Endogenous Fluorophores
A number of endogenous fluorophores e.g., NADH, flavins producing autofluorescence have proved extremely useful for bioimaging. While using exogenous fluorophores, the autofluorescence produced by the endogenous fluorophores gives rise to undesirable background. But in some cases, endogenous fluorophores prove to be useful for monitoring cellular processes. Some of them also exhibit sufficiently strong two-photon excited emission, making them useful for cellular bioimaging. Some of the most...
Photonics And Biomaterials
The previous chapters have dealt with the applications of interactions of light with biological materials for optical diagnostics and light activated therapy. In Introduction to Biophotonics, by Paras N. Prasad. ISBN 0-471-28770-9 Copyright 2003 John Wiley amp Sons, Inc. Figure 16.1. Interconnection of the various key areas to produce paradigms for new generation information technology. Figure 16.1. Interconnection of the various key areas to produce paradigms for new generation information...
u y u y u
Q Biological Q Anti-biologic IgG d IgG Fiber Figure 9.22. Schematics of a Mach-Zehnder interferometer biosensor. where M is the modulation factor and AO is the relative phase shift between the two arms. If the initial conditions are adjusted so that the relative phase shift AO is zero, the binding of an analyte to the sensing layer on the sample arm channel waveguide introduces an additional phase shift AOsens, given as Heideman and Lambeck, 1999 where Lint is the interaction length of the...
DarkField Microscopy
Another technique commonly used for contrast enhancement is dark-field illumination Abramowitz, 1987b Davidson, 1999 . In this case, the sample is illuminated at an angle that cannot be accepted by the objective's aperture. In this case only the highly diffracted rays enter the objective. Hence only highly scattering or diffracting structures can be observed using this technique. Dark-field illumination requires blocking off the central light that ordinarily passes through and around the...
Twophoton Photodynamic Therapy
As discussed in Section 12.2 on photosensitizers, currently most photosensi-tizers in clinical applications are being photoactivated using a light source in the range 630-690nm. At this wavelength, the tissue penetration defined by 1 e loss of intensity see Chapter 6 is about 2-4 mm and the photodynamic effect is generally seen up to 2-3 times deeper than that. As a result, the largest attainable depth of PDT-induced cellular changes could reach up to 15 mm, but in most cases it is much less...
Organization Of Cells Into Tissues
A tissue is a multicellular bioassembly in which cells specialized to perform a particular task contact tightly and interact specifically with each other. The functions of many types of cells within tissues are coordinated, which collectively allows an organism to perform a very diverse set of functions such as its ability to move, metabolize, reproduce, and conduct other essential functions. The various constituents forming tissue are shown in Table 3.6. Figure 3.21 presents a schematic view...
References Asf
Andronov, A., Gilat, S. L., Frechet, J. M. J., Ohta, K., Neuwahl, F. V. R., and Fleming, G. R., Light Harvesting and Energy Transfer in Laser Dye-Labeled Poly Aryl Ether Dendrimers, J. Am. Chem. Soc. 122,1175-1185 2000 . Bigham, S. R., and Coffer, J. L., The Influence of Adenine Content on the Properties of Q-CdS Clusters Stabilized by Polynucleotides, Colloids and Surfaces A 95, 211-219 1995 . Birge, R. R., Protein-Based Optical Computing and Memories, IEEE Comput. 25, 56-67 1992 . Birge, R....
NearField Imaging
Near-field scanning optical microscopy NSOM can provide images with a resolution less than the wavelength of light. Therefore, in principle, it is of great value in studies of biological structures such as viruses and bacteria, whose dimensions are often in submicrons. Conventional optical microscopy, with its inherent limitations on resolution, and electron microscopy SEM and TEM , with rigid sample preparation requirements, cannot be used to image biological samples in in vitro conditions and...
Imaging of Nucleic acids
Nucleic acid stains are usually cationic dyes that bind with the polyanionic nucleic acid, thus showing specificity for DNA and RNA. Furthermore, hydrophilic hydrophobic interactions play an important role in a providing some dyes with the ability to intercalate between the nucleic acid bases and b providing other dyes with the ability to bind with double-stranded nucleic acids in grooves. In double-stranded DNA, the alignment of the strands is antiparallel and asymmetric along the axis. This...
NATURE OF LIGHT 211 Dual Character of Light
The description of the nature of light provided here is at the very basic level. It serves to review the concepts that most readers already may be familiar with. Light is an electromagnetic field consisting of oscillating electric and magnetic disturbances that can propagate as a wave through a vacuum as well as through a medium. However, modern theory, quantum mechanics, also imparts a particle-like description of light as energy packets called photons or quanta Atkins and dePaula, 2002 . This...
