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Confocal Fluorescence Microscopy

Optical Imaging and Spectroscopy

  1. Oleg D. Lavrentovich

Published Online: 25 JUN 2012

DOI: 10.1002/0471266965.com127

Characterization of Materials

Characterization of Materials

How to Cite

Lavrentovich, O. D. 2012. Confocal Fluorescence Microscopy. Characterization of Materials. 1–15.

Author Information

  1. Kent State University, Kent, OH, USA

Publication History

  1. Published Online: 25 JUN 2012

1 Introduction

  1. Top of page
  2. Introduction
  3. Principles and Practical Aspects of Confocal Fluorescence Microscopy
  4. Examples of Practical Applications: Sample Preparation
  5. Conclusion
  6. Acknowledgments
  7. Abbreviations
  8. Literature Cited
  9. Key References

Much of the modern understanding of materials is based on optical microscopy (OM). For some materials, the optical contrast is achieved by light reflection, scattering, and absorption. For others, especially those of soft nature, such as polymers, colloids, liquid crystals, biological cells and tissues, these effects are not always sufficient to get an insight into the structural details. Fluorescence microscopy (FM) is one of the historically earliest and most successful approaches to generate a high-contrast image of organic and inorganic materials. It is based on the phenomenon of fluorescence and phosphorescence, that is, the property of some atoms and molecules to absorb light at a particular wavelength and then emit a lower energy light of a longer wavelength. The difference is called the Stokes shift, after the name of George Stokes who discovered the effect in 1852 (Masters, 2005). The Stokes shift is a key to the enhanced contrast of FM as it allows one to filter out the excitation light from the fluorescent signal. The image is created by the emitted fluorescent light, that is, the fluorescent sample itself serves as a light source. FM guarantees a high contrast, as the stained object appears on a dark background. This contrast is typically much better than a contrast created through light absorbance, since for small objects, the amount of light absorbed becomes only a small fraction of the background (Lichtman and Conchello, 2005).

The FM has been dramatically improved with the invention of confocal mode, which allows one to optically slice the specimens and to create high-contrast images in three dimensions (3D). The confocal fluorescence microscope registers only the signal originating from the tightly focused small region of space and rejects light that does not come from this region. The effect is achieved by using an opaque disk with a pinhole that plays the role of spatial filter. The CFP images 3D distribution of fluorescent markers in the material and is especially useful in studies of heterogeneous and composite materials, comprised of components with different affinity to the fluorescent markers, such as colloids, block copolymers, polymer solutions, and phase separating systems. By using polarized light probing and staining with sufficiently anisometric fluorescent markers, one designs a fluorescence confocal polarizing microscope (FCPM) that is capable of 3D imaging of orientational order pertinent to materials such as liquid crystals.

The most dramatic recent development of FM is in breaking the diffraction limit in far-field optical imaging. By using fluorescent probes that can be controllably switched between a dark (D) and bright (B) states, and by limiting the size of the fluorescent regions, one can resolve features in the range of few nanometers. This area of far-field optical nanoscopy (Hell, 2010) or “super-resolution fluorescence microscopy” (SRFM) (Huang et al., 2009) is rapidly developing, filling the gap between the regular diffraction-limited FM and the electron microscopy, (Electron Techniques, Introduction).

This review oversees the basic principles of FM, its confocal modes CFM and FCPM; for an at-depth consideration of SRFM, see the article Super-Resolution Optical Microscopy by J. Werner and A. Shreve.

2 Principles and Practical Aspects of Confocal Fluorescence Microscopy

  1. Top of page
  2. Introduction
  3. Principles and Practical Aspects of Confocal Fluorescence Microscopy
  4. Examples of Practical Applications: Sample Preparation
  5. Conclusion
  6. Acknowledgments
  7. Abbreviations
  8. Literature Cited
  9. Key References

2.1 Fluorescence

A light energy (photons) absorbed by a molecule alters its electronic, vibrational, and rotational states. It can cause an electron in the outermost orbitals to move into a more distant orbital. Fluorescence (=spontaneous emission) occurs when such an electron returns back to its ground state, by emitting a photon. The energy loss typically also involves nonradiative processes, such as vibrational relaxation. The emitted light is thus of a longer wavelength. Correspondence between the energy of photons and the characteristic absorption and fluorescence spectra is illustrated schematically in Figure 1.

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Figure 1. Schematic diagram of fluorescence. (a) Energy levels corresponding to the ground state inline image and the excited singlet state inline image of a molecule; the photon excitation to different vibrational states of inline image is shown by the upward blue arrows and the fluorescence, or spontaneous emission of light is shown by the downward red arrows. The black arrows show relaxation between vibrational levels. (b) A corresponding spectrum of absorption and emission of a fluorescent molecule.

It is customary to distinguish four basic steps in fluorescence emission (Nie and Zare, 1997): (1) electron transition from the ground electronic state inline image to an excited electronic state; (2) internal relaxation in the exited electronic state, through transitions from higher energy electronic, vibrational, and rotational states to lower energy excited states; (3) radiative or nonradiative decay from the excited state to the ground state; (4) internal relaxation in the ground states. In Figure 1, the process (1) is shown by thick grey upward arrows, the process (3) by thick grey downward arrows, and the internal relaxation through vibrational states (2, 4) is shown by small black downward arrows; rotational levels are not shown for simplicity. Steps (2) and (4) are the reason for the Stokes shift.

The steps (1–4) have different lifetime. Transitions between the vibrational (and rotational) states are very fast, on the order of picoseconds for small molecules in condensed phases. The lifetime of the excited state is longer, in the subnano- and nanosecond range (Nie and Zare, 1997). This time depends not only on the molecular structure but also on the environment and how easily and quickly this environment can absorb the energy from the exited molecule. Fluorescence lifetime imaging microscopy (FLIM) uses this dependency to study interactions between fluorescently active molecules and their immediate surrounding. If the interacting partners are themselves fluorescent, with a suitable absorption spectrum, a fluorescence resonance energy transfer (FRET) occurs, which can be used for trace changes in the intermolecular separations that are much smaller than the resolution limit of a regular light microscope (Jares-Erijman and Jovin, 2003).

