Cell Structure and Function by Microspectrofluorometry

By Elli Kohen

Cell Structure and Function by Microspectrofluorometry provides an overview of the state of knowledge in the study of cellular structure and function using microspectrofluorometry. The book is organized into six parts. Part I begins by tracing the origins of modern fluorescence microscopy and fluorescent probes. Part II discusses methods such as microspectroscopy and flow cytometry; the fluorescence spectroscopy of solutions; and the quantitative implementation of fluorescence resonance energy transfer (FRET) in the light microscope. Part III presents studies on metabolism, including the mechanism of action of xenobiotics; biochemical analysis of unpigmented single cells; and cell-to-cell communication in the endocrine and the exocrine pancreas. Part IV focuses on applications of fluorescent probes. Part V deals with cytometry and cell sorting. It includes studies on principles and characteristics of flow cytometry as a method for studying receptor-mediated endocytosis; and flow cytometric measurements of physiologic cell responses. Part VI on bioluminescence discusses approaches to measuring chemiluminescence or bioluminescence in a single cell and measuring light emitted by living cells.

• Language: English
• Publisher: Academic Press
• Release date: Jun 28, 2014
• ISBN: 9781483269733

Book preview

3135–3139.

PART I

HISTORY

Outline

Chapter 1: The Origins of Modern Fluorescence Microscopy and Fluorescent Probes

1

The Origins of Modern Fluorescence Microscopy and Fluorescent Probes

FREDERICK H. KASTEN,     Department of Anatomy, Louisiana State University Medical Center, New Orleans, Louisiana

This review of the history of fluorescence microscopy and fluorescent probes emphasizes the roots of modern work in the field and contemporary lines of research. It centers on significant accomplishments and the pioneers involved. The contribution is presented as a survey rather than an exhaustive review. To conserve space, bibliographic citations are restricted largely to a small number of significant articles and reviews. Further details and references are given in The Development of Fluorescence Microscopy up through World War II (21). Other fundamental sources of information are De Ment’s volume on Fluorochemistry (5) and Radley and Grant’s Flourescence Analysis in Ultra-Violet Light (34). The volumes by Wang and Taylor (41a, 42c) give a modern perspective of fluorescence microscopic technology applied to living cells.

I Introduction

II The First Fluorescence Microscopes

III Technical Progress

IV Advances in Biomedical Applications

A Autofluorescence 15

B Vital and Supravital Fluorochroming 21

C Intravital Fluorochroming 22

D Introduction of Fluorescent Dyes in Histology and Cytology. 24

V Modern Fluorescence Microscopy in Cell and Molecular Biology

A Acridine Orange in Cell Biology

B Other Fluorescent Probes

C Flow Cytometry

VI Development of Immunofluorescence

References

I INTRODUCTION

The description of the phenomenon of fluorescence dates back to 1838. At that time, David Brewster, a Scottish preacher and experimentalist in the field of optics, observed internal dispersion from the mineral fluorspar, a natural fluoride of calcium, and from solutions of quinine and chlorophyll. The term fluorescence was coined by George Stokes, a physicist and professor of mathematics at Cambridge. Fluorescence is ordinarily considered to be light emission induced during excitation. The related term phosphorescence refers to light that persists after the exciting light is turned off. In his famous monograph of 1852, Stokes described the results of his pioneering and ingenious experiments to reveal refrangible radiations from many biological materials. He used mirrors to direct sunlight, the source of ultraviolet (UV) light, through a solution of cuprammoniam (cupric hydroxide in ammonia water) as the primary filter, onto the specimen, and then through a yellow barrier filter created by a potassium dichromate solution. What is known today as Stokes’s law states that the fluorescent light is always of a longer wavelength than the exciting light. This law was extended in 1875 by Eugen Lommel, a physics professor from Munich, who stated that a body must first absorb radiation in order to exhibit fluorescence.

Other nineteenth century physicists who contributed to the emerging field were Edmond Becquerel and Eilhart Wiedemann. By 1868, it was clear that fluorescent spectra were characteristic of specific substances. The term Fluoreszenzanalyses was devised at this time by F. Goppelröder, and spectral characterization by fluorescence analysis was quickly accepted by organic chemists. Two important volumes that contributed to the adoption of the technique were those by Peter Pringsheim, Fluoreszenz und Phosphoreszenz im Lichte der neueren Atom-theorie, and P. W. Danckwortt, Lumineszenz-Analyse im Filtrierten Ultravioletten Licht, both of which went through numerous editions.