Photosynthesis
Photosynthesis is another example of Nature's utilization of biophotonics in a very clever way to harness light and utilize photon energy for conducting various cellular processes to sustain life. Photosynthesis occurs in plants, algae, and a variety of bacteria these organisms utilize sunlight to power various cellular processes. A good general coverage of the topic of photosynthesis is provided by Ksenzhek and Volkov 1998 . A true appreciation for this marvelous photosynthetic machinery of...
Immobilization
The various physical or chemical methods used to immobilize a biorecogni-tion element an enzyme, antibody, etc. are discussed extensively in the literature Boisde and Harmer, 1996 Kuswandi et al., 2001 . A brief discussion of this topic is presented here. Physical Methods. The simplest physical method is containment within semipermeable membranes. A number of optical fiber sensors have utilized this technique. Another method calls for adsorption on a solid support. Depending on the nature of...
Preface
Biophotonics deals with interactions between light and biological matter. It is an exciting frontier which involves a fusion of photonics and biology. It offers great hope for the early detection of diseases and for new modalities of light-guided and light-activated therapies. Also, biology is advancing photonics, since biomaterials are showing promise in the development of new photonic media for technological applications. Biophotonics creates many opportunities for chemists, physicists,...
Bioimaging Principles and Techniques
Bioimaging using optical methods forms a major thrust of biophotonics. Optical bioimaging can be used to study a wide range of biological specimens, from cells to ex vivo tissue samples, to in vivo imaging of live objects. Optical bioimaging also covers a broad range of length scale, from submicron size viruses and bacteria, to macroscopic-sized live biological species. This chapter describes the basic principles and techniques used for optical bioimaging. Thus it is intended to provide the...
Metallic Nanoparticles And Nanorods For Biosensing
Other types of materials used for biosensing are in the form of metal nanopar-ticles and nanorods. Storhoff and Mirkin 1999 linked a single-stranded DNA, modified with a thiol group at one terminal, to a gold nanoparticle 15nm in diameter via strong gold-sulfur interactions, discussed in Chapter 9. The 15-nm-diameter gold particles exhibit well-defined surface plasmon resonance, a topic also discussed in Chapter 9. Due to this resonance, the individual gold particles, even when attached to DNA,...
Biophotonicsa New Frontier
We live in an era of technological revolutions that continue to impact our lives and constantly redefine the breadth of our social interactions. The past century has witnessed many technological breakthroughs, one of which is photonics. Photonics utilizes photons instead of electrons to transmit, process, and store information and thus provides a tremendous gain in capacity and speed in information technology. Photonics is an all-encompassing light-based optical technology that is being hailed...
TwoPhoton Fluorophores
Availability of high-peak power pulse laser sources led to pioneering work by a number of groups on multiphoton proceses in organic systems Rentzepis et al., 1970 Fredrich and McClarin, 1980 Birge, 1986 . Since the original work of Denk et al. 1990 , the field of two-photon laser scanning microscopy has witnessed phenomenal growth. Much of the impetus has been derived from the availability of new fluorophores with considerably enhanced two-photon emission efficiency Bhawalkar et al., 1996 . In...
Numerical Aperture and Resolution
The resolution of a microscope is its ability to distinguish between the smallest possible objects. This is directly related to the cone of light entering the objective from the sample. But, this optical resolution is limited by the diffraction of light occurring from the object, due to the wave nature of light. This principle can be understood by looking at a beam of light passing through a pinhole Abbe, 1873 Born and Wolf, 1999 . The image produced by the light passing through a pinhole and...
Data Manipulation And Presentation
This section describes the steps of data acquisition, storage, analysis, and display. Important steps in data acquisition also include calibration of the instrument, spectral compensation, and, sometimes, the use of gating. These topics are also discussed here. Calibration. Often the performance of a flow cytometer is calibrated by using inert and stable standards that are nonbiological particles of dimensions comparable to biological cells. For this purpose, fluorescently labeled micro-beads...
Biosensors An Introduction
Biosensors are analytical devices that can detect chemical or biological species or a microorganism. They can be used to monitor the changes in the in vivo concentrations of an endogenous specie as a function of a physiological change induced internally or by invasion of a microbe. Of even more recent interest is the use of biosensors to detect toxins, bacteria, and viruses because of the danger posed by chemical and biological terrorism. Biosensors thus find a wide range of applications...
