3.3E: Confocal Micropscopy - Biology

3.3E: Confocal Micropscopy - Biology

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Learning Objectives

  • Compare and contrast confocal and fluorescence microscopy

Confocal microscopy is a non-invasive fluorescent imaging technique that uses lasers of various colors to scan across a specimen with the aid of scanning mirrors. The point of illumination is brought to focus in the specimen by the objective lens. The scanning process uses a device that is under computer control. The sequences of points of light from the specimen are detected by a photomultiplier tube through a pinhole. The output is built into an image and transferred onto a digital computer screen for further analysis. The technique employs optical sectioning to take serial slices of the image. The slices are then stacked (Z-stack) to reconstruct the three-dimensional image of the biological sample. Optical sectioning is useful in determining cellular localization of targets. The biological sample to be studied is stained with antibodies chemically bound to fluorescent dyes similar to the method employed in fluorescence microscopy. Unlike in conventional fluorescence microscopy where the fluorescence is emitted along the entire illuminated cone creating a hazy image, in confocal microscopy the pinhole is added to allow passing of light that comes from a specific focal point on the sample and not the other. The light detected creates an image that is in focus with the original sample. Confocal microscopy has multiple applications in microbiology such as the study of biofilms and antibiotic-resistant strains of bacteria. Development of modern confocal microscopes has been accelerated by new advances in computer and storage technology, laser systems, detectors, interference filters, and fluorophores for highly specific targets.

Key Points

  • Confocal microscopy requires immunoflurescence staining of biological samples.
  • Confocal microscopy serves to control depth of field, eliminate background, and collect optical sections.
  • The use of confocal microscopy has expanded to study both fixed and live cells with the ability to quantify targets.

Key Terms

  • photomultiplier tube: A vacuum tube that detects ultraviolet, visible, and near infrared light and multiplies it 100 million times.

NMR Microscopy

Michal Neeman , Laurel O. Sillerud , in NMR in Physiology and Biomedicine , 1994


The main attractions of NMR microscopy are its noninvasiveness, the ability to probe inner layers of opaque samples that cannot be approached by confocal microscopy , and its sensitivity to unique parameters such as diffusion, perfusion, flow, and metabolism. Resolution of NMR microscopy is at the lower end of the optical microscopic methods, with the best images obtained so far showing in plane resolution of around 5 μm. In this section we look at the current limitations to image resolution and possible avenues for breakthrough.

Spatial encoding in NMR microscopy is obtained by tagging spins with a position-dependent phase shift through the application of time-dependent orthogonal magnetic field gradients. In contrast with light and electron microscopy, resolution is not related to the wavelength of the electromagnetic radiation. Resolution depends on the smallest phase shift that can be measured. However, the phase shift difference between two points depends on the applied magnetic field gradient. Thus, by increasing the magnetic field gradient it is possible to improve the spatial resolution.

Is there a fundamental limit to spatial resolution in NMR microscopy? Sensitivity is the dominant practical limit to spatial resolution. Resolution loss due to line broadening by relaxation, bulk susceptibility, and diffusion imposes a more fundamental limit to resolution but occurs slower than the irreversible loss of signal due to reduction in pixel size and phase dispersion by diffusion in the presence of large field gradients ( Ahn and Cho, 1989 Cho et al., 1988 McFarland, 1992 Callaghan and Eccles, 1987 , 1988 Callaghan, 1990 ). The rate of signal loss depends heavily on the experimental pulse sequence and is generally higher for frequency encoding than for phase encoding. Thus 3D phase encoding is probably the most sensitive method that promises the highest spatial resolution albeit at the cost of longer acquisition times.

Calculations of the fundamental diffusion limit to resolution predicted a value of about 10 μm for free water ( Callaghan and Eccles, 1987 ). This prediction ignored the obvious resolution enhancement due to hindered or restricted diffusion in most cases in which resolution has any practical meaning. This important point has been raised in a number of studies ( Hyslop and Lauterbur, 1991 Zawodzinski et al., 1992 ). In biological and medical applications we are interested in resolving structures that are physically “different.” That would imply a change in relaxation, diffusion, or chemical composition, or a presence of a membrane between the two structures. Any of these changes will immediately affect the image in a way that will enhance the distinction between the two structures. In addition, intracellular water has diffusion coefficients significantly lower than those of free water reducing the effect of diffusion “smearing” even more.

Since sensitivity is the dominant practical limitation to spatial resolution, we can see a trend to higher magnetic fields. The gain in resolution due to the increase in signal intensity is somewhat lost by the increased magnetic susceptibility. This line-broadening effect can be partially overcome by application of higher magnetic field gradients at the expense of further dephasing and signal attenuation due to molecular diffusion.

The application of high magnetic fields in conjunction with small sample volumes and small radiofrequency coils implies that the noise due to the receiving coil and the electronics dominates, in contrast to the large sample noise in clinical imaging. Thus a significant improvement in signal to noise can be obtained by reducing the noise due to the rf coil. An excellent example for a possible breakthrough in this direction may be in the application of cool high-temperature superconducting rf coils. This approach has already demonstrated a 10-fold improvement over conventional room-temperature copper coils ( Black et al., 1993 ).

Image resolution is also limited by the ability to resolve structures with sufficient contrast. In that sense NMR provides us with a wide array of possible contrast mechanisms. Contrast due to variations in spin density and due to T1 and T2 relaxation affects NMR microscopy in a manner similar to their effects in conventional MRI and are not discussed here. Due to the large magnetic field gradients used in NMR microscopy and the large variations in motional freedom of biological systems, diffusion is a dominant contrast mechanism and is dealt with in detail in the next section. The gradients used in most of the NMR microscopy studies of 10–50 G/cm cause significant attenuation of signal intensity in regions of free water, such as the interstitial space and vacuoles. Regions with slow water diffusion, on the other hand, appear bright. It should be noted that the routine method for T2-weighted imaging, namely long TE, results frequently also in increased diffusion weighting. The effects of T2 and diffusion can be opposite and care must be taken to prevent loss of contrast due to signal attenuation of immobile species by T2 and loss of signal from mobile species by diffusion.