With the startup of the synthetic dye industry by the English teen-aged chemist William Perkin in 1856, new fluorescent dyes were synthesized as well (22). Listed in Table I are the biologically important fluorochromes, dates when they were first synthesized, and applications in fluorescence microscopy and biology.

Table I

FLUOROCHROMES USED IN BIOLOGICAL MICROSCOPYa

aThe word fluorochrome was coined by Haitinger in 1934 to denote fluorescent dyes used in biological staining to induce secondary fluorescence in tissues. Data listed above were derived from many sources. In addition to obtaining information from published research articles, other material was assembled from the Colour Index (3rd ed., 1971–1976), Reichert’s Fluorescence Microscopy with Fluorochromes. Recipes and Tables (2nd ed., 1952), Conn’s Biological Stains (R. D. Lillie, 9th ed., 1977), Histochemistry Theoretical and Applied (29), Staining Procedures (20), Handbuch der Farbstoffe für die Mikroskopie (H. Harms, 1965), and catalogues of the Aldrich Chemical Co., Eastman Kodak Co., Polysciences, Inc., and Sigma Chemical Co.

II THE FIRST FLUORESCENCE MICROSCOPES

Fluorescence microscopy was made possible because of the ultraviolet absorption microscope, which was invented by August Köhler of Zeiss/Jena in 1904 (Fig. 1). Quartz monochromatic UV objectives had been developed a few years earlier by Moritz von Rohr, and Köhler constructed the microscope and photographic systems. The duo aimed to improve resolution by utilizing UV absorption by cells (Fig. 2). Köhler noted the emission of fluorescence in the visible range when a crystal of barium platinum cyanide was used as a test object for fluorescence in the microscope. However, the optical results were defective because the monochromatic lenses were designed for work in the UV rather than the visible range. At a vacation course on scientific microscopy held in Vienna in 1908, Köhler and Siedentopf put together an improved microscope to demonstrate fluorescence. This ad hoc instrument, which represented a first attempt at a fluorescence microscope, used for illumination a line of 275 nm from a cadmium spark. Although Köhler recognized the long-term potential of his preliminary observations, he did not pursue this line of work. Nevertheless, Köhler should be credited with laying the groundwork for fluorescence microscopy.

Fig. 1 August Köhler, inventor of the ultraviolet microscope, forerunner of the fluorescence microscope.

Fig. 2 Photomicrographs of diatoms taken with the ultraviolet microscope by Köhler, using von Rohr’s monochromatic objectives to demonstrate resolution.

The breakthrough and stimulus for the first fluorescence microscope was the discovery in 1903 by Robert Wood (Fig. 3), professor of experimental physics at Johns Hopkins University, that a band of longwave UV radiation (approximately 300–400 nm) could be isolated from arc lamps by a dye solution of nitrosodimethylaniline.² In 1910, H. Lehmann of Carl Zeiss/Jena combined Wood’s filter solution with gelatin, added a copper sulfate solution to a separate chamber, and surrounded the two parts with Jena blue–Uviol glass. When placed in front of an iron arc lamp, the entire assembly transmitted a rich spectrum of longwave UV (relatively free of visible light). Carl Zeiss/Jena supplied Lehmanns filter to many researchers. Indeed, a competitor, Carl Reichert of Vienna, recognized the possibilities created by Lehmann’s modification of Wood’s filter. In the following year, Reichert promoted the development of a fluorescence microscope built around a new dark-field quartz condenser. The details of this newly constructed Reichert fluorescence microscope were described by O. Heimstädt in 1911 (Fig. 4). The microscope utilized an iron carbide or carbon arc lamp as a source of UV excitation, a quartz lens to focus the light on the quartz condenser through Wood’s liquid filter, a quartz slide with a conventional coverslip, and an objective and eyepiece. The condenser and objective were immersed in glycerine.

