the tandem scanner does and how it works. • GaAsP photocathode. The 405 nanometer (nm) UV laser is commonly added to image counter stains such as DAPI. Based on your experimental needs. DETECTION When evaluating detection systems. multiphoton experiments. Special Accessories for Deep Tissue Imaging When you’re regularly imaging thick specimens such as a living organism or brain tissue. The tunability and flexibility of a spectral detector makes your system ready for new dyes and markers today and in the future. Each confocal brand has a slightly different definition of tandem scanning. the power of today’s confocal technology is quickly making compromise a thing of the past. multiphoton microscopes are the answer. fixed filter. 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. • W hite light laser. Captures more light to record the faintest structures from deep tissue sections. Keep in mind that there are always tradeoffs. • Tandem scanner. Need speed? The resonant scanner is an alternative to the conventional scanner that’s usually included in a confocal microscope. The best of both worlds. A favorable option when you’re seeking higher sensitivity than what’s possible with the standard photomultiplier tube. EXCITATION SOURCES • U V laser. SCANNERS • Resonant scanner. IR lasers are used for deep tissue imaging since there is less scattering with longer wavelengths.UPGRADES THAT MATTER Let’s take a look at the most common upgrades to configurable confocal microscopes. in fact. In general. • Infrared (IR) laser. but operates with the sample noise characteristics of a standard photomultiplier tube due to traditional dynode amplification. A tandem scanner allows you to have two imaging scanners in one system. This is the detector choice for multiphoton microscopy. if you need high resolution. 7 . and allows for elaborate multicolor. it’s a good idea to weigh the benefits of investing in a mutiphoton confocal instrument. you can switch between a conventional scanner and a resonant scanner. many systems offer more than one combination of these features. plus the additional benefit of pulsed emission. • Spectral detection. you may have to compromise on speed. Researchers with high-speed requirements often choose a resonant scanner. and the conventional scanner can be used to capture a large field with image formats up to 8k x 8k. Especially if deep tissue imaging is central to your research. Multiphoton systems combine advanced infrared lasers and non-descanned detectors for elaborate multicolor multiphoton experiments. Other common uses for this wavelength include photoactivation or uncaging. • Non-descanned detectors. For better signal-to-noise ratio. For example.

OTHER CONSIDERATIONS UPRIGHT OR INVERTED Most confocal microscopes are available in two configurations: upright or inverted. learn what software features are part of the core package versus what’s considered an add-on. test a resonant scanner to see whether it will help you accomplish your research goals. protein trafficking or interaction. • Inverted microscopes position the imaging objective below the sample. Spinning disk confocal microscopes take a parallel approach to point scanning by using rotating Nipkow disks in the excitation and emission paths. or any sample that you do not want to disturb. In one. and vesicle movement in live cells. be sure to consider what software is available for your application and how easy it is to use. and the software's features and capabilities are important. In the other type. On the other hand. a fixed stage is best. Spinning disk systems have been used historically to image highly dynamic processes such as cell division. the stage is used to move the sample up or down for focus. a core lab with dozens or more users might desire a static interface that’s extremely easy to use – even for beginners. They are most commonly used in applications that involve studying cell cultures in liquid. the stage is fixed. You will be interpreting and managing an immense amount of data. user-friendly workflow. each with tens of thousands of pinholes. Consider your lab's preferences as you begin the buying process. Look for an intuitive. DATA MANAGEMENT — WHAT YOU NEED TO KNOW When evaluating confocal options. • Upright microscopes allow you to view a specimen from above. superresolution tandem scanner optics resonant scanner 3D imaging infrared laser UV Laser 8 . • Most confocal microscopes come with proprietary software. 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. 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. If you commonly image larger organisms such as a whole mouse. and test it out during your product demonstration. So if fast live cell imaging is your priority. Resonant scanners offer the speed advantage of spinning disk technology with the added benefit of simultaneous multicolor acquisition. so be sure that you (and other microscope users) are comfortable with the interface. An EM-CCD or sCMOS camera is used for detection. • A smaller imaging group of confocal experts is more likely to benefit from sophisticated and customized data analysis features. Now there’s another option to consider: Resonant scanners. but the objective nosepiece moves up and down. • To get an accurate pricing picture. There are two types of upright models.