Although many organic compounds are capable of intrinsic fluorescence (autofluorescence), modern FM relies on the carefully designed fluorophores that are being added to the material of interest. Effective fluorophores are typically characterized by a molecular structure with conjugated double bounds and polyaromatic structure, which facilitates a distribution of the outer electrons over a large area of the fluorophore, Figure 2. This makes the energy between the ground and the exited states small enough to allow an excitation by a relatively low-energy photons, including the ones that correspond to the visible part of spectrum (Lichtman and Conchello, 2005). Note that the excitation can be caused by two (or more) photons of a low energy rather by one photon of a high energy. The multiple photons should be in the same place and practically the same time (the absorption time is short), which implies sufficiently high light intensities. This effect is used in multiphoton excitation microscopy to be discussed later.

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Figure 2. Molecular structures of fluorescent markers (a) Acridine orange, (b) N,N'-Bis(2,5-di-tert-butylphenyl)-3,4,9,10-perylenedicarboximide (BTBP), and (c) fluorescent label fluorescein isocyanate (FITC) covalently bound to poly(ethylene glycol).

The Stokes shift allows one to build a simple FM around four basic elements, Figure 3: (1) an arc lamp to excite the specimen (it can be a Xenon or Mercury arc-discharge lamp with a proper heat filter to block infrared irradiation), (2) a filter to separate emitted light from the excitation, (3) an objective, and (4) a detector of fluorescent light. The most popular arrangement of these elements is the so-called epi-illumination FM. In this scheme, the objective plays a dual role, as a magnifying element and as a condensor to illuminate the sample. Such a combination implies that the probing and emitted lights share some portion of the optical path. To separate them, one uses a dichroic beam splitter, Figure 3, placed in the pathway at 45°. The dichroic mirror reflects light of shorter wavelength coming from the light source and transmits light of longer wavelength originating in the fluorescent sample. The dichroic beam splitters are accompanied by additional filters to improve the signal/noise ratio; these additional elements are of crucial importance in detecting weak signals, such as those produced by single fluorescent molecules.

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Figure 3. A principal scheme of a fluorescence microscope with epi-illumination.

A wide-field illumination FM produces good quality images when the samples are relatively thin, a micrometer or so. For thick samples, the image is blurred. The reason is that the excitation light illuminates all regions of the sample along the optical path and causes fluorescence not only from the focus plane but also from the regions below and above the focus plane. In some techniques, such as polarizing microscopy (PM), this integration of the optical information along the pathway is beneficial. For a birefringent sample viewed between two crossed linear polarizers, a PM converts the optical phase retardation inline image integrated over the pathway of light into the light intensity, inline image (Bellare et al., 1990; Kleman and Lavrentovich, 2003). Here, inline image is the effective value of birefringence, determined by the ordinary inline image and extraordinary inline image refractive indices, orientation angle inline image of the local optic axis that might vary along the coordinate inline image parallel to the optical axis of the PM; inline image is the thickness of the birefringent element, and inline image is the wavelength of the probing light. An advanced implementation of PM, the so-called LC-PolScope uses a switchable compensator representing a liquid crystal slab with the electrically controlled inline image, to determine the local values of optical retardance and establish the orientation of the optic axis in the inline image plane of the sample (Oldenbourg and Mei, 1995).

In FM, the out-of-focus background signal coming from the essentially 3D specimen is detrimental, as it reduces the contrast of the in-focus image. The problem can be partially mitigated by using low light intensities. The highest excitation power is in the center of the focused beam and the fluorescent contribution from the regions below and above it might be somewhat reduced. A much better approach to eliminate the out-of-focus signal is to use the confocal mode of observation.

2.2 Confocal Mode of Fluorescent Microscopy

The confocal mode by Marvin Minski 1957 allows one to measure the optical signal that is uniquely defined by the given illuminated point of the sample. First consider the transmission type of a confocal microscope, Figure 4a. The first distinctive element is an opaque disk with a pinhole 1 placed in front of the light source in order to form a point-like source. This point light source is refocused into the sample by a focusing lens labeled as “Condensor” in Figure 4a, that is actually a microscope objective, (see also Optical Microscopy). The second focusing lens, labeled as “Objective” in Figure 4a, brings the optical signal from the illuminated “voxel” (an elementary 3D region of the sample, derived from the word pixel in a 2D display) into the detector. However, the detector would detect also some signal from out-of-focus points, Figure 4a. The key feature that blocks the detector from the undesired out-of-focus signal is the second pinhole 2 located in the optically conjugated plane in front of the detector. This confocal arrangement greatly enhances the optical resolution in the direction of light propagation (Webb, 2000; Pawley, 2006). It allows one to acquire in-focus images from a preselected level of depth in the sample and then reconstruct the 3D image by combining optical “slices” registered at different depths. The “thickness” of each optical slice in case of the isotropic (nonbirefringent) media is roughly equal the wavelength of light divided by the numerical aperture of the objective.

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Figure 4. Confocal microscope for observation in the transmitted (a) and reflected (b) light. Note the role of opaque disk with pinholes as a spatial filter that blocks light originating from out-of-focus regions.

The same confocal principle can be applied to the reflective mode of observation, Figure 4b, where only one objective and one pinhole are used. The reflective scheme is more popular as for the same quality of imaging it eliminates one of the two objectives and can be used to image the top regions of thick specimens that cannot transmit light and thus cannot be viewed in the transmission mode.

Since the confocal microscope blocks a significant fraction of light that comes out of focus, the measured signal is usually weak and implies long exposures. Combining the confocal model and fluorescence into the confocal fluorescence microscopy (CFM) overcomes the problem. By selecting an appropriate fluorescent dye, one selectively stains different regions of the specimen. The fluorescent signal intensity is proportional to the concentration of fluorescent dye. Another measure to overcome the light-limiting feature of the pinhole is to use a laser as a light source with a high intensity narrow beam. The laser source generates a monochromatic light of a certain wavelength that should match the absorption band of the used fluorescent dye.

2.3 Confocal Laser Scanning Microscopy

In CFM, the sample is illuminated by a tightly focused beam, thus only one voxel is imaged at a time. To obtain a full image, the sample should be scanned in the x,y plane normal to the pathway of light and along the depth direction z. In the confocal laser scanning microscope (CLSM), the sample is scanned in the x,y plane with a laser beam that is deflected by two sets of mirrors (x-scanning and y-scanning). The mirrors are installed on a shaft of a servo motor; the frequency of the mechanical response of the scanner is several kHz, which determines the rate of scanning.