We felt that the sensitivity of the NMR signal to molecular diffusion offers a unique opportunity to study systems in which diffusion plays a vital role. For that end we looked in detail at the effects of diffusion on NMR signal intensity in single and multicompartment systems and in systems with anisotropic diffusion.

Microscopy and Imaging Facility

Located on the first floor of Langley Hall, the department's Microscopy and Imaging Facility is equipped with state-of-the-art facilities for light, confocal, and electron microscopy digital image and video manipulation and large-format printing. The suite houses both scanning and transmission electron microscopes as well as a complete system for laser scanning confocal microscopy with 3D image reconstruction using a graphics workstation. This facility is staffed by a full-time microscopist, Mr. Tom Harper, to assist students and researchers with their projects and is located on the first floor of Langley Hall.

The Leica TCS SP5 confocal and multi-photon microscope is a state-of-the-art laser scanning microscope that features broad band wavelength tuning of emission and detection wavelengths, as well as high scan speeds and multi-photon imaging. The mciroscope is ideal for live specimens and deeper fluorescent imaging.

The Olympus FV1000 Fluoview laser scanning biological microscope is a highly evolved confocal platform that utilizes an array of stable and cool diode lasers, multiple channels of simultaneous dye imaging, and quality optics. The microscope provides excellent resolution, high sensitivity and accurate quantification.

Department of Biology

The UNCG confocal microscopy facility is located in SSB 353. The facility provides access to confocal microscopy equipment and image processing resources. Access is on a fee for use basis.

This confocal microscope was purchased and supported by grant DBI-0319021 from the National Science Foundation, grant 2003-IDG-1011 from the North Carolina Biotechnology Center, and by funds from UNCG Department of Biology and Office of the Provost.

Facility Resources

Available Objectives:
Position 1: UPlanApo 10x/0.40 infinity 8/0.17
Position 2: UPlanApo 20x/0.70 infinty 8/0.17
Position 3: UPlanFL 40x/1.30 Oil infinity 8/0.17
Position 4: PlanApo 60x/1.40 Oil infinity 8/0.17
Position 5: Open
Position 6: UPlanApo 40x/0.85 infinity 8 0.11-0.23
Available for use upon request:
UplanApo 60x/1.20 Water infinity 8 0.13-0.21

UIS Fluorescence Mirror units:

Available lasers:
Multi-line Argon laser (457nm, 488nm, 514nm)
Green Helium Neon laser (543nm)
Red Helium Neon laser (633nm)
Blue Diode laser (405nm)

Supplemental Analysis Software:
Olympus Microsuite FIVE

A separate imaging workstation with Fluoview review software and Microsuite software is available for use.


Sign up policies for internal and extramural users:
All policies are subject to change without notice.

All extramural users are required to make payment arrangements, booking arrangements and submit a usage agreement prior to their first session. All extramural user time must be negotiated prior to their first booking.

The confocal committee will negotiate all usage problems.

Internal and extramural users may sign up for available time slots a maximum of 48 hours prior to use. Sign up for a time slot by contacting the facility manager with your request. If you are unable to utilize your reserved time please inform the facility manager immediately so you can be removed from the schedule.

When booking available time slots please include your name, your PI name (if applicable) and a telephone number where you can be reached to confirm your appointment.

All students and Researchers must complete a training session(s) specific to our facility to be approved to use the facility, in addition to acquiring approval from the facility manager prior to requesting time.

Facility hours of operation are Monday-Friday 8:00 AM – 5:00 PM. The facility is closed during all of the recognized university holidays. After hours access to the facility is available to approved users only.

UNCG Faculty and all Extramural Researchers are fully responsible for their students and or technical staff using the facility.

Please report any mechanical or technical issues with facility equipment to the facility manager immediately via voice mail. This applies to all emergencies occurring at anytime. Dial (336) 256-0574 (6-0574)

Some general confocal facility Do’s & Don’ts that should be practiced:

Do not touch anything on the shelf the multi line laser is sitting on
Do cover the scope after use! Do not cover the epifluorescent unit until it is completely cool!
Do not change any instrument settings in the software that will not be reset to the default value after closing the program
Do be careful with the use of immersion oil. The 40x & 60x located in positions 3 & 4 on the nosepiece are both oil immersion objectives. The 40x in position 6 is a high dry.
Do leave the multi line argon laser cooling fan on. This will cut itself off after 3 minutes. Then the toggle switch may be turned off.
Please leave the facility as you found it. Clean and Straight

If you are unsure about the proper use of any of the equipment in the confocal microscopy facility please ask some one before you make a costly assumption.

Facility resource fees

All outside users must make arrangements for scheduling, billing and payment prior to using the facility.
All fees are charged on a per quarter hour basis.
The fees listed below are charged to UNCG user accounts quarterly as of 01/01/2010

User Hourly Rate To include:
UNCG Internal $30.00 Limited technical instruction and supervision
UNCG Internal $60.00 Technical assistance
Extramural user $110.00 Limited technical instruction and supervision
Extramural user $170.00 Technical assistance

Resource booking

If you are an internal user interested in booking time please contact the facility manager to do so.

When booking available time slots through Calcium please include your name, your PI name (if applicable) and a phone number where you can be reached to confirm your appointment.

Facility contacts

Head of Confocal Committee: John Tomkiel Dean, Ph.D.