and GSDIM.1 ic mm ros c op e icr os co pe M icr os co pe s improved the understanding of cellular dynamics at the molecular level. Speed and z-resolution are two other key considerations when selecting your super-resolution approach.1 ectr nm on M . Remember.IS SUPER-RESOLUTION RIGHT FOR YOU? For many researchers today. Known commercially as PALM. Widefield Microscope Super 1 cm 1 mm 100 µ m 1 µm 100 nm 1 nm 0.1 nm Resolution Choosing the best super-resolution system requires an understanding of how each imaging method varies in performance with various sample types.2 solu 00 t nm ion M 20 Su El 0. STORM. lateral resolution isn’t always the most important factor. Known commercially as STED. Benefit: Simplest transition from traditional light microscopy. 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. super-resolution microscopy has dramatically H >1 uma mm n E ye C 20 onf 0 n oca m la -1 nd 00 W mm ide fie ld pe r nm -Re . 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. the answer is yes. Benefit: Live cell and video rate capabilities (28 frames/second). Known commercially as SIM. 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. no post-processing required 9 . With optical resolution down to 20 nanometers.

Look at the big picture.Step 3. Explore the service options and different ways you can buy it. 10 Get Creative About Funding Look beyond the National Institutes of Health and the National Science Foundation. workflow improvements. But beware: If a system claims to be modular. 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. rank features relative to the experiments you have planned right now. Budget Your Purchase: A Holistic View When shopping for a confocal microscope. 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. work within your budget and prioritize features based on your most common application needs. find out what that means. Seek help from the companies who make and sell confocal microscopes. Today’s tougher funding climate means you have to prepare a compelling grant application. with an eye for systems that can grow with your research by adding new modules catered to your applications. Don’t forget to factor in the service contract. both of which have seen serious budget cuts. Understand the benefits of modularity. Researchers who must adhere to a limited budget don’t have to sacrifice quality. You’ll want to provide the committee with evidence that the instrument will play a central role in solving an important research question. Seek out the best possible instrument you can afford. 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. Pay attention to factors such as maintenance. if you’re seeking funding approval from within an institution or company. . ask for tips and resources that you can use in your applications. 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. you’ll need to show and tell a compelling story about how your new confocal microscope will help your organization accomplish key goals. Likewise. you’re over budget. To prioritize your needs.. 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. Consider other grant-writing organizations that would support the discoveries you’re pursuing.

and routine maintenance. Scientists are always inventing new ways of doing things. as well as ongoing follow-up to make sure that everything is working as planned. Learn About Reputation & Support Just as with most significant purchases. Investigate the vendors you’re considering: What is the company's history of innovation? And perhaps most importantly. Engage with this community to learn about others’ experiences using the systems you’re considering. This is where a company’s strong reputation. what level of service will the company offer to its customers in the days. Explore the level of scientific expertise the vendor offers in-house. Look for a well defined. weeks. and responsive service support is an advantage. Ask the sales representative whether the company offers extra training or webinars to help keep your knowledge fresh. an active community offers the benefit of deep knowledge and innovative ideas.Step 4. Is there an established community? Look for a group of users who specialize in your key applications. 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. personalized onboarding process. not just drop off an instrument at your lab. months. 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. If your lab lacks this internal support. you’ll want to feel confident in the brand you select. it’s especially important to have a reliable tech support system to call for even simple questions. or even non-urgent ones. 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). 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. What are the company's high-tech support capabilities? Remote access is an absolute must today. trouble shooting. established community of users. 11 .