The fluorescent signal from the sample returns back through the same mirror system (the step is called descanning) and then passes directly through dichroic beam splitter and is routed into a detector. Typically, in CLSM, one uses a photomultiplying tube (PMT) as a detector, for sensitive and fast single point intensity registration. Since the light signal comes from a pinhole, such a detector does not require good spatial discrimination, but should respond quickly to a varying light intensity. In a PMT, the incoming photons are converted into electrons by a photocathode; the signal is multiplied and is read-out as an electronic output at the anode (Spring, 2001; Hergert, 2001; Claxton et al., 2005). The image is thus reconstructed from the recorded point intensities mapped as a function of spatial coordinates and is not directly visible to an eye.

The point-by-point scanning performed by a single laser beam is slow. With conventional galvano scanners, it takes about 1–10 s to construct a 1000 × 1000 pixel image of a single horizontal plane. The z-scanning is performed by either moving the microscope stage or the objective, which is also slow.

2.4 Confocal Fluorescence Microscopy with Nipkow Disk

The rate of confocal imaging can be increased if one uses multiple beams to scan the sample simultaneously. The structural element allowing a simultaneous scanning is a rotating Nipkow disk with thousands of pinholes, a very old invention, used in early-stages television. The pinholes are arranged into Archimedes spirals. Each consecutive pinhole is placed at an increased radial distance from the center of the disk and is shifted by a constant angle with respect to its neighbors. When the Nipkow disk rotates, such an array of pinholes scans the sample in a raster pattern, increasing the rate of imaging. The approach suffers from a very low fraction (a few percent) of light that is transmitted by the pinholes. To overcome the problem, Tanaami et al. 2002, proposed to supplement the Nipkow disk with a coaxial disk containing an array of micro-lenses at positions corresponding to the pinholes, Figure 5. The two disks are mechanically connected and are rotated together by an electric motor. The micro-lenses illuminated by an expanded laser beam focus the resulting multiple beams onto the pinholes. The images of pinholes are focused into the sample by an objective. The fluorescent signal is reflected by a dichroic mirror located between the two rotating disks and is focused onto a charge-coupled camera (charge-coupled device (CCD)) that serves as a detector (instead of a PMT). The dichroic beam splitter is different from the one used in a conventional FM: It transmits shorter wavelengths and reflects longer wavelengths.

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Figure 5. Confocal microscopy with coaxial lenslets and Nipkow disks.

The sample is scanned by thousands of beams at once, thus representing an alternative to the slower CLSM. Yokogawa Electric Corporation designed a number of confocal scanning units (CSU) such as CSU-10 and CSU-X1. CSU-10 achieved the scanning/imaging speed of 1 ms/frame while CSU-X1 is twice as fast. There are about 20,000 pinholes of diameter 50 μm each, about 1,000 of them are in the illuminated area. The CSU enables real-time observation of many dynamical processes, including those in living cells. Since the intensity of each beam in the light bundle is low, on the order of microwatts, the approach reduces fluorescence photobleaching and specimen damage (Wang et al., 2005).

A Nipkow disk principle does not require a laser as a light source and can operate with a mercury or xenon lamp commonly used in epi-FM. These sources feature full-spectrum capabilities and can deliver high intensities sufficient for registration by the high quantum efficiency cooled CCDs. The full-spectrum feature makes it easier to choose the best fluorescent tags for the specimens, as the appropriate wavelength of probing light can be selected by a simple filter. Of course, if the fluorescent signal is very weak, the intense laser illumination in CLSM might be the only option for imaging.

The size of pinholes in CFM is an important factor controlling the quality of imaging. To adjust the pinhole size, several apertures of various diameter are placed on the same touret. A smaller pinhole diameter reduces the effective thickness of the optical “slice” of the specimen, but at the same time, reduces the signal strength. A wider pinhole allows one to obtain a brighter signal, at the expense of a thicker optical section and reduced resolution. In Nipkow disk configurations, the pinholes are typically of diameter 50–80 μm.

If the standard CLSM can be called a 3D microscope then a fast scanning microscope with a wide selection of wavelength can be called a five-dimensional microscope, time and wavelength being the fourth and the fifth dimensions. Microscopes with a full-spectrum illumination allow one to explore specimens with many different fluorescent probes. An especially interesting development in this direction started when the green fluorescent protein (GFP) was isolated from the jellyfish Aequorea victoria. This protein shows bright green fluorescence when exposed to blue light. Development of numerous mutant spectral derivatives of GFP dramatically improved the ability of confocal fluorescent microscopy in biological imaging, as these markers are much less toxic to living cells and organisms as compared to the traditional dyes such as fluorescein isothiocyanate (Tsien, 2005; Lippincott-Schwartz and Patterson, 2003). Multiple fluorescent proteins have been used, for example, to image neuronal network architecture with 90 different colors (Livet et al., 2006).

Another class of fluorescent probes is quantum dots, that is, nanometer-sized crystals of purified semiconductors. These nanoparticles are covered with an appropriate organic coat, suitable for integration with the studied specimen. The quantum dots are excited by photons. The absorbed photon generates an electron-hole pair that emits light upon recombination. The energy of the emitted photons depends strongly on the quantum dot size. The absorption spectrum of quantum dots does not have a well-defined peak, and the emission spectrum has the same profile for different wavelength of excitation, thus opening the door for multicolor labeling of specimens (Lacoste et al., 2000).

To provide the ability of multiwavelength illumination, many modern confocal microscopes are equipped with more than one laser source. The control of intensity and wavelength in these microscopes is typically achieved with the acousto-optic tunable filters (Chang, 1995).

2.5 Fluorescence Confocal Polarizing Microscopy

Many materials feature an orientational order of the building units. The simplest examples are liquid crystals (LCs) and block copolymers. For example, uniaxial nematic LCs (NLCs) used in the production of liquid crystal displays (LCDs), are comprised of elongated rod-like molecules about 2–3 nm long and 0.5 nm wide. The rods are on average align along a common direction called a director inline image. In a uniaxial NLC, inline image is simultaneously the local optical axis. By realigning inline image by the external fields, one alters the optical appearance of the material, which explains the numerous applications of LCs. For many years, the LCs were studied by a regular wide-field PM that allows one to discriminate between different orientations of inline image, but only “on average,” without resolving the sample's depth. A PM image of a LC bears only 2D information, integrating the 3D pattern of optical birefringence over the path of light. A regular CFM is not capable to image how the director field inline image is distorted, as director deformations are smooth and do not alter mass density of the LC. Two modifications to CFM convert it into a fluorescent confocal polarizing microscope (FCPM), a tool for 3D imaging of orientational order and the director field, Figure 6 (Smalyukh et al., 2001; Lavrentovich, 2003). First, the material is doped with fluorescent molecular markers that are strongly anisometric (say, rod-like); these molecules are aligned by the LC host. Second, the sample is probed with a (linearly) polarized light.