References Muo
Aubin, J. E., Autofluorescence of Viable Cultured Mammalian Cells, J. Histochem. Cytochem. 27,36-43 1979 . Bassnett, S., Reinisch, L., and Beebe, D. C., Intracellular pH Measurement Using Single Excitation-Dual Emission Fluorescence Ratios, Am. J. Physiol. 258, C171-C178 1990 . Bastiaens, P. I., and Squire, A., Fluorescence Lifetime Imaging Microscopy Spatial Resolution of Biochemical Processes in the Cell, Trends Cell Biol. 9, 48-52 1999 . Bhawalkar, J. D., He, G. S., and Prasad, P. N.,...
Optical Aberrations and Different Types of Objectives
The two major distortions or aberrations Davidson and Abramowitz, 1999 in optical microscopy are i chromatic aberration which is due to the different refractive indices of the glass optical element such as a lens for different wavelengths and ii geometrical or spherical aberrations due to the shape of the lens, in which the rays from the edges of the lens don't get focused at the same point where the axial rays focus. Both these aberrations can be corrected by using lens doublets consisting of...
Laser Tissue Regeneration
Laser-induced tissue regeneration is an exciting prospect to repair tissue damage after an injury. Since the early report of low-level light therapy for wound healing Mester et al., 1971 , there have been numerous reports of effects of light on wound healing and tissue regeneration Basford, 1996 . Many investigators report visible and IR radiation at relatively low fluences irradiation densities of 1-4J cm2 stimulates capillary growth and granulation of tissue formation Basford, 1986 . However,...
Differential Interference Contrast Microscopy DIC
Differential interference contrast is a technique that converts specimen optical path gradients into amplitude differences that can be visualized as improved contrast in the image. This is accomplished by using a set of modified Wollaston prisms Abramowitz, 1987b Davidson, 1999 . In this technique, living or stained specimens, which often yield poor images when viewed in bright-field illumination, are made clearly visible. Today there are several implementations of this design, which are...
In Vivo Imaging
Optical techniques like fluorescence imaging and optical coherence tomography, adapted for in vivo imaging, can complement other techniques such as MRI, CT, and ultrasound, for diagnosis and therapy. Imaging technologies that can provide the same kinds of cellular and in vivo molecular information, currently available only from in vitro techniques, would be very useful for molecular profiling of diseases and for monitoring their progression. A major thrust of in vivo imaging is in the early...
Optical Biosensors
The field of biosensors has emerged as a topic of great interest because of the great need in medical diagnostics and, more recently, the worldwide concern of the threat of chemical and bioterrorism. The constant health danger posed by new strands of microbial organisms and spread of infectious diseases is another concern requiring biosensing for detecting and identifying them rapidly. Optical biosensors utilize optical techniques to detect and identify chemical or biological species. They...
Future Directions Dvb
Flow cytometry is a rapidly expanding field worldwide where an enormous increase in its capability can be expected over the coming years. As indicated in the beginning of this chapter, there has been renewed interest in flow cytom-etry from the point of view of research where a major impetus is derived from its applications to genomics and proteomics. Recent advances in solid-state lasers, microfluidics, microarray technology, micro-optics, and miniaturized detectors provide challenging...
The Components of a Flow Cytometer
A flow cytometer consists of a light source, which in all modern flow cytome-ters is a laser, and illumination optics to focus the laser beam on to a flowing biological cell or a polystyrene microsphere, which is fluorescently labeled. The scattered laser light and the fluorescence response are separated and focused onto photodetectors. A special electronics processes the optical response and controls a sorter if it is provided. These components of a flow cytometer are described in detail here....
Principles of Laser Action
The basic principle of the laser, as the name light amplification by stimulated emission of radiation indicates, is based on stimulated emission from a higher level f to a lower level i not necessarily the ground state Svelto, 1998 . As discussed in Chapter 4, this will require that a population inversion is created so that the number of atoms or molecules in level f is higher than that in level i i.e., Nf gt N . Most lasers utilize electronic levels for laser actions. A widely used carbon...
Laser Tissue Contouring And Restructuring
The two specific applications discussed here are for the use of lasers in der-matologic and ophthalmologic procedures. The theory of selective photother-molysis, introduced by Anderson and Parrish in 1981, is the basis for much advancement in dermatological lasers Anderson and Parrish, 1983 . It allows for highly localized destruction of light absorbing targets in skin, with minimal damage to the surrounding tissue. To achieve selective photother- molysis, an appropriate wavelength, exposure...