Fig. 3 Robert Wood, a physicist at Johns Hopkins University at the beginning of this century. Wood, an expert in physical optics, developed the so-called Wood’s filter used to isolate ultraviolet light from arc lamps. He is shown here in his personal barn laboratory working on a mercury telescope. Photograph supplied by Marilyn E. Moran of Department of Physics, The Johns Hopkins University.

Fig. 4 Heimstädt’s fluorescence microscope, first developed at the Reichert Company in 1911. Note the enclosed carbon arc lamp at the left (from O. Heimstädt, 1911. Z. wiss. Mikr. 28:330–337).

Early in 1912, about the same time that the Reichert microscope came on the market, H. Lehmann described and demonstrated a brand new Carl Zeiss/Jena Lumineszenzmikroskop at a meeting in Münster. The following year, Lehmann published a long article in which he discussed the current state of knowledge with respect to fluorescence, described the new fluorescence microscope, and pointed out its potential applications.

Both Lehmann and Heimstädt utilized powerful Siemens arc lamps requiring 2000 or 3000 W of power in their fluorescence microscopes. This enormous expenditure of energy utilized expensive, cumbersome power equipment that produced much heat and noise. Also, there were potentially lethal electrical and UV hazards to microscopists. The Reichert and Zeiss microscopes were very similar with respect to light source, the employment of quartz lenses in the illuminating apparatus, normal glass lenses in the microscope, and their use only with transmitted light. The main difference was that Zeiss used a bright-field condenser. The dark-field condenser of Reichert had the advantage of blocking the ultraviolet from entering the objective. However, it had the disadvantage of cutting down the energy concentrated onto the specimen.

In his seminal work on the chemotherapy of infectious diseases, Paul Ehrlich employed fluorescent dyes as part of the chemical armamentarium in his search for magic bullets. The action of fluorescent dyes in sensitizing organisms to light was a subject of great interest to pharmacologists and experimental therapeutists. Research on light inactivation by fluorescent dyes (photodynamische Wirkung) was heightened with the publication in 1907 of the book Die Sensibilisierende Wirkung Fluoreszierender Substanzen by H. von Tappeiner and A. Jodlbauer. Werbitzki showed in 1909 that acridine dyes were effective in treating trypanosomes.

With the availability of the new fluorescence microscope, it was only a matter of time before someone would use this new instrument in conjunction with fluorochromes to study dye influence on living microorganisms. In 1914, von Provazek added various fluorescent dyes and drugs (fluorescein, eosin, neutral red, quinine) to cultures of the ciliated protozoan Colpidia and observed the induced fluorescence of living cells in the microscope. In Provazek’s own words, the object was:

To introduce into the cell certain substances of different types, without regard to whether they are stains or colorless drugs, on the assumption that they follow definite distribution laws and collect under certain circumstances in particular functional elements inside the cell so that they effectively illuminate the partial functions of the cell in the dark field of the fluorescence microscope.

These prophetic words heralded a new approach in understanding cell structure and function. A few physiologists recognized the value of fluorescence microscopy. For example, W. M. Bayliss in his 1920 textbook on Principles of General Physiology remarked that by means of fluorescence observations, previously invisible materials in the cell could be revealed.

The years between the two World Wars represented a period of great advancement in the field of fluorescence microscopy. There were substantial developments in components and apparatus and in biomedical applications, notably vital and supravital fluorochroming, intravital fluorochroming of organ microcirculation, autofluorescence detection of tissue components (porphyrins and other animal pigments, vitamins), and use of fluorochromes in histology and microbiology. After the Second World War, there followed metachromatic and cytochemical applications of acridine orange, development of other fluorescent probes, video intensification microscopy, the confocal laser scan microscope, flow cytometry, and immunofluorescence. A summary of these accomplishments follows.

III TECHNICAL PROGRESS

The Wratten and Wainwright firm of England was creating color filters early in this century. Their Wratten filters had been produced under this name already in 1907. Charles E. K. Mees, a scientist associated with the company, wrote a book in 1909, Atlas of Absorption Spectra. The following year, he and his company published a manual on photomicrography in which the use of color filters was discussed. In 1912, the Wratten and Wainwright concern was bought out by the Eastman Kodak Company, who continued to produce the well-known Wratten filters and filter booklets that later included detailed absorption and fluorescence spectra.