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Figure 6. Fluorescence confocal polarizing microscope with the elongated fluorescent molecules (such as BTBP, Figure 2b) aligned by the molecules of the LC; the sample is probed with a linearly polarized focused beam.

To understand the principle, consider a NLC doped with anisometric molecules of a fluorescent dye, such as N,N′-Bis(2,5-di-tert-butylphenyl)-3,3,9,10-perylenedicarboximide (BTBP), Figure 2. The transition dipoles of both excitation and fluorescence are along the long axis of the dye molecule. The anisometric “guest” markers are aligned by the nematic “host,” Figure 7. For the linearly polarized probing beam, the probability of photon absorption depends on the angle inline image between the beam polarization inline image and the direction of the transition dipole of the dye, as inline image, Figure 7, where the brackets denote an average of all orientation of the transition dipoles in the illuminated region. In epi-illumination mode, the probability of the emitted photon to reach the detector through the very same polarizer is inline image, where inline image is the value of inline image at the moment of emission. Even when the probing and the emitted light beams are passing through the same polarizer, inline image might be different from inline image. The reason is that the time delay inline image between the acts of adsorption and fluorescence emission might be longer than the characteristic time inline image of rotational relaxation of the dye in the LC. Typically, inline image, while inline image for BTBP molecules in different organic solvents is smaller, inline image (Ford and Kamat, 1987). If inline image then inline image, and the detected fluorescence intensity is related to the angle between inline image and the polarizer direction inline image, as inline image (Shiyanovskii et al., 2001; Smalyukh and Lavrentovich, 2002). Note that the fluorescence lifetime is usually short enough to assure that the excited dye molecule remains within the given voxel. In LCs, the diffusion coefficient for most dye molecules is of the order of inline image (Blinov and Chigrinov, 1993); therefore, to travel the distance of the order of l ∼ 1 μm, the dye molecule would need time inline image, which is much larger than inline image. The fluorescent molecule will emit within the same voxel in which it was excited.

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Figure 7. Principle of visualizing the director in FCPM. The fluorescent molecules (ellipsoids) are aligned by the LC. Polarized probing beam excites the dye and cause fluorescence. The efficiency of excitation depends on the angle between the transition dipole of dye molecule and polarization of probing beam. The efficiency is minimum in (a) and (b), intermediate in (c), and maximum in (d). The detected signal will be maximum when the polarization of fluorescent light is parallel to the direction of polarizer.

The whole idea of CFM and FCPM makes sense only if the probing beam can be focused into a small micron-size voxel. In birefringent LCs and polymers, the probing beam gives rise to ordinary and extraordinary waves, propagating with different velocities and thus converging in different regions of the sample. This birefringence-induced defocusing will cause some blurring. This problem can be mitigated, however. The spatial defocusing of the ordinary and extraordinary modes is roughly inline image, where inline image is the depth of scanning, inline image is the difference in the refractive indices for extraordinary and ordinary waves, inline image is the average refractive index of the LC, and inline image is a number of the order of unity that depends on geometry of light propagation, director field, objective, and so on. For d = 20 μm and inline image, the defocusing is of the order of 1 μm; the lower the birefringence inline image, the better. Therefore, one can always obtain a suitable resolution of the order of 1 μm by working either at a relatively shallow scanning depth or choosing a LC with a small inline image.

An important feature of FCPM is that it is free of inline image ambiguity known for PM observations. In PM, the optical phase shift is zero whenever the director is parallel or perpendicular to inline image. Because of this, the PM image cannot distinguish between two mutually perpendicular director configurations without additional elements, such as waveplates. In FCPM, the ambiguity is avoided: if inline image is parallel to inline image, the intensity is maximum; if perpendicular, it is minimum.

2.6 Multiphoton Microscopy

In the conventional confocal schemes considered so far, the optical slicing ability is achieved by using a pinhole that performs as a spatial filter by blocking fluorescent light that comes out of focus. However, the specimen is illuminated not only in the region of focus but also in the entire double-cone region extending above and below the focus. This exposure results in photobleaching, photodamaging, and additional absorption of light. These shortcomings are overcome in the two-photon and multiphoton excitation microscopy (Diaspro, 2002).

If a sample is illuminated by a tightly focused beam emitting a long wavelength light, there is a probability that two or more coherent photons can excite molecules at a fraction (half or less) of the wavelength of light. Processes such as single-photon absorption and fluorescence depend linearly on the light intensity, but the multiphoton effects belong to the rich family of the nonlinear optical effects, that depend on the light intensity in a nonlinear manner. The probability of two-photon absorption is proportional to the square of light intensity. The interaction of the two photons and the atom must occur within a short period of time, corresponding to the lifetime of the virtual state of the atom, about 1 fs. The first photon excites the atom from the ground state into the virtual state and the second photon elevates the atom to the excited state.

Although the nonlinear processes have been known theoretically since 1920s, and widely explored in nonlinear spectroscopy after the invention of laser, their applications in optical microscopy started only in 1990s, with the demonstration of a two-photon laser microscope (Denk et al., 1990). The sample was illuminated with a Gaussian laser beam at 700 nm, tightly focused to a diffraction-limited waist of less than 1 μm. In addition, the laser irradiation was temporally compressed to produce short (subpicosecond) pulses with a very high peak intensity. In the focus region, the light intensity is sufficiently high to produce molecular excitations in the material through the simultaneous absorption of two photons. Importantly, the average power of irradiation was only 25 mW, which is safe for most hard and soft materials and for living cells and tissues. In the regions far away from the focus, the intensity of light is too low for noticeable two-photon effects. Therefore, the two-photon microscopy is intrinsically capable of optical slicing. No pinhole-based spatial filter is needed to block the signal from outside the focus, as these regions simply do not produce any signal. Since the probing light in the two-photon microscopy is of a longer wavelength, it is less scattered than the probing light of shorter wavelength in regular confocal microscopes and allows one to explore samples at a deeper penetration length. The quadratic dependence of the signal on the light intensity allows one to use moderate mean power that does not damage the specimen. Since the probing and fluorescent light have very different wavelengths, their separation is much easier than in the case of a regular FM with a small Stokes shift. Similarly, three-photon absorption is possible, allowing one to use IR irradiation to excite ultraviolet (UV) fluorescence. The intensity of fluorescence in this case scales with the cube of the illumination intensity.