Technical Staff / Facility Manager / Emergency Contact
Lee Griffin
Office Phone (336) 334-4976

3.3E: Confocal Micropscopy - Biology

Confocal microscopy offers several advantages over conventional optical microscopy, including controllable depth of field, the elimination of image degrading out-of-focus information, and the ability to collect serial optical sections from thick specimens. The key to the confocal approach is the use of spatial filtering to eliminate out-of-focus light or flare in specimens that are thicker than the plane of focus. There has been a tremendous explosion in the popularity of confocal microscopy in recent years, due in part to the relative ease with which extremely high-quality images can be obtained from specimens prepared for conventional optical microscopy, and in its great number of applications in many areas of current research interest. Visit the Molecular Expressions and Nikon MicroscopyU articles, galleries, interactive tutorials, and Web references using the links provided below.

Laser Scanning Confocal Microscope Simulator - Perhaps the most significant advance in optical microscopy during the past decade has been the refinement of mainstream laser scanning confocal microscope ( LSCM ) techniques using improved synthetic fluorescent probes and genetically engineered proteins, a wider spectrum of laser light sources coupled to highly accurate acousto-optic tunable filter control, and the combination of more advanced software packages with modern high-performance computers. This interactive tutorial explores multi-laser fluorescence and differential interference contrast ( DIC ) confocal imaging using the Olympus FluoView FV1000 confocal microscope software interface as a model.

Introduction to Confocal Microscopy - Confocal microscopy offers several advantages over conventional widefield optical microscopy, including the ability to control depth of field, elimination or reduction of background information away from the focal plane (that leads to image degradation), and the capability to collect serial optical sections from thick specimens. The basic key to the confocal approach is the use of spatial filtering techniques to eliminate out-of-focus light or glare in specimens whose thickness exceeds the immediate plane of focus. There has been a tremendous explosion in the popularity of confocal microscopy in recent years, due in part to the relative ease with which extremely high-quality images can be obtained from specimens prepared for conventional fluorescence microscopy, and the growing number of applications in cell biology that rely on imaging both fixed and living cells and tissues. In fact, confocal technology is proving to be one of the most important advances ever achieved in optical microscopy.

Basic Concepts - Current instruments are highly evolved from the earliest versions, but the principle of confocal imaging advanced by Marvin Minsky, and patented in 1957, is employed in all modern confocal microscopes. In a conventional widefield microscope, the entire specimen is bathed in light from a mercury or xenon source, and the image can be viewed directly by eye or projected onto an image capture device or photographic film. In contrast, the method of image formation in a confocal microscope is fundamentally different. Illumination is achieved by scanning one or more focused beams of light, usually from a laser or arc-discharge source, across the specimen. This point of illumination is brought to focus in the specimen by the objective lens, and laterally scanned using some form of scanning device under computer control. The sequences of points of light from the specimen are detected by a photomultiplier tube ( PMT ) through a pinhole (or in some cases, a slit), and the output from the PMT is built into an image and displayed by the computer. Although unstained specimens can be viewed using light reflected back from the specimen, they usually are labeled with one or more fluorescent probes.

Imaging Modes - A number of different imaging modes are used in the application of confocal microscopy to a vast variety of specimen types. They all rely on the ability of the technique to produce high-resolution images, termed optical sections , in sequence through relatively thick sections or whole-mount specimens. Based on the optical section as the basic image unit, data can be collected from fixed and stained specimens in single, double, triple, or multiple-wavelength illumination modes, and the images collected with the various illumination and labeling strategies will be in register with each other. Live cell imaging and time-lapse sequences are possible, and digital image processing methods applied to sequences of images allow z-series and three-dimensional representation of specimens, as well as the time-sequence presentation of 3D data as four-dimensional imaging. Reflected light imaging was the mode used in early confocal instruments, but any of the transmitted light imaging modes commonly employed in microscopy can be utilized in the laser scanning confocal microscope.

Specimen Preparation and Imaging - The procedures for preparing and imaging specimens in the confocal microscope are largely derived from those that have been developed over many years for use with the conventional wide field microscope. In the biomedical sciences, a major application of confocal microscopy involves imaging either fixed or living cells and tissues that have usually been labeled with one or more fluorescent probes. A large number of fluorescent probes are available that, when incorporated in relatively simple protocols, specifically stain certain cellular organelles and structures. Among the plethora of available probes are dyes that label nuclei, the Golgi apparatus, the endoplasmic reticulum, and mitochondria, and also dyes such as fluorescently labeled phalloidins that target polymerized actin in cells. Regardless of the specimen preparation protocol employed, a primary benefit of the manner in which confocal microscopy is carried out is the flexibility in image display and analysis that results from the simultaneous collection of multiple images, in digital form, into a computer.

Fluorophores for Confocal Microscopy - Biological laser scanning confocal microscopy relies heavily on fluorescence as an imaging mode, primarily due to the high degree of sensitivity afforded by the technique coupled with the ability to specifically target structural components and dynamic processes in chemically fixed as well as living cells and tissues. Many fluorescent probes are constructed around synthetic aromatic organic chemicals designed to bind with a biological macromolecule (for example, a protein or nucleic acid) or to localize within a specific structural region, such as the cytoskeleton, mitochondria, Golgi apparatus, endoplasmic reticulum, and nucleus. Other probes are employed to monitor dynamic processes and localized environmental variables, including concentrations of inorganic metallic ions, pH, reactive oxygen species, and membrane potential. Fluorescent dyes are also useful in monitoring cellular integrity (live versus dead and apoptosis), endocytosis, exocytosis, membrane fluidity, protein trafficking, signal transduction, and enzymatic activity. In addition, fluorescent probes have been widely applied to genetic mapping and chromosome analysis in the field of molecular genetics.

Spectral Bleed-Through Artifacts in Confocal Microscopy - The spectral bleed-through of fluorescence emission (often termed crossover or crosstalk ), which occurs due to the very broad bandwidths and asymmetrical spectral profiles exhibited by many of the common fluorophores, is a fundamental problem that must be addressed in both widefield and laser scanning confocal fluorescence microscopy. The phenomenon is usually manifested by the emission of one fluorophore being detected in the photomultiplier channel or through the filter combination reserved for a second fluorophore. Bleed-through artifacts often complicate the interpretation of experimental results, particularly if subcellular colocalization of fluorophores is under investigation or quantitative measurements are necessary, such as in resonance energy transfer ( FRET ) and photobleaching ( FRAP ) studies.