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. the resolution of an optical system is limited. Point-scanning confocal systems typically use galvanometric mirrors to raster scan the laser across the sample. 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. In fluorescence microscopy. making it a highly customizable beam splitter. the extremely narrow reflection band around each selected laser line increases the transmission of emission light. Resonant scanner. In microscopy. Also. Acousto Optical Beam Splitter technology is used in confocal microscopy as a replacement for dichroic beam splitters. The numerical aperture (NA) of an optical system characterizes the range of angles over which the system can collect or focus light. FEATURES & HARDWARE AOBS. this translates into the minimum lateral spacing that can be resolved by an optical system. Due to the process of diffraction. Know the Terms: Glossary for Confocal Microscopy As you and your team compare your options. Diffraction limit. AOBS is capable of dynamically tuning reflection or suppression of multiple. as well as the numerical aperture of the objective used. or beam splitters. Airy pattern represents the ideal focused spot of light that a perfect lens with a circular aperture can make. Dichroic mirrors. limited by the diffraction of light. 12 . Due to the wave nature of light. they are used to reflect the excitation light into the sample and transmit emitted fluorescence to the detector. This is dependent on the wavelength of light used. Diffraction.Step 5. spectrally separate light by transmitting or reflecting light as a function of wavelength. This fundamental limitation is known as the diffraction limit. HyD. simultaneous laser lines. Due to this process. Lateral resolution. This dimensionless number directly relates to the resolving power of a lens. Dichroic beam splitters. HyDs are capable of photon counting for quantitative imaging applications. Unlike fixed dichroic splitters. and the index of refraction of medium between the lens and sample. The minimum distance at which two airy patterns of sufficient contrast can be distinguished as distinct objects. which interfere with each other. The reduced pixel dwell time during resonant scanning reduces photo-stress on fluorochoromes. Numerical aperture. a beam of light passing through an optical element such as a lens spreads out as it propagates. The resonant scanner can allow line frequencies up to 24 kHz (as compared to the 3 kHz that non-resonant scanners can achieve). MICROSCOPY TERMS Airy pattern. It is the product of the sin of the half angle of the cone of light emerging from the objective. make sure that everyone is up to speed on the common terminology and how these terms relate to your purchase. a propagating light beam acts as infinite point sources. which can be added to the confocal microscope to provide the speed necessary for real-time imaging of live cells. A resonant scanner is a special variant of such galvo scanners.

this technique enables virtually unlimited resolution. Supercontinuum light source (white light laser). This technique uses laser wavelengths in the infrared to reduce scattering and eliminate out-of-focus fluorophore excitation. Coherent Anti-Stokes Raman Scattering (CARS) is a label-free imaging method. Ground State Depletion (GSD) technology produces 2D and 3D super-resolution images with a precise localization microscopy method. Multiple wavelengths can be freely selected to optimize the simultaneous excitation of dye combinations with reduced cross-excitation. A data set of spatially ordered optical sections that represent a 3D volume. WLLs emit a continuous spectrum of light between the range of 470 nm to 670 nm. The specimen is generally moved using motorized stages with high precision. 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. GSD works directly on standard fluorophores to achieve super-resolution images with a resolution down to 20 nm. 13 . Supercontinuum lasers. A tunable beam splitter. compromising the sectioning performance in comparison to true confocal scanning (single point scanning) to increase frame rate. also know as White Light Lasers (WLLs). Based on the localization microscopy technique GSDIM (Ground State Depletion microscopy followed by Individual Molecule return). which rejects light originating from regions that are out of focus. A widefield fluorescence microscope is a conventional type of microscope in which the full field is illuminated and imaged. Widefield. CARS allows the analysis of samples based on intrinsic vibrational contrast with specificity to molecular bonds within the sample. such as an AOBS. one that excites and the other that de-excites fluorochromes. Applications range from whole brain imaging to imaging real-time processes within organs. SIM. A drawback of widefield microscopy is that fluorescence emitted by the specimen above and below the focal plane interferes with the resolution. It differs from a confocal. PALM. Multiphoton microscopy enables researchers to image deep into thick samples. 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. STORM. are an alternative to conventional lasers used in confocal microscopy. complements the tunable output of a WLL. A localization-based super-resolution method that uses photoactivatable fluorophore constructs to stochastically achieve super-resolution images with a resolution down to 20 nm. Z-stack. STED. Multiphoton. METHODS & PLATFORMS CARS. which laser-scans and images the specimen point by point. Since no fluorophores are required. labeling steps can be bypassed to image the sample in a minimally invasive manner. Spinning disk systems use many pinholes arranged on rotating disks (or equivalent arrangements). GSD. A super-resolution method that uses combined activator/reporter dye pairs to stochastically achieve super-resolution images with a resolution down to 20 nm. The geometry of the confocal pinhole determines the diffraction pattern in the intermediate image plane. Employing two different diffraction patterns. STimulated Emission Depletion Microscopy (STED) is a method that resolves structures below optical resolution and is therefore attributed to super-resolution.Square pinhole. An approach for imaging a large specimen by assembling many single images to form one large composite image. Spinning disk. Tile scanning. The square pinhole is beneficial because it improves spectral separation by reducing the overlap of colors along a linear detection axis.