The most challenging technical part of the multiphoton microscopy is in the development of the laser sources that yield sufficiently high-powered pulses of irradiation; these sources are significantly more expensive than the cw type of lasers used in standard confocal microscopy. The most widely used are titanium-sapphire lasers with a broad tunability in the range of 700–1100 nm (which allows one to cover the absorption bands of many fluorescent markers), high average power (1 W), short width of temporarily compressed pulses (80–150 fs) and high repetition rate (100 MHz) (see, e.g., Masters, 2005). Besides imaging, the multiphoton microscopy is successfully used to produce microstructures through photopolymerization.

2.7 Diffraction-Limited Resolution

The resolving power of an Optical Microscopy, including the CFM, can be considered by analyzing the image of an infinitely small (in practice, much smaller than the wavelength of light) luminous object. A visible image of such a point source represents a complex three-dimensional diffraction pattern, called an intensity point spread function. In the plane perpendicular to the direction of light propagation, the two-dimensional intensity distribution is a circular Airy disk, with a bright central circular area surrounded by progressively weaker concentric dark and bright circles. The radius of the first dark ring depends on the wavelength inline image of light (measured in vacuum) and the numerical aperture (NA) of the objective:

  • mathml alt image(1)

where inline image is defined by the refractive index inline image of the medium in which light propagates and by the half-angle inline image of the maximum cone of light that can enter or exit the lens. The Rayleigh criterion for resolution states that the two equally bright spots are resolved when their separation is equal or larger than inline image; that is, when the bright center of one Airy disk is located on the first dark circle of the second Airy disk (Spring and Inoué, 1997). When imaging with a visible light, say, at 500 nm, using an immersion oil with inline image = 1.5, and a high NA = 1.4 objective, one can achieve a resolution of about 200 nm.

In the axial direction, along the propagation direction, the distance between the bright central portion of the image and the first minimum is

  • mathml alt image(2)

The latter expression can be used as a practical measure of the axial resolution of the microscope, about 500 nm for visible light. Note that the numerical aperture of the objective has a very strong effect on the axial resolution of the microscope, inline image and that the ratio inline image is substantially larger than 1.

Confocal fluorescent microscopy has somewhat better resolution as compared to the conventional wide-field mode, as it employs a single-point scanning and single-point detection, with the two corresponding point spread functions (PSFs) being multiplied, which reduces the width of the resulting point spread function, and improves resolution by a factor of inline image (Wilson, 1990; Stelzer, 1997). The greatest advantage of the confocal mode is not so much in the lateral and actual axial resolution, but in the optical sectioning capability enabling 3D imaging, guaranteed by blocking the out-of-focus light.

The axial resolution can be additionally improved by the schemes in which the effective NA is increased. In a regular microscopic observation, the objective collects light only from one side of the sample. By using two opposing objectives for excitation and/or detection, one improves the axial resolution to about 100 nm. The two techniques using these set-ups are called 4Pi microscopy (Hell and Stelzer, 1992) and I5M (Gustafsson et al., 1999). The abbreviation I5M represents a combination of abbreviations for I2M (illumination interference microscopy) and I3M (incoherent imaging interference microscopy). Furthermore, the resolving power can be enhanced by using a structured-illumination microscopy (SIM). The specimen is illuminated with a patterned light field. The high-frequency sample features mix with the spatial frequency of the illuminating pattern, shifting the resulting frequency to the lower values that can be detected by the microscope. SIM improves the resolution by combining the two diffraction-limited sources of information (Gustafsson, 2000), without changing any specific fluorescence features. As will be discussed below, by involving nonlinear photophysics and photochemistry of fluorophores, one can break the diffraction limit of resolution by at least one order of magnitude.

2.8 Super Resolution Fluorescence Microscopy

The Rayleigh principle applied to standard Optical Microscopy sets the lateral resolution at about 200 nm and the axial resolution at about 500 nm. Near-field scanning optical microscopy (NSOM); see also Scanning Near-Field Optical Microscopy) can yield a better resolution, down to tens of nanometers, but it can image only surface features: NSOM relies on the nonpropagating evanescent fight field fading out exponentially within a inline image distance from the surface. Similarly, the evanescent waves are used in the imaging principle of a “perfect” lens made of a metamaterial with a negative index refraction (Pendry, 2000), but in most cases, especially in lossy materials, the resolving power degrades in the far-field (Podolskiy and Narimanov, 2005). In the so-called hyperlens, the evanescent waves are converted into the propagating waves by employing an optically anisotropic metamaterial with a hyperbolic dispersion which constitutes nonfluorescent approach to the far-field imaging beyond the diffraction limit, based on optical metamaterials (Jacob et al., 2006; Salandrino and Engheta, 2006). The viability of the metamaterials approach has been already demonstrated (Liu et al., 2007; Smolyaninov et al., 2007) and much is expected in the future; for the detailed description of the current status, see the monograph by Cai and Shalaev 2009.

Breaking the optical diffraction limit in 3D fluorescent imaging has been achieved within the last decade, see the reviews by Dedecker et al. 2008, Hell 2010, and Huang et al. 2009. The new approach is based on nonlinear fluorescence related to photochemistry and photophysics of the fluorescent probes rather than on manipulation of optical beam propagation. The experimental and even commercially available devices have already demonstrated super-resolution that is at least one order of magnitude better than the diffraction-limited values. The FM with subdiffraction resolution is called “far-field optical nanoscopy” (Hell, 2010), “SRFM” (Huang et al., 2009), or “diffraction-unlimited optical microscopy” (Dedecker et al., 2008). The unifying feature of many independently developed approaches to SRFM is that in all of them, one uses two states of the fluorescent markers, namely, the bright (B) and dark (D) states. The idea of overcoming the diffraction limit is to create small islands of subdiffraction size, of the B-state, embedded into the sea of the D state. These islands are separated by distances larger than the diffraction limit inline image, so that their images do not overlap with each other. Although the image size of a small light source cannot be smaller than the diffraction limit, the position of this source can be determined with a resolution well below inline image, by determining the centroid of the emission. Finding the positions of fluorescent markers allows one to reconstruct the image of the entire sample stained with fluorescent molecules, with a resolution below the diffraction limit. The main difficulty is to simultaneously satisfy two requirements: To separate the B islands by a large distances, inline image, and to have a sufficient number of these islands to reconstruct structural features smaller than inline image. The requirements can be satisfied by separating the A and B species either in space or in time, according to which one distinguishes two types of SRFM:

  1. SRFM of the First Kind (SFRM-I)

    Separation of fluorescent markers in space is used to sharpen the point spread function below the diffraction-limited size; these approaches include stimulated emission depletion (STED) microscopy, ground-state depletion (GSD) microscopy, and related techniques of saturated structured illumination microscopy (SSIM) and reversible saturable optically linear fluorescence transition (RESOLFT).