Choosing Fluorophore Combinations for Confocal Microscopy - In planning multiple label fluorescence staining protocols for widefield and laser scanning confocal fluorescence microscopy experiments, the judicious choice of probes is paramount in obtaining the best target signal while simultaneously minimizing bleed-through artifacts. This interactive tutorial is designed to explore the matching of dual fluorophores with efficient laser excitation lines, calculation of emission spectral overlap values, and determination of the approximate bleed-through level that can be expected as a function of the detection window wavelength profiles.

Laser Systems for Optical Microscopy - The lasers commonly employed in optical microscopy are high-intensity monochromatic light sources, which are useful as tools for a variety of techniques including optical trapping, lifetime imaging studies, photobleaching recovery, and total internal reflection fluorescence. In addition, lasers are also the most common light source for scanning confocal fluorescence microscopy, and have been utilized, although less frequently, in conventional widefield fluorescence investigations.

Laser Safety - The two major concerns in safe laser operation are exposure to the beam and the electrical hazards associated with high voltages within the laser and its power supply. While there are no known cases of a laser beam contributing to a person's death, there have been several instances of deaths attributable to contact with high voltage laser-related components. Beams of sufficiently high power can burn the skin, or in some cases create a hazard by burning or damaging other materials, but the primary concern with regard to the laser beam is potential damage to the eyes, which are the part of the body most sensitive to light.

Acousto-Optic Tunable Filters (AOTFs) - Several benefits of the AOTF combine to greatly enhance the versatility of the latest generation of confocal instruments, and these devices are becoming increasing popular for control of excitation wavelength ranges and intensity. The primary characteristic that facilitates nearly every advantage of the AOTF is its capability to allow the microscopist control of the intensity and/or illumination wavelength on a pixel-by-pixel basis while maintaining a high scan rate. This single feature translates into a wide variety of useful analytical microscopy tools, which are even further enhanced in flexibility when laser illumination is employed.

Resolution and Contrast in Confocal Microscopy - All optical microscopes, including conventional widefield, confocal, and two-photon instruments are limited by fundamental physical factors in the resolution that they can achieve. In a perfect optical system, resolution is limited by numerical aperture of the optical components and by the wavelength of the light, both incident and detected. The concept of resolution is inseparable from contrast, and is defined as the minimum separation between two points that results in a certain contrast between them. In a real fluorescence microscope, contrast is determined by the number of photons collected from the specimen, the dynamic range of the signal, optical aberrations of the imaging system, and the number of picture elements ( pixels ) per unit area.

Non-Coherent Light Sources for Confocal Microscopy - The traditional illumination system in the modern widefield microscope utilizes a tungsten-halogen source for transmitted light and a short-arc lamp for fluorescence excitation. Various lasers have been utilized as a light source for widefield observation by a few investigators, but the advent of the confocal microscope vastly increased laser use in microscopy. This discussion reviews the merits and limitations of non-coherent (or non-laser) light sources in confocal microscopy, both as light sources for confocal illumination and as secondary sources for widefield microscopy in confocal microscopes. Two initial issues frequently arise when illumination systems for confocal microscopes are considered, and these have a direct bearing on the choice of light sources for a particular instrument.

Confocal Microscope Objectives - For any conventional optical microscope configuration, the objective is the most critical component of the system in determining the information content of the image. The contrast and resolution of fine specimen detail, the depth within the specimen from which information can be obtained, and the lateral extent of the image field are all determined by the design of the objective and its performance under the specific conditions employed for the observation. Additional demands are imposed on the objective in scanning confocal techniques, in which this crucial imaging component also serves as the illumination condenser and is often required to perform with high precision at a wide range of wavelengths and at very low light levels without introducing unacceptable image-degrading noise.

Confocal Microscope Scanning Systems - Confocal imaging relies upon the sequential collection of light from spatially filtered individual specimen points, followed by electronic signal processing and ultimately, the visual display as corresponding image points. The point-by-point signal collection process requires a mechanism for scanning the focused illuminating beam through the specimen volume under observation. Three principal scanning variations are commonly employed to produce confocal microscope images. Fundamentally equivalent confocal operation can be achieved by employing a laterally translating specimen stage coupled to a stationary illuminating light beam ( stage scanning ), a scanned light beam with a stationary stage ( beam scanning ), or by maintaining both the stage and light source stationary while scanning the specimen with an array of light points transmitted through apertures in a spinning Nipkow disk . Each technique has performance features that make it advantageous for specific confocal applications, but that limit the usefulness in others.

Signal-to-Noise Considerations - In any quantitative assessment of imaging capabilities utilizing digital microscopy techniques, including confocal methods, the effect of signal sampling on contrast and resolution must be considered. The measured signal level values do not directly represent the number of photons emitted or scattered by the specimen, but are proportional to that number. Furthermore, each individual sample of signal intensity is only an approximation of the number of collected photons, and will vary with repeated measurement. The variation, referred to as noise , imparts an uncertainty in the quantification of intensity, and therefore in the contrast and resolution of the image data.

Electronic Light Detectors: Photomultipliers - In modern widefield fluorescence and laser scanning confocal optical microscopy, the collection and measurement of secondary emission gathered by the objective can be accomplished by several classes of photosensitive detectors, including photomultipliers, photodiodes, and solid-state charge-coupled devices ( CCDs ). In confocal microscopy, fluorescence emission is directed through a pinhole aperture positioned near the image plane to exclude light from fluorescent structures located away from the objective focal plane, thus reducing the amount of light available for image formation. As a result, the exceedingly low light levels most often encountered in confocal microscopy necessitate the use of highly sensitive photon detectors that do not require spatial discrimination, but instead respond very quickly with a high level of sensitivity to a continuous flux of varying light intensity.

