Circular polarization is when the electric field of light is made up of two linear components perpendicular to one another, of the same amplitude, but with a phase difference of π/2. The electric field that results will rotate in a circle about the propagation direction and, depending on the rotation direction, is referred to as right- or left-hand circularly polarized light.

The process which causes light waves to vibrate in a single plane is called polarization of light.

This essential explains what distinguishes light sheet microscopy from ordinary light microscopy. The author briefly exa

A new fluorescence microscopy technique has come to the fore that is faster and less phototoxic than other microscopy techniques.

Light sheet microscopy

H90: Closed transmittance, k1k2, is the transmittance of two polarizers oriented for minimum transmission in unpolarized incident light.

Polarizing Cube Beamsplitters consist of a pair of precision right-angle prisms carefully optically contacted or cemented together to minimize wavefront distortion. A dielectric coating is placed onto the hypotenuse of one of the prisms. Polarizing beamsplitters are designed to split the light into two –reflected S-polarized and transmitted P-polarized beams. They can be used to split unpolarized light at a ratio of 50/50, as well as for polarization separation applications, including optical isolation.

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Elliptical polarization is when light’s electric field describes an ellipse. This is caused by a combination of two linear components with different amplitudes or a phase difference that isn’t π/2. Elliptical polarization is the most common description of polarized light, while circular and linear polarized light can be looked at versions of elliptically polarized light.

Precision linear polarizers are constructed by laminating a thin, stretched and dyed polymer polarizing film between two high-precision AR coated glass or fused silica windows. The polymer has been stretched and stressed in one direction to align the long polymer molecules to create a filtering effect, which allows light waves oscillating parallel to the direction of the stress to pass through, while blocking their polarization. The compact component that results is ideal for flux densities below 1 W/cm2. Polymer polarizers are used throughout the visible spectrum.

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Superior high-energy polarizer performance is achieved through advanced coating design and meticulous production procedures. These optics have been developed for use in some of the most demanding lasers in the world. Some polarizer coatings have been optimized for use with Nd:YAG lasers. When these polarizers are mounted at Brewster’s angle, extinction ratios exceed 100:1. Thin film polarizers have