Benzoporphyrin Derivatives
Benzoporphyrins are fused-ring chlorin derivatives produced by cycloaddition reactions see Structure 12.3 . The mono-acid benzoporphyrin derivative, called veteroporfin also labeled BPD-MA , absorbs at 690 nm with a molar extinction coefficient of 35,000M-1cm-1 Sternberg et al., 1998 . Phase I and II clinical trials of this photosensitizer show rapid tumor accumulation and reduced skin photosensitivity. A benzoporphyrin derivative has been approved for the treatment of age-related macular...
Biomaterials for Photonics
Photonics, which utilizes light-matter interactions for information processing, transmission, data storage, and display, is being hailed as the dominant technology for the 21st century. The continued development of photonics technology is crucially dependent on the availability of suitable optical materials. Biomaterials are emerging as an important class of materials for a variety of photonics applications. This chapter describes potential applications of various types of biomaterials for...
Bioderived Materials
This section presents selected examples of naturally occurring biomaterials or their chemically derivatized forms that have been investigated for photonics. Among those are Bacteriorhodopsin for holographic memory Green fluorescent proteins for photosensitization Biocolloids for photonics crystal media Bacteriorhodopsin. Bacteriorhodopsin often abbreviated as bR grows in the purple membrane of a salt marsh bacterium known as Halobacterium salinarium or Halobacterium halobium Birge et al., 1999...
Kohler Illumination
One of the most commonly used illumination system in a transmission microscope is Kohler illumination, which provides an evenly illuminated field of view with a bright image, without glare and minimum heating of the specimen. Furthermore, as discussed below, it is important to have an illumination scheme where the sample is illuminated with a cone of light as wide as possible to achieve the best resolution possible. This feature is realized in the Kohler illumination scheme as shown in Figure...
Design Of A Laser Tweezer
Block 1998 provides a good description of the construction of optical tweezers. The single-beam optical trapping system used at our Institute for Lasers, Photonics, and Biophotonics is described here as an example of a basic design for a laser tweezer. The optic layout of this laser trap unit is shown in Figure 14.4. The main components of an optical trap used in this design are as follows 1. A Microscope Either Upright or Inverted with a High-Numerical-Aperture Objective Lens. Some typical...
The Human Eye and Vision
Structure. At a very simple level, the visual system is comprised of the eye Figure 6.6 and a long chain of neural connections. The pattern of excitation within the retina is processed by a neuronal machinery to create sensory perception in our brain. The light from an object enters the eye through the clear and transparent cornea. The pupil, an opening of the iris, in the front of the eye regulates the amount of light allowed to enter the eye. Much like the diaphragm in a camera, the iris...
Nanochemistry
Nanochemistry is an active new field that deals with confinement of chemical reactions on nanometer length scale to produce chemical products that are of nanometer dimensions generally in the range of 1-100 nm Murray et al., 2000 . The challenge is to be able to use chemical approaches that would reproducibly provide a precise control of composition, size, and shape of the nano-objects formed. These nanomaterials exhibit new electronic, optical, and other physical properties that depend on...
Cellular Interactions Probed by FRETFLIM Imaging
FRET, as discussed in Chapter 7, is a phenomenon that occurs when two different chromophores with overlapping emission donor and absorption acceptor spectra are separated by a distance in the range 10-80 A. Recently, the introduction of the green fluorescent protein GFP to FRET-based imaging has provided great impetus to the study of noncovalent molecular interactions inside cells Jovin and Arndt-Jovin, 1989 Herman, 1989 Gadella et al., 1999 Bastiaens and Squire, 1999 . Mutagenic variants of...
Fluorescence Lifetime Imaging Microscopy Flim
Fluorescence lifetime imaging microscopy, often abbreviated as FLIM, provides a spatial lifetime map of a fluorophore within a cell or a tissue Tadrous, FLUORESCENCE LIFETIME IMAGING MICROSCOPY FLIM 239 2000 Bastiaens and Squire, 1999 . The use of fluorescence lifetime of a fluo-rophore as an imaging contrast mechanism offers a number of advantages over steady-state fluorescence microscopy. First, the fluorescence lifetime is a highly sensitive probe of the local environment of the fluorophore....
Light Absorption in Cells
Proteins are the most abundant chemical species in biological cells. They also are the most diversified chemical unit in living systems, from smaller-sized enzymes to larger proteins and from colorless to highly colored systems. They also exhibit a diversity of functions, from carrying oxygen to providing a light-induced neurological response for vision. The basic constituents building blocks of proteins are amino acids, which can be aliphatic or aromatic containing benzene or fused benzene...