Critical Aspects of Confocal Microscopy - Quantitative three-dimensional imaging in fluorescence microscopy is often complicated by artifacts due to specimen preparation, controllable and uncontrollable experimental variables, or configuration problems with the microscope. This article, written by Dr. James B. Pawley, catalogs the most common extraneous factors that often serve to obscure results collected in fluorescence widefield and confocal microscopy. Among the topics discussed are the laser system, optical component alignment, objective magnification, bleaching artifacts, aberrations, immersion oil, coverslip thickness, quantum efficiency, and the specimen embedding medium.

Aberrations in Multicolor Confocal Microscopy - Refinements in design have simplified confocal microscopy to the extent that it has become a standard research tool in cell biology. However, as confocal microscopes have become more powerful, they have also become more demanding of their optical components. In fact, optical aberrations that cause subtle defects in image quality in widefield microscopy can have devastating effects in confocal microscopy. Unfortunately, the exacting optical requirements of confocal microscopy are often hidden by the optical system that guarantees a sharp image, even when the microscope is performing poorly. Optics manufacturers provide a wide range of microscope objectives, each designed for specific applications. This report demonstrates how the trade-offs involved in objective design can affect confocal microscopy.

Three-Color Imaging for Confocal Microscopy - The laser scanning confocal microscope ( LSCM ) is routinely used to produce digital images of single-, double-, and triple-labeled fluorescent samples. The use of red, green and blue ( RGB ) color is most informative for displaying the distribution of up to three fluorescent probes labeling a cell, where any colocalization is observed as a different additive color when the images are colorized and merged into a single three-color image. In this section we present a simplified version of a previously published method for producing three-color confocal images using the popular image manipulation program, Adobe Photoshop. In addition, several applications of the three-color merging protocol for displaying confocal images are discussed. Note that these digital methods are not confined to images produced using the LSCM and can be applied to digital images imported into Photoshop from many different sources.

Basics of Confocal Reflection Microscopy - Confocal reflection microscopy can be utilized to gather additional information from a specimen with relatively little extra effort, since the technique requires minimum specimen preparation and instrument re-configuration. In addition, information from unstained tissues is readily available with confocal reflection microscopy, as is data from tissues labeled with probes that reflect light. The method can also be utilized in combination with more common classical fluorescence techniques. Examples of the latter application are detection of unlabeled cells in a population of fluorescently labeled cells and for imaging the interactions between fluorescently labeled cells growing on opaque, patterned substrata.

Applications in Confocal Microscopy - The broad range of applications available to laser scanning confocal microscopy includes a wide variety of studies in neuroanatomy and neurophysiology, as well as morphological studies of a wide spectrum of cells and tissues. In addition, the growing use of new fluorescent proteins is rapidly expanding the number of original research reports coupling these useful tools to modern microscopic investigations. Other applications include resonance energy transfer, stem cell research, photobleaching studies, lifetime imaging, multiphoton microscopy, total internal reflection, DNA hybridization, membrane and ion probes, bioluminescent proteins, and epitope tagging. Many of these powerful techniques are described in these reviews.

Confocal Microscopy Image Gallery - The Nikon MicroscopyU Confocal Image Gallery features digital image sequences captured using a Nikon PCM-2000 confocal microscope scanning system coupled to an Eclipse E-600 upright microscope. Successive serial optical sections were recorded along the optical axis of the microscope over a range of specimen planes. These sequences are presented as interactive Java tutorials that allow the visitor to either "play" the series of sections automatically, or to utilize a slider to scroll back and forth through the images.

Olympus FluoView Laser Scanning Confocal Microscopy - The new Olympus FluoView TM FV1000 is the latest in point-scanning, point-detection, confocal laser scanning microscopes designed for today's intensive and demanding biological research investigations. Excellent resolution, bright and crisp optics, and high efficiency of excitation, coupled to an intuitive user interface and affordability are key characteristics of this state-of-the-art optical microscopy system.

Marvin Lee Minsky (1927-Present) - While at Harvard University, Marvin Minsky made his primary contribution to the field of optics by inventing the confocal scanning microscope. Despite the theoretical benefits of the confocal approach for biological purposes, Minsky's microscope originally generated little interest. In hindsight it has become apparent that the technology of the period limited Minsky's demonstration of the potential of the confocal approach. Yet, years later, with the advent of such applicable devices as lasers, sensitive low-noise photodetectors, and fast microcomputers with image processing capabilities, Minsky's microscopy technique has become widespread in biological research.

Interactive Java Tutorials

Laser Scanning Confocal Microscopy - (approximately a 30 second download on 28.8K modems) Several methods have been developed to overcome the poor contrast inherent with imaging thick specimens in a conventional microscope. Specimens having a moderate degree of thickness (5 to 15 microns) will produce dramatically improved images with either with confocal or deconvolution techniques. The thickest specimens (20 microns and above) will suffer from a tremendous amount of extraneous light in out-of-focus regions, and are probably best-imaged using confocal techniques. This tutorial explores imaging specimens through serial z-axis optical sections utilizing a virtual confocal microscope.

Comparing Confocal and Widefield Fluorescence Microscopy - Confocal microscopy offers several distinct advantages over traditional widefield fluorescence microscopy, including the ability to control depth of field, elimination or reduction of background information away from the focal plane (that leads to image degradation), and the capability to collect serial optical sections from thick specimens. The basic key to the confocal approach is the use of spatial filtering techniques to eliminate out-of-focus light or glare in specimens whose thickness exceeds the dimensions of the focal plane. This interactive tutorial explores and compares the differences between specimens when viewed in a confocal versus a widefield fluorescence microscope.