During the 1920s, various technical advancements were made by Metzner, a microscopist from Greifswald. He replaced Lehmann’s UV filter with an improved glass filter, introduced a quartz prism in the system, and transferred the pale yellow Euphos coverslip to the ocular as a more convenient place for the barrier filter. His contributions are summarized in his book, Das Mikroskop (26). In France, several research workers (Albert Policard, Eugene Derrien, Jean Turchini) made fluorescence microscopic observations at low magnifications using a Greenough observation microscope (binocular dissecting-type microscope) in conjunction with a mercury vapor lamp. Minor modifications introduced during the early 1930s were a water-filled flask to intensify and focus light, a Reichert incident reflecting condenser, an annular illuminating tube, and oblique illumination.

The ordinary UV light sources available were the Hanau low-pressure mercury vapor lamp and the Osram point lamp. The mercury lamps were sufficient to illuminate microscope areas at low magnifications up to about 160× but failed to induce sufficient fluorescence at higher magnifications to allow resolution of cytologic details. More expensive arc lamps used metallic carbon and iron arcs, which were included in redesigned lamp assemblies of new Zeiss and Reichert fluorescence microscopes (1929–1931). In 1932, following the reports by Ellinger and Hirt of their innovative intravital microscope, Leitz/Wetzler produced a new fluorescence microscope, known as the Ultropak. This used a lateral illumination system for incident light observation.

New high-pressure mercury-vapor arc lamps were introduced by the General Electric Company, beginning about 1935. They were placed in special housings for microscope use by the Bausch & Lomb and the American Optical companies. The power ratings of these new lamps were 100 W (AH-4), 250 W (AH-5), and 1000 W (AH-6, water-cooled). High-pressure mercury arc lamps were produced as well by Osram (HBO-200), Phillips (CS 150), and Hanovia. Other ultraviolet arc sources included mercury-xenon, mercury cold cathode, xenon, and zirconium or hafnium concentrated arcs. When the fluorochrome acridine orange was introduced into cytology in 1940 by Siegfried Strugger, he showed that it was unnecessary for UV light to be used: blue light was sufficient to excite the cellular-bound dye and to induce fluorescence. This made it possible to use tungsten filament lamps for fluorescence microscopy. Modern epi-fluorescence microscopes utilize halogen (100, 250 W), mercury (HBO 50, 100, 200), xenon (XBO 75, 150), or tungsten filament (100 W) as illumination sources. The confocal laser scan microscope employs an argon laser as a light source.

In 1938, just prior to the Second World War, modern primary, or excitation (UV-transmitting), and secondary, or barrier (UV-absorbing), filters were on the market. UV-transmitting filters, referred to as black glass filters, were available from Corning, Hanauer, Kodak, Sendungen/Berlin, and Schott/Jena (UG1, UG2). Schott/Jena also supplied a popular GG4 pale yellow barrier filter for the eyepiece. Wood’s liquid filter, which had served so well in the early days of fluorescence microscopy, became a relic of the past.

This section would be incomplete if the work of Barnard and Welch were not mentioned. In 1936 and 1937, they demonstrated both UV absorption and fluorescence results attainable at medium and oil-immersion magnifications using state of the art equipment. Apparently sparing no expense, they employed ideal high-intensity lamps and power supply together with quartz lenses, slides, and coverslips to permit observations. Although the emphasis of their work was on absorption, they demonstrated more cytologic detail in autofluorescing bacteria and fluorochromed tissues by fluorescence photomicrographs than could be observed by UV-absorption, dark-field, or light-transmission microscopy. However, the introduction of fluorescent dyes into histology and cytology made it feasible to use longwave UV and violet-blue visible light as excitatory wavelengths. Other investigators found it practical to use standardized, less expensive commercial fluorescence microscopes with glass lenses for this research. Monocular microscopes were generally used, because the intensity of fluorescence was insufficient for observations with the binocular system. Later, anti-reflection films were coated on air-glass surfaces to reduce loss of light. As a consequence, binocular fluorescence microscopes became practical after the Second World War.