  2. SRFM of the Second Kind (SFRM-II)

    Separation of fluorescent markers in time and finding the location of individual emitting molecule; these approaches are called stochastic optical reconstruction microscopy (STORM), photoactivated localization microscopy (PALM), and fluorescence photoactivation localization microscopy (FPALM).

It is important to remember that the super-resolution is brought about by nonlinear regimes of fluorescence and thus requires relatively high intensities of irradiation. Effects such as photosaturation and photobleaching might damage the sample before one reaches the intensity needed to obtain super resolution. Thus, SRFM approaches require a careful selection of the dyes and adjustment of irradiation regimes used.

3 Examples of Practical Applications: Sample Preparation

  1. Top of page
  2. Introduction
  3. Principles and Practical Aspects of Confocal Fluorescence Microscopy
  4. Examples of Practical Applications: Sample Preparation
  5. Conclusion
  6. Acknowledgments
  7. Abbreviations
  8. Literature Cited
  9. Key References

3.1 General Considerations and Imaging with Confocal Fluorescence Microscopy

In practical applications, one should be aware that the CFM probes the fluorescent ingredients of the material under study rather than the material itself. Preparation of samples thus involves doping with an appropriate fluorescent label. The latter can be selected on the ground of its compatibility with the material under study (e.g., hydrophobic versus hydrophilic labels; disk-like or rod-like anisometric dyes in the imaging of LCs), spectral properties (the absorption band should match the available laser wavelength), efficiency, and so on. Typically, the number density of the fluorescent probes is relatively small, much less than 1%. For example, in FCPM, to resolve the orientation of the LC samples, one needs 0.01 wt.% (or less) of fluorescent molecules added to the LC (Smalyukh et al., 2001).

In all types of FM, spatial density, size of the fluorescent markers, and their excitation and de-excitation kinetics play a crucial role in setting the resolution. Lower densities might lead to artifacts, for example, a continuous polymer fiber stained sparsely with a fluorophore, would appear as a discontinuous object. The so-called Nyquist criterion states that the structural features are resolvable if they are larger than twice the distance between the fluorophores (Huang et al., 2009). A high density of labels might be also detrimental. First, it can modify the structure of the material. Second, there is always some probability of the dye to fluoresce, even in the absence of the excitation beam. The resolution limitation associated with the size of the fluorescent label is an important factor in biomedical sciences, but in the study of materials, one can bypass it by using organic dyes of relatively low molecular weight with a molecular size of 1–10 nm.

If the specimen is heterogeneous, the concentration of the fluorescent probe is coordinate-dependent because of its different affinity to different components. CFM has been proven to be an excellent tool in exploring materials such as colloids and polymers (White and Wiltzius, 1990; Chestnut, 1997; Held et al., 1997; Tata and Raj, 1997; Nephew et al., 1998; Korlach et al., 1999; Anderson et al., 2001; Srinivasarao et al., 2001; Prasad et al., 2007).

The fluorescent label can be attached covalently, as in Figure 2c that shows fluorescein isothiocyanate (FITC) attached to poly(ethylene glycol) (PEG). FITC-PEG helps one to determine the spatial location of different components in phase separating multicomponent system containing a water solution of disodium cromoglycate (DSCG) and a condensing agent PEG, a neutral polymer, Figure 8 (Tortora et al., 2010). The two complementary textures in Figure 8 show a CLSM view and a PM view. The fluorescent image maps the concentration of the fluorescent FITC-PEG. The dark regions in Figure 8a are deprived of FITC-PEG but rich in DSCG. DSCG condenses into the LC phase that is easily seen in PM as a birefringent bundled texture, Figure 8b. The birefringent regions in (b) match the dark regions in (a) and the isotropic dark regions in (b) correspond to PEG and FITC-PEG regions in (a).

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Figure 8. CLSM (a) and PM (b) views of a phase separating multicomponent mixture with fluorescent FITC-PEG and PEG as condensing agents and a LC material DSCG. FITC-PEG and PEG accumulate in the isotropic regions seen as bright fluorescing areas in (a) and dark areas in PM in (b); FITC-PEG and PEG act as crowding agents that condense DSCG into a LC state, seen as nonfluorescent areas in (a) and as bright birefringent bundles in (b); the crowding agents are expelled from the LC regions.

In the studies of colloids, CFM combined with the particle tracking algorithms, allows one to directly image the 3D bulk microscopic structures and dynamics (van Blaaderen et al., 1995; van Blaaderen and Wiltzius, 2005; Weeks et al., 2005). The colloidal particles, such as spheres, are fluorescently labeled in a variety of ways. For example, one can covalently incorporate FITC inside the silica spheres, making either a small spherical core or a thin spherical shell fluorescent, as described by van Blaaderen et al. 1995 and reviewed by Schartl 2000. In the exploration of very dense colloidal systems in which one needs to monitor the particles touching each other, one would prefer volume-labeled spheres. One technique is to use an organic solvent that swells the polymer (e.g., polymethyl methacrylate (PMMA)) spheres of interest and fill them with the dissolved fluorescent dye (Dinsmore et al., 2001). In other approaches, one covalently links the reactive dye (such as methacryloylated dye) to the monomer of interest, such as methyl methacrylate (Bosma et al., 2002), or simply adds a nonpolymerizable dye such as Acridine orange or Nile red to the monomers during the polymerization process (Campbell and Bartlett, 2002). Fluorescent colloidal particles of ellipsoidal shape can also be prepared (Sacanna et al., 2006). Note that the fluorescently labeled colloidal particles can be used as sensors, if the fluorescence of the particle depends on the environment, for example, pH of the solvent, see, for example, Kainz et al. 2010.