Colocalization of Fluorophores in Confocal Microscopy - Two or more fluorescence emission signals can often overlap in digital images recorded by confocal microscopy due to their close proximity within the specimen. This effect is known as colocalization and usually occurs when fluorescently labeled molecules bind to targets that lie in very close or identical spatial positions. This interactive tutorial explores the quantitative analysis of colocalization in a wide spectrum of specimens that were specifically designed either to demonstrate the phenomenon, or to alternatively provide examples of fluorophore targets that lack any significant degree of colocalization.

Reflected Confocal Microscopy: Integrated Circuit Inspection - Examine individual layers on the surface of integrated circuits with this interactive tutorial. Digital images for the tutorial were collected with a Nikon Optiphot C200 reflected light confocal microscope. For each sequence, a series of z-axis optical sections was recorded as the microscope was successively focused (at 1-micrometer steps) deeper within the patchwork of circuitry on the surface of the silicon chips.

Excitation Photobleaching Patterns - Multiphoton fluorescence microscopy utilizes diffraction-limited focusing by a high numerical aperture objective to localize the spatial concentration of excitation light to narrow region near the focal point. In contrast, the excitation region of a laser scanning confocal microscope is similar to that of a widefield microscope. This tutorial compares excitation-induced photobleaching patterns that occur near the focal region in both multiphoton and confocal microscopy systems.

Olympus FluoView Resource Center Interactive Java Tutorials - Explanations for many of the exceedingly complex concepts in laser scanning confocal microscopy can significantly benefit from the assistance of interactive tutorials that enable the student to obtain instanteous (real-time) response to changes in variables. The tutorials in section address the basic aspects of confocal microscopy instrumentation, laser systems, detectors, image processing, resolution, contrast, and many other aspects of the technique. All interactive Java tutorials require the Java Virtual Machine, which is available without cost as a browser plug-in from Sun Microsystems.

References and Resources

Recommended Books on Confocal Microscopy - A surprisingly limited number of books dealing with various aspects of laser scanning and spinning disk confocal microscopy and related techniques are currently available from the booksellers. This section lists the FluoView Resource Center website development team's top 12 recommended books. Although the volumes listed in this section deal pricipally with confocal microscopy and related methodology, there exist a number of additional books that contain focused treatments of the materials described below, and these should also be consulted for specific techniques and timely review articles.

ZEISS Campus Confocal Microscopy Reference Library - A majority of the literature pertaining to review articles on laser scanning confocal microscopy has been published in textbooks, edited article collections, and symposia, with only an intermittent sprinkling of papers in the scientific journals. The reviews listed in this section should be available to students and investigators who have access to subscriptions through their host institutions.

Confocal Microscopy Web Resources - Laser scanning confocal microscopy ( LSCM ), a tool that has been extensively utilized for inspection of semiconductors, is now becoming a mainstream application in cell biology. The links provided in this section from the Molecular Expressions web site offer tutorials, instrumentation, application notes, technical support, glossaries, and reference materials on confocal microscopy and related techniques.

Basic Concepts in Laser Scanning Confocal Microscopy (PDF 2.8 Mb) - Laser scanning confocal microscopy has become an invaluable tool for a wide range of investigations in the biological and medical sciences for imaging thin optical sections in living and fixed specimens ranging in thickness up to 100 micrometers. Modern instruments are equipped with 3-5 laser systems controlled by high-speed acousto-optic tunable filters (AOTFs), which allow very precise regulation of wavelength and excitation intensity. Coupled with photomultipliers that have high quantum efficiency in the near-ultraviolet, visible and near-infrared spectral regions, these microscopes are capable of examining fluorescence emission ranging from 400 to 750 nanometers. Download this review article to learn more.

Kenneth W. Dunn and Exing Wang - Department of Medicine, Indiana University, School of Medicine, 1120 South Drive, FH115, Indianapolis, Indiana 46202-5116.

John M. Murray - Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, 19104.

Stephen W. Paddock , Eric J. Hazen , and Peter J. DeVries - Laboratory of Molecular Biology, Howard Hughes Medical Institute, University of Wisconsin, Madison, Wisconsin 53706.

James B. Pawley - Department of Zoology, 1117 W. Johnson Dr., University of Wisconsin, Madison, Wisconsin 53706.

David W. Piston - Department of Molecular Physiology and Biophysics, Vanderbilt University, 702 Light Hall, Nashville, Tennessee, 37212.

Kenneth R. Spring - Scientific Consultant, Lusby, Maryland, 20657.

Matthew J. Parry-Hill , Thomas J. Fellers , and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.

Key Features

See More of the Specimen

Whole mouse bladder optically cleared with iDISCO and acquired at 8192 x 8192 pixels using a 2x Plan Apo objective, effective pixel size 0.6 μm (over 5x the spatial resolution of a typical monochrome CMOS camera).
Courtesy of Dr. Gerry Apodaca, Integrative Systems Biology, Department of Medicine, University of Pittsburgh in collaboration with Dr. Alan Watson at the Center for Biological Imaging, University of Pittsburgh.

With the largest field-of-view on both inverted and upright microscope stands available (25mm diagonal), more specimens fit in one FOV with more objective lens choices than ever before.

Coupled with scanning sizes up to 8192 x 8192 pixels, sampling beyond the optical diffraction limit is possible even at low magnifications with the AX/AX R.

Using lower magnifications with longer working distances and high numerical apertures enables more flexible specimen preparations to be used, while the large FOV allows simultaneous high resolution in one image. Collect more data in every image, and at faster rates.

The AX/AX R has a 25mm diagonal FOV, much larger than other confocal instruments.

Cleared adult mouse brain acquired with 1x objective in one acquisition of one FOV*

Drosophila sp. Embryo development easily fits within the FOV using a high NA 25x SIL 1.05 NA objective*

* It would not be possible to capture this sample in one FOV or at this resolution with other commercial confocal systems

Observe with Minimal Disturbance

Laser scanning confocal imaging is principally challenging on specimen viability, as it applies focused laser illumination point by point on a sample.