Filter combinations are now provided as changeable components of modern fluorescence microscopes. These are designed to transmit the greatest amount of excitation light to the object and of emission light to the eyepiece or film. If the absorption and emission peaks of the fluorochrome are not well separated, as with fluorescein isothiocyanate (FITC)-stained preparations, the transmission curves of the primary and secondary filters must have sharp slopes with little overlap. Both glass color filters and multilayer interference filters are available with sharp cutoff features.

With narrow-band filter combinations, good contrast is obtainable between the fluorescing specimen and the background because of the half-width of 25 nm, but at the expense of total intensity. Wideband filters with half-widths of more than 50 nm may also be usable, according to the spectral characteristics of the fluorochrome. Microscopes today generally come equipped with filters that permit viewing of both the green fluorescence from FITC-stained material and red fluorescence from Rhodamine or Texas red-stained material. These dyes are the ones most commonly used in the immunofluorescence detection of antigens. Immunocytochemical applications probably account for at least 50% of all fluorescent studies. In addition to these filter combinations, special ones are available for specific fluorochromes. Heat-absorbing filters are placed in front of all the other filters to block the infrared portion of the light.

One of the major technical advances in the incident-light fluorescence microscope is the introduction by Brumberg in 1948 and subsequently by Ploem in 1967 (29b) of the dichroic mirror or dichromatic beam-splitting plate. With this device in the system, the exciting light is deflected through 90° and directed through the objective lens and on the object. The longer wavelength fluorescent light emitted by the specimen is transmitted back through the objective and the beam-splitting dichroic mirror into the viewing tube. The original source light is returned in the direction of the source. There is no condenser in the system and the dichroic mirror with interference coatings serves also as a barrier and excitation filter. The Brumberg-Ploem beam splitter and filter system is highly efficient and is present as an integral component in modern epi-illumination fluorescence microscopes.

Lamps in common use in modern fluorescence microscopes are high-pressure mercury arcs, xenon arcs, and tungsten-halogen lamps. The mercury arc lamps emit their energy at specific wavelengths corresponding to lines of the mercury spectrum. Xenon arc lamps have a continuous spectrum and are helpful for fluorophores whose excitation peaks do not coincide with a mercury line, as with FITC. Tungsten-halogen lamps are relatively cheap but do not give off intense light.

With the fluorescence phenomenon light is absorbed by a fluorophore and immediately re-emitted in about 10−8 sec. Phosphorescence is similar to fluorescence but emitted light may last from 10−3 sec or longer. The detection and measurement of cellular phosphorescence has been described by Polyakov in 1967 with a phosphorescence microscope referred to as a microspectrophosphorimeter. There was one other report of a phosphorescence microscope by Parker in 1969. A number of observations were noted with the Soviet instrument based on ultraviolet excitation of cells and recordings of visible phosphorescence spectra and decay curves at room temperature and −180°C. It was reported that the spectrum of microorganisms is specific for each species (Zotikoff and Polyakov). Cultured human fibroblasts exhibit a phosphorescence peak at 455 nm with deep cooling, which corresponds to the maximum of phosphorescence of DNA (44).

The reviews on fluorescence microscopy by Philipp Ellinger (9) and Oscar Richards (35) are rich sources of detailed information on the older literature concerned with microscope equipment, filters, light sources, tissue preparation, and evaluation and applications. Other summaries of the method are given in Kasten’s review of the history of fluorescence microscopy (21). The chapter by Rost (35a) should be consulted for a recent discussion of fluorescence microscope technology and applications.

IV ADVANCES IN BIOMEDICAL APPLICATIONS

A AUTOFLUORESCENCE

The earliest microscopic observations of autofluorescence (Eigenfluoreszenz) were done by Hans Stübel in 1911. Stübel, a physiologist at Jena University, was apparently the first person to make use of the newly invented fluorescence microscope. He systematically investigated the autofluorescence of bacteria, protozoa, and various animal organs such as teeth. Also, he surveyed many organic substances of biological origin, such as albumin, chitin, elastin, gelatin, glycogen, hemoglobin, keratin, and melanin. He confirmed that hemoglobin and melanin did not fluoresce. Lehmann, a co-inventor of the fluorescence microscope, reported in 1913 its use for examining ashed materials. Another early study was that of R. Wasicky, who published in 1913 on the fluorescence properties of different natural fibers and plant products. Using the Zeiss/Jena luminescence microscope, Robert Heller reported in 1916 that individual alkaloids could be identified by their characteristic fluorescence. Other investigators noted the autofluorescence of plant tissues, chlorophyll, elastic fibers, lens of the eye, and thyroid colloid. Attempts were reported by C. Kaiserling between 1917 and 1938 to discriminate between different bacterial pathogens based on their autofluorescent properties.