In recent years, with the development of fast CFMs, many research projects focus on dynamics of colloids. Aarts and Lekkerkerker 2004 used CLSM and fluorescent spheres to monitor phase separation in the colloid–polymer mixtures and to measure the colloid diffusion coefficients, by performing real space fluorescence recovery after photobleaching of the dye. Wu et al. 2005 used a fast CLSM (a commercial model Leica TCS-SP2) to explore the dynamics of silica and polymer spheres of diameter 1 μm, during melting and crystallization of colloidal crystals subject to a shear flow in a plate-cone shear cell. The set-up allowed one to trace the particles individually and to determine their coordinates using approach similar to that one described by Crocker and Grier 1996. The colloidal particles were stained with the fluorescent dyes, either FITC (in the case of silica) or rhodamine (in the case of PMMA). One can also use an inverse staining of the colloidal system. For example, a fluorescein sodium salt can be added to the water–glycerine mixture that serves as a dispersive medium for silica spheres, in which case the spheres appear dark on a bright background in CLSM (Cheng et al., 2011).

We briefly comment on the applications of SRFM, mostly its STED mode, in materials science. STED microscopy resolved dynamic assembly of fluorescent polymer colloidal particles (Lauterbach et al., 2010; Harke et al., 2008) and lithographic nanostructures (Westphal et al., 1995) with features at the length scale ranging from 30 to 80 nm. Friedemann et al. 2011 imaged nanostructured poly(vinyl alcohol) fibers containing nanoinclusions of fluorescent polystyrene, with a resolution down to 50 nm. In-plane resolution of 5.8 nm was achieved in the imaging of color centers in bulk diamond (Rittweger et al., 2009). These nitrogen-vacancy centers were shown to be an excellent source of luminescence, with no observable photobleaching. It is clear that the materials science will greatly benefit from further developments in the field of SRFM, in revealing complex structures with both positional and orientational order at the scale of nanometers, while using conventional lenses and propagating light.

3.2 Imaging Orientational Order

FCPM or its fast version with the Nipkow disk (Lavrentovich, 2003; Gu et al., 2006) has been successful in the studies of numerous systems containing LCs. The preparation of samples in most cases implies a simple mixing of a LC of interest with a fluorescent dye that is sufficiently anisometric to be aligned by the LC. Most of the studies of thermotropic LCs use BTBP dye (Smalyukh et al., 2001) and Nile red (Smalyukh et al., 2004; Sengupta et al., 2011) as both dissolves well in nonpolar organic solvents and can be excited with the laser beams incorporated into the commercially available confocal microscopes such as Olympus Fluoview BX50 or Leica TCS-SP2. In some LCs, the molecular orientation is established along two different directions (rather than along one director, as in a uniaxial NLC). These two different directors can be selectively imaged by doping the LC with two different dyes, in which the transition dipoles have different orientation with respect to the LC host (Smalyukh et al., 2005). Other dyes can be used to dope the thermotropic LCs in multicomponent systems. For example, fluorescent surfactants (such as BODIPY C5) can be used to label LC droplet in coexistence with glycerine (Lavrentovich, 2003). For lyotropic LCs, formed by polar organic molecules in water, one can use Acridine orange (Smalyukh et al., 2006; Nazarenko et al., 2010).

The FCPM technique was used to reconstruct the 3D structure and dynamics of line defects such as dislocations (Smalyukh and Lavrentovich, 2003) and disclinations (Vella et al., 2002; Sengupta et al., 2011); to explore dynamics of undulations in lamellar materials (Senyuk et al., 2006), director structures in LCs with colloidal inclusions (Liao et al., 2005; Pishnyak et al., 2007), including patterns of phase separation in LCs driven by the director gradients (Voloschenko et al., 2005). Figure 9a shows an FCPM image of a small colloidal particle levitating in the nematic LC bulk thanks to the elastic forces that repel it from the bottom and top plates (Pishnyak et al., 2007). The LC is doped with the dye BTPT, Figure 2 that is aligned by the NLC host parallel to inline image. The colloidal inclusion is not fluorescent and appears dark. The surrounding LC director is distorted as shown in Figure 9b; the distortions are readily visualized by FCPM including the vertical optical slice as shown in Figure 9a. Figure 10 shows an FCPM image of a cholesteric liquid crystal in which inline image is helicoidally twisted, remaining perpendicular to the helicoid axis. The helicoidal structure is seen as alternating bright (inline image is parallel to the polarization of probing beam) and dark (inline image is perpendicular to the polarization) stripes. The images capture the dynamics of an edge dislocation (Smalyukh and Lavrentovich, 2002) and patterns of undulations caused by the applied electric field (Senyuk et al., 2006). Recently, the FCPM technique has been applied to explore the dynamic behavior of LCs in microfluidic devices (Sengupta et al., 2011) and to the studies of LCs doped with metal nanoparticles and with CdSe quantum dots (Urbanski et al., 2010). By registering the FCPM signal at inline image = 540 nm, which corresponds to the fluorescence of the BTBP dye, Urbanski et al. 2010 first deciphered the director distortions in the sample and then tuned the FCPM to a different wavelength inline image = 610 nm to cause luminescence of the quantum dots and to demonstrate that the quantum dots are gathered at the ends of the disclination lines, apparently reducing the elastic energy of the deformed LC (Voloschenko et al., 2002).

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Figure 9. FCPM image of a nematic LC slab containing a levitating glass sphere of diameter 4.9 μm (a) with the corresponding director distortions (b).

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Figure 10. FCPM textures of the vertical cross section of a cholesteric LC with an edge dislocation (a) and cholecteric layers distorted by an electric field applied perpendicularly to the bounding plates (b).

The list below discusses other important requirements specific to the FCPM imaging of the LC director field (Smalyukh et al., 2001; Shiyanovskii et al., 2001).

  1. The concentration of dye and the intensity of light should be low enough to avoid light-induced director distortions in dye-doped LCs, the so-called Janossy effect (Janossy, 1994). A high-contrast imaging of LCs usually requires only small quantities of fluorescent dye, 0.01% (by weight), or less.