a demo is to determine whether you can fully • Request a demo. The clearer you can state those goals. do some digging to find another researcher who works with similar applications. and explain your specific needs. • Make your decision! After you’ve struck a deal. Talk about your sample material. desired frame rate. Contact the manufacturer’s support team to answer any questions that you have. Contact the sales representative for the instruments you’re considering. how many channels. the better the rep can provide you with the right solution. 14 complete your experiments to your satisfaction. desire for 3D images. Ask each company to fulfill the same request so that you can compare options with the greatest ease. 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. The goal of questions and learn more. perform working demonstrations with a variety of technologies. etc. become the expert on your new system. how many colors you’ll use. Your next steps: • Reach out to a company salesperson. . and get active in the community of users.Step 6. Tell the sales rep exactly what you need for the experiment you’d like to perform using the confocal microscope. Make a Confident Decision Now that you understand your needs and have evaluated the options. Don’t forget to try out the software. Before making a confocal purchase. the science is really what you do with the image after it’s been acquired. and use your pending purchase as an excuse to make a new connection in your field. Formally express your interest in the brand and set an appointment to ask TIP: Be specific or be sorry. The rep should be able to configure a system and tell you a preliminary price before the demo.

If you will be performing live cell imaging and require advanced spectral capabilities. allowing incredible new discoveries. The lowest cost models have limited upgrade flexibility. Schedule an appointment to ask questions and learn more. • W hat happens after the purchase? You will want help to get up and running. What do they love about their confocal microscope? What do they wish it could do? • Reach out to a company salesperson. Step 3 Budget Your Purchase: A Holistic View Prioritize features based on your most common application needs. • Talk to colleagues. • Understand the benefits of future upgrades to see how the microscope can grow with your research needs. 15 . (See full glossary in Buyer’s Guide for more. you’ll want to feel confident in the brand you select. • Explore the service options and types of contracts. •C  onfigurable models. • A re the in-house service experts qualified? Seek accessible and knowledgeable application support personnel. but can still produce spectacular images. Step 4 Learn About Reputation & Support As with any significant purchase. and non-descanned detection. 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. The most common upgrades are excitation sources (UV lasers. as well as those in years to come. and on what scale? Step 2 Understand the Options • Basic models. Use this checklist to evaluate your options and guide your purchasing decision. GaAsP photocathode. Get your hands on the systems before buying and test your own samples. this is the right choice.) Step 6 Make a Confident Decision Do your research and make an informed decision. workflow improvements. • Pay attention to maintenance. IR lasers). Step 5 Know the Terms Make sure that you are up to speed on common microscopy terms and how they relate to your purchase. • Request a demo.Confocal Microscope Buyer's Guide Checklist: Images captured with today’s confocal microscopes go well beyond what can be seen through a conventional microscope. and detection systems. • Is field support available? Find out how long it may take to get support when needed. These models allow greater flexibility for simultaneous multicolor imaging. scanners (resonant and tandem). which offer any combination of options such as spectral. and sample prep costs. both virtual and on-site. • Upgrades. Step 1 Evaluate Your Research Needs—Present and Future Evaluate your current research needs. Copyright © by Leica Microsystems 2014 .www.