1 Porphyrins

An interesting series of investigations during the 1920s was done on porphyrin pigments. Iron-containing respiratory pigments of animals (hemoglobin, myoglobin) fail to fluoresce, whereas their iron-free breakdown derivatives (protoporphyrin, hematoporphyrin, uroporphyrin, coproporphyria and phylloporphrin) emit a striking red fluorescence, confirming an observation made in the nineteenth century by Felix Hoppe-Seyler, the father of biochemistry.

Two different French groups, led by Eugène Derrien and Jean Turchini³ of the Faculty of Medicine at Montpellier and by Albert Policard⁴ of the Faculty of Medicine at Lyon, carried out extensive fluorescence microscopic observations of animal porphyrins in the 1920s. Since specific microscopic identification of porphyrins was generally lacking at the time, the occurrence of red fluorescence in tissues was considered diagnostic. A review of tissue fluoroscopy, emphasizing the French studies, appeared in 1925 (31). Further investigations on tissue porphyrins were carried forward in 1929 (1b) by Bommer, who detected porphyrin-like fluorescence in sebaceous plugs of human hair follicles, and in 1934 (35d) by Seggel, who observed that a small percentage of circulating erythrocytes contained protoporphyrin. Other fluorescence studies during the 1930s centered on the detection of porphyrins in patients with porphyria diseases. Later, Frank Figge and Allan Grafflin reexamined porphyrin fluorescence in the Harderian gland of rodents, previously discovered by Derrien and Turchini. Porphyrins were investigated in tumors by Frank Figge.

Specific chemical identification of individual porphyrins rested on fluorescence spectroscopic analysis of pigments extracted from tissues. Pioneering work on this group of compounds was done by Charles Dhéré⁵ of the Institute of Physiology in Fribourg, Switzerland. Dhéré had already begun his momentous work on the porphyrins in 1909, with his thesis for the degree of doctor of natural science, Récherches spectrographiques sur l’absorption des rayons ultra-violets par les albuminoides, les protéides et leurs dérivés (University of Paris). His researches in optical chemistry culminated in a remarkable book, La Fluorescence en Biochimie (6). The data obtained by Dhéré made it possible to diagnose the porphyrial diseases through urine analysis and formed the basis for an important piece of work done by M. Borst and H. Königsdörffer, Jr., of Munich. They carried out exhaustive microchemical and microspectrographic analyses of tissues from a single patient, M. Petry, who had died of congenital porphyria. The tissues were examined with a Heimstädt-Reichert fluorescence microscope that was modified and adapted for visual, microspectrographic, and microspectrophotometric measurements using a commercial microspectrograph (Fig. 5). Individual porphyrins were fingerprinted in tissue sections by their identifying fluorescent spectra, which had been obtained photometrically from a photographic plate. This outstanding work by Borst and Königsdörffer was described in detail in their book, Untersuchungen über Porphyrie mit Besonderer Berücksichtingung der Porphyria congenita (2). This sophisticated microspectrographic approach to the differentiation of cellular porphyrins antedated by 6–13 years the microspectrophotometric studies of von Euler and co-workers on riboflavin fluorescence (9a), Caspersson’s work on the ultraviolet absorption analyses of nucleic acids and proteins (3a), and the fluorescence histospectrophotometric investigations of deLerma (4h). Although many pathologists were certainly aware of Borst and Königsdörffer’s new analytical approaches to cellular disease, the work was essentially ignored or overlooked by histochemists and by quantitative cytochemists of the next era. Beginning in 1959, kinetic studies of reduced coenzyme systems were investigated in mitochondria of living cells with the aid of a differential microfluorimeter and flow cytofluorometer (Chance and Thorell; Kohen). The spatiotemporal organization of cell metabolism has been investigated by Kohen et al.