The AX R’s high speed resonant scanning, which decreases the illumination time by more than 20x typical confocal scanning times, greatly reduces biases caused by merely acquiring images. Reducing the acquisition time also allows for extremely high-speed imaging (up to 720 fps @ 2048 x 16).

The result: longer imaging and/or more frequent imaging at high speed of living samples which allows capture of dynamic events, but also allows longer time-lapse imaging or significantly faster collection times on fixed specimens.

Time-lapse Z series maximum intensity projection images of a developing Drosophila embryo expressing PLC-PH::GFP (PIP2) acquired every 10 minutes for 12 hours at 2K x 1K pixels using a 25x silicone immersion objective.
Courtesy of Yang Hong Laboratory, Department of Cell Biology, University of Pittsburgh in collaboration with the Center for Biological Imaging.

Acquire More Rapidly

Confocal imaging, notoriously slow because of its point-scanning requirement for high quality 3-dimensional imaging at high resolution, is greatly changed by fast imaging with the AX R’s resonant scanning capabilities.

Utilizing 2048 x2048 pixel resonant scanning and a 25mm FOV on a large intestinal sample montage, acquiring 25 high-resolution images and merging them in under 2 minutes.

Ultrafine Detail

With a full 25mm FOV, up to 8192 x 8192 pixels, and the capability for supravideo frame rates, the AX/AX R allows for spectacular imaging with high resolution, at both low and high magnifications.

The entire range of a whole organism or system biology down to intracellular imaging is achievable on one instrument.

Mouse muscle acquired with a 25x SIL immersion objective using 2048 x 2048 pixel resonant scanning

Detectors In Tune with Labels

Maximum intensity projection of Z stack images of marmoset brain acquired with a 60x 1.27 NA water immersion objective using 2048 x 2048 pixel resonant scanning and a DUX-VB detector with user-defined emission bands.

The AX/AX R’s all new DUX-VB detector custom-tunes emission bandwidths to a library of labels and probes, and provides the freedom to fine-tune emission bands to minimize unwanted fluorescence.

Simply select the number of labels in your specimen and their catalog names. Alternatively, you can define the desired emission ranges, or even simply the emission color: the AX/AX R and NIS-Elements software does the rest, including optimizing the dichroic mirror and laser excitation choices best suited for imaging. Or, acquire hyperspectral images in up to 66 emission channels for unmixing.

Optionally, the AX/AX R’s base DUX-ST detector allows up to 12 discreet bandpasses of emission, upgradable to 18.

And all detector systems can be customized with high sensitivity and low noise GaAsP or Multi-alkali PMT detectors to provide the best detector for sensitivity and wavelength response requirements as well as budgets.

Superior optical technologies to support all confocal applications

Nikon provides a broad range of high-NA objectives with unrivaled optical quality to redefine the boundaries of confocal imaging. Options include silicone oil immersion objectives for thick live cell imaging, large-FOV low-magnification objectives and easy-to-use dry objectives. Chromatic aberrations are corrected from ultraviolet to near infrared range, enabling excellent multicolor imaging.

Maximum intensity projection of Z stack images, color-coded by depth, of vascular development in embryonic zebrafish acquired with a 10X 0.45 NA Plan Apo Lambda S objective using 1024x2048 pixel resonant scanning. Courtesy of Erika Driekorn and Dr. Beth Roman, Department of Human Genetics, University of Pittsburgh Graduate School of Public Health.

Optional Components

Water Immersion Dispenser

A software-controlled, automatic water dispenser enables long-term time-lapse imaging using refractive-index matching water immersion objectives in any environment, including incubation.

Automatic Correction Collar

Moves the objective correction collar to the optimum position for best resolution both remotely and by software control. Motorized collars allow users to adjust the correction collar without disturbing the specimen position, even in incubated enclosures or environmental chambers.

Multiple Modalites

Ti2-LAPP Modular Illumination System

The Ti2-E microscope supports up to 5 episcopic illumination sources, which can be used in tandem with AX/AX R confocal imaging: total internal reflection fluorescence (TIRF), point, raster or field stimulation devices, and fluorescence light sources can all be integrated onto the same microscope stand, and used in the same experiments.

Total Internal Reflection Fluorescence (TIRF)

The incident angle of a laser and corresponding penetration depth of the evanescent field can be controlled via NIS-Elements software. When multiple TIRF modules are mounted, the penetration depth can be independently set for each wavelength.

Photostimulation: Point and Raster Scanner

The XY galvano scanning unit can stimulate the desired area of a sample using laser point scanning. It allows simultaneous photostimulation and confocal imaging.

Photostimulation: Digital Micromirror Device (DMD)

The DMD module enables photoactivation of user-specified patterns rather than photoactivation of a single spot. This allows stimulation of multiple points and tracking of their behavior. The DMD module can be used with either laser illumination or less phototoxic LED illumination.

Confocal Microscopy

In Confocal Microscopy Methods and Protocols, Stephen Paddock and a highly skilled panel of experts lead the researcher using confocal techniques from the bench top, through the imaging process, to the journal page. They concisely describe all the key stages of confocal imaging-from tissue sampling methods, through the staining process, to the manipulation, presentation, and publication of the realized image. Written in a user-friendly, nontechnical style, the methods specifically cover most of the commonly used model organisms: worms, sea urchins, flies, plants, yeast, frogs, and zebrafish.

Centered in the many biological applications of the confocal microscope, the book makes possible the successful imaging of both fixed and living specimens using primarily the laser scanning confocal microscope. The powerful hands-on methods collected in Confocal Microscopy Methods and Protocols will help even the novice to produce first-class cover-quality confocal images.