Molecular Beacons
The molecular beacon approach is also based on the electronic energy transfer scheme between a fluorescent unit Fl and a fluorescent quencher Q Tan et al., 2000 . A molecular beacon consists of a loop and a stem. The loop structure involves a single-stranded oligonucleotide in a specific sequence. The stem usually consists of five to seven complementary base pairs. The two ends of the stem consist of a fluorophore Fl and a fluorescence quencher Q . In the absence of the analyte, the stem is...
Some Recent Novel Sensing Methods
Photonic Crystals Sensors. Photonic crystals are ordered dielectric arrays that diffract light at wavelengths determined by the lattice spacing between the arrays and the average refractive index of a structure Carlson and Asher, 1984 Asher, 1986 John, 1987 Yablonovitch, 1987 . One example of a photonic crystal is a closely packed colloidal array as shown in Figure 9.26. This crystal was prepared at our Institute, using 200-nm polystyrene spheres. These spheres were floated, as a suspension,...
References Byk
Abbe, E., Beitrage zur Theorie des Mikroskops der microskopischen Wahrnehmung, Schultzes Arch. Mikr. Anat. 9, 413-468 1873 . Abramowitz, M., Microscope Basics and Beyond, Olympus Corporation Publishing, New York, 1987a. Abramowitz, M., Contrast Methods in Microscopy Transmitted Light, Olympus Corporation Publishing, New York, 1987b. Abramowitz, M., Fluorescence Microscopy The Essentials, Olympus America, New York, 1993. Amos, W. B., White, J. G., Fordham, M., Use of Confocal Imaging in the...
Hollow Tube Waveguides
Another light delivery system involves a hollow tube made of a metal, ceramic, or plastic Cossman et al., 1995 . The inside wall of the tube is coated with a high reflector. The laser light is propagated down the tube by reflection from the inner wall. The advantage of using this type of plastic waveguide over an articulated arm is its semiflexibility. The plastic waveguides generally have an inner diameter of 1mm and an outer diameter of 2mm. An important recent development in this area is the...
J
Virus Prokaryotic cells Bacteria Size scale 20-200nm Size scale 1-10mm Structure Single- or double- Structure Single-cell organism consisting of stranded RNA or DNA single closed compartment that lacks a Function Infectious but defined nucleus not self-replicating Function Free-living and self-replicating Eukaryotic cells Animal plant cells Size scale 10-100mm Structure Complex structure surrounded by a lipid membrane, contains an organized nuclear structure Function Self-replicating and able...
LightInduced Cellular Processes
Cells exhibit a wide variety of photophysical, as well as photochemical, processes followed by light absorption. Some of these processes are shown in Table 6.2. A number of cellular constituents fluoresce when excited directly or excited by energy transfer from another constituent Wagnieres et al., 1998 . This fluorescence is called endogenous fluorescence or autofluorescence, and the emitting constituent is called an endogenous fluorophore also called fluorochrome . As discussed in Chapter 4,...
Femtolaser Surgery
An area of growing interest is the use of ultra-short pulsed femtoseconds lasers for surgery and tissue ablation Juhasz et al., 2002 . The advantages offered by these ultra-short pulses are that cuts or ablations can be made more precisely, with very little collateral damage. The mechanism of laser-tissue interactions that occur using ultra-short laser pulses is also different from the photothermal and photoablation mechanisms discussed above because they pertain to tissue contouring and...
Highlights Of The Chapter Fxs
Bionanophotonics refers to research and applications that involve both biomedical sciences and nanophotonics. Nanophotonics involves light-matter interactions on nanoscale. It is another exciting frontier dealing with nanoscale optical science and technology. Nanoscale light-matter interactions can be manifested in two ways i by confining the light on nanoscale with the use of a near-field geometry such as that in near-field microscopy, discussed in Chapters 7 and 8, and ii by confining the...
Vibrational Spectroscopy
Vibrational spectroscopy comprises IR spectroscopy and Raman spectroscopy Chalmers and Griffiths, 2002 . In IR spectroscopy, the absorption of an IR or far IR photon produces a change in vibrational levels. The selection rule for a vibrational transition using a harmonic oscillator model discussed above is Dv 1 for any vibrational mode. Overtone Dv gt 1 absorption is possible, but it is much weaker. The overtone absorption in water is, however, important in some wavelength ranges i.e., 1.9 im....
Laser Safety
A laser as a high-intensity light source providing concentrated photon energy in a small beam size also poses potential safety hazards. Therefore, it requires a laser operator to be aware of these hazards and to exercise appropriate caution while using lasers. A good source of information on laser safety is the booklet Laser Safety Guide Marshall and Sliney, 2000 , published by the Laser Institute of America. This section only provides a brief discussion of the possible laser hazards and some...