  2. Adiabatic following polarization. In twisted nematic cells or in chiral LCs such as cholesterics, polarization of both ordinary and extraordinary waves follows the local director (the so-called Mauguin regime) (Kleman and Lavrentovich, 2003). This effect must be taken into account while interpreting the FCPM images for samples with twist deformations, especially when the twist scale is supramicron and light propagates along the twist axis. To avoid this regime, one should reduce the Mauguin parameter

    • mathml alt image(3)

    down to ∼0.1; here inline image is the cholesteric pitch.

  3. Light scattering. Because of director fluctuations, light scattering in LCs is rather strong and leads to losses of intensity of both exciting and fluorescent light. Using LCs with small inline image helps to reduce light scattering.

  4. Concentration gradients. Even in a one-component anisotropic medium, there is a possibility that the dye concentration would be spatially nonuniform as the strong director gradients (e.g., at the cores of topological defects such as dislocations and disclinations) might cause concentration gradients of the dyes. On the positive side, this feature might be helpful in locating the defect cores.

4 Conclusion

  1. Top of page
  2. Introduction
  3. Principles and Practical Aspects of Confocal Fluorescence Microscopy
  4. Examples of Practical Applications: Sample Preparation
  5. Conclusion
  6. Acknowledgments
  7. Abbreviations
  8. Literature Cited
  9. Key References

FM, CFM, and their numerous modifications considered above, play an increasingly important role in the materials science. The most important advance of the last decade is in breaking the diffraction limit in the far-field FM that is bound to see numerous applications for decades to come. The physical phenomenon of fluorescence that makes possible the very operation of any FM, is also a limiting factor, as an addition of fluorescent elements modifies the specimen. Other problems are photobleaching, necessity to adjust the spectral performance of the dye and/or the illumination and detecting systems. Ideally, one would like to get rid of any staining dopants in the system under study. This is why, in parallel with the breathtaking developments of FM, research efforts also concentrate on label-free approaches based on optical metamaterials and on nonfluorescent nonlinear optical effects such as coherent anti-Stokes Raman scattering (CARS) microscopy, reviewed by Cheng and Xie 2004. CARS microscopy produces 3D images of materials such as thermotropic LCs (Saar et al., 2007; Kachynski et al., 2007) without fluorescent labels. In the studies of modern complex materials, multimode microscopy that combines FM with other modes, such as CARS, become increasingly popular (see, e.g., Lee et al., 2010).

5 Acknowledgments

  1. Top of page
  2. Introduction
  3. Principles and Practical Aspects of Confocal Fluorescence Microscopy
  4. Examples of Practical Applications: Sample Preparation
  5. Conclusion
  6. Acknowledgments
  7. Abbreviations
  8. Literature Cited
  9. Key References

I am thankful to I. I. Smalyukh, B. Senyuk, D. Voloschenko, S. V. Shiyanovskii, D.-K. Yang, H.-S. Park, L. Tortora, J. Xiang, and I. Lazo for useful discussions and cooperation. The work was supported by NSF grants DMR 1104850 and DMR 1121288 CEMRI UWM.

6 Abbreviations

  1. Top of page
  2. Introduction
  3. Principles and Practical Aspects of Confocal Fluorescence Microscopy
  4. Examples of Practical Applications: Sample Preparation
  5. Conclusion
  6. Acknowledgments
  7. Abbreviations
  8. Literature Cited
  9. Key References
BTBP

N,N′-Bis(2,5-di-tert-butylphenyl)-3,3,9,10-perylenedicarboximide (anisometric fluorescent dye used in FCPM of LCs)

CARS

Coherent anti-Stokes Raman scattering

CCD

Charge-coupled device

CFM

Confocal fluorescence microscopy

CSU

Confocal scanning unit

DSCG

Disodium cromoglycate (forming LC phases when dissolved in water)

FPALM

Fluorescence photoactivated localization microscopy

FITC

Fluorescein isothiocyanate

FLIM

Fluorescence lifetime imaging microscopy

FM

Fluorescence microscopy

FRET

Fluorescence resonance energy transfer

GFP

Green fluorescent protein

GSD

Ground-state depletion

I5M

A combination of abbreviations for I2M (illumination interference microscopy) and I3M (incoherent imaging interference microscopy)

LC

Liquid crystal

LCD

Liquid crystal display

NA

Numerical aperture

NLC

Nematic liquid crystal

NSOM

Near-field scanning optical microscopy

OM

Optical microscopy

PALM

Photoactivated localization microscopy

PEG

Poly(ethylene glycol)

PM

Polarizing microscopy

PMMA

Polymethyl methacrylate

PMT

Photomultiplying tube

PSF

Point spread function

RESOLFT

Reversible saturable optically linear fluorescence transition

SIM

Structured-illumination microscopy

SRFM

Super-resolution fluorescence microscopy

SSIM

Saturated structured illumination microscopy

STORM

Stochastic optical reconstruction microscopy

STED

Stimulated emission depletion

UV

Ultraviolet

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  4. Examples of Practical Applications: Sample Preparation
  5. Conclusion
  6. Acknowledgments
  7. Abbreviations
  8. Literature Cited
  9. Key References
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Key References

  1. Top of page
  2. Introduction
  3. Principles and Practical Aspects of Confocal Fluorescence Microscopy
  4. Examples of Practical Applications: Sample Preparation
  5. Conclusion
  6. Acknowledgments
  7. Abbreviations
  8. Literature Cited
  9. Key References
  • Masters, B. R. 2005. Confocal Microscopy and Multiphoton Excitation Microscopy. SPIE Press, Bellingham, Washington.
  • An excellent introduction into the principles and applications of confocal and multiphoton microscopy.
  • Claxton, N. S., Fellers, T. J., Davidson, M. W. Laser scanning confocal microscopy. Available at http://www.olympusfluoview.com/theory/LSCMIntro.pdf. Accessed November 2, 2011.
  • Richly illustrated at-depth discussion of LSCM.
  • Prasad, V., Semwogerere, and Weeks, E. R. 2007. Confocal microscopy of colloids. J. Phys. Condens. Matter 19: 113102, 25 pp.
  • A comprehensive review of the confocal 3D imaging of colloidal systems.
  • Smalyukh, I. I., Shiyanovskii, S. V., and Lavrentovich, O. D. 2001. Three-dimensional imaging of orientational order by fluorescence confocal polarizing microscopy. Chem. Phys. Lett. 336: 8896.
  • A description of the FCPM technique for imaging of the orientationally ordered materials such as liquid crystals.