"This book is an essential reference for anyone who is involved in the production of laser scanning confocal (LSCM) images. . . Exceptionally useful information on a great variety of methodologies that can be employed with LSCM is provided. . . In consideration of the vast quantity of useful information on all aspects of LSCM that is presented in this volume, and the fact that it is the first comprehensive book on this topic, it is highly recommended."-Quarterly Review of Biology

". almost every research institute has a confocal microscope, and many are developing much larger multi-user biomedical imaging facilities. The publication of this collection of methods and protocols for confocal microscopy is therefore very timely. The tips and tricks used on the various systems should prove useful to a diverse range of confocal microscope users and would be a beneficial reference volume for any imaging unit and reasonable value for money."-Cell Biology International

" This is an excellent source for anyone who wants to explore techniques in confocal microscopy. It is a practical manual with many gems that will help both new and experienced users. The principles are efficiently and accessibly explained and there is a useful chapter on fluorescent probes. there is a wealth of ideas here waiting to be applied to prokaryotic biology and many would be equally useful for simple fluorescence rather than confocal studies. I would certainly recommend it to any lab that uses fluorescence microscopy extensively."-Microbiology Today

". The texts are aimed at the biological user. for the user for whom they are intended, these essays contain a wealth of down-to-earth practical information. The book is well-produced and contains colour illustrations. "-Ultramicroscopy

". excellent information as an introductory basis for confocal microscopy."-Cellular and Molecular Biology

  • Microscope stand: Nikon Ti Eclipse inverted
  • Features:
    • Motorized XY Stage, allowing mosaic images and multi-point time-lapse imaging
    • Fast live imaging with resonant scanner and piezoelectric focus
    • Spectral unmixing of overlapping fluorophores or autofluorescence
    • Autofocus using the Perfect Focus System
    • Tokai Hit stage-top incubator and objective heater
    • Targeted photobleaching, FRAP, FRET and FLIP capability
    • 2-photon microscopy with non-descanned detector: Best for deep (up to 1mm) or long-term imaging
    • Spectral fingerprinting, multiphoton excitation fingerprinting, unmixing of multiple fluorophores/autofluorescence sources
    • 1-photon: 458, 488, 514, 561, 639 nm
    • 2-photon: Software-tunable Chameleon II laser (680-1040 nm)
    • 1-photon: Similar to DAPI, DyLight 405, CFP, FITC, YFP, mOrange, TRITC, Cy5
    • 2-photon: Compatible with most red, green and blue fluorophores
    • 1-photon: Nikon Objectives
    • 2-photon: 25x/1.10 coverslip-corrected water-immersion IR lens
    • Microscope standNikon Ti Eclipse inverted
    • Features:
      • SIM provides double the resolution of the conventional confocal microscope (lateral

      Confocal 1 - Zeiss LSM 880 with Airyscan on an Axio Observer.Z1 invert (B/K035)

      • Airyscan detector for high resolution imaging
      • fully automated microscope
      • 4 independent lasers, 6 laser lines including (405, 458, 488, 514, 561, and 633 nm)
      • Spectral head for spectral imaging - allowing the easy differentiation of very similar fluorescence dyes and fluorescence from autofluorescence
      • an array of lenses ranging from 100 x oil down to 10 x air to suit most needs
      • stage adapted for use with Incubator S system for live cell imaging
      • ZEN 2.1 software

      Confocal 2 - Zeiss LSM 780 multiphoton on an Axio Observer.Z1 invert (B/K029)

      • fully automated microscope
      • Coherent Chameleon pulsed Ti:Sa IR Laser (range 690-1040nm)
      • 4 independent lasers, 6 laser lines including (405, 458, 488, 514, 561 and 633 nm)
      • 4 external non-descanned detectors (NDD) for increased sensitivity at greater depth
      • Spectral head for spectral imaging - allowing the easy differentiation of very similar fluorescence dyes and fluorescence from autofluorescence
      • Objective lenses ranging from 10x air through to 63x oil as well as 40x/1.1 and 63x/1.2 water immersion and 20x/1.0 and 40x/0.8 water dipping objectives ideal for deep tissue imaging
      • Solent light tight incubation chamber with full temperature and CO2 control
      • ZEN 2010 software

      Zeiss LSM 780 multiphoton

      Confocal 3 - Zeiss LSM 710 on an AxioImager.M2 (B/K034)

      • fully automated upright microscope
      • 5 independent lasers, 7 laser lines including (405, 458, 488, 514, 561, 594 and 633 nm)
      • Spectral head for spectral imaging - allowing the easy differentiation of very similar fluorescence dyes and fluorescence from autofluorescence
      • an array of lenses ranging from 100 x oil down to 10 x air to suit most needs
      • stage adapted for use with Incubator S system for live cell imaging
      • ZEN 2011 software

      Confocal 4 - Zeiss LSM 510 meta on an Axiovert 200M (B/K037)

      • fully automated microscope
      • 3 independent lasers, 6 laser lines including (458, 477, 488, 514, 543 and 633 nm)
      • Spectral head for spectral imaging - allowing the easy differentiation of very similar fluorescence dyes and fluorescence from autofluorescence
      • an array of lenses ranging from 100 x oil down to 10 x air to suit most needs
      • stage adapted for use with Incubator S system for live cell imaging
      • LSM510 and ZEN 2009 software

      Zeiss LSM 510 meta invert

      Zeiss on-stage incubation system

      Confocal Microscope

      The UCA Department of Biology received a National Science Foundation grant in June 2002 to equip the facilities with a confocal laser scanning microscope (CLSM). This system provides the new means for research in biochemistry, cell biology and neuroscience for faculty and students.

      Various avenues of research include studies of the neural networks in mammalian brains, monitoring intracellular pH in cancer cells, and tracking mitochondrial proteins in yeast and slime mold. Visiting researchers from the University of Arkansas for Medical Sciences have been studying effects of ethanol on early neural development. As of spring 2005 we have started working with the “Saturdays with SEM” program to introduce high school students and educators to confocal microscopy. Here, visitors spend the day working with both the Scanning Electron Microscope (SEM) and the CLSM. During the day, they are taught the principles microscope operation and are allowed to visualize samples with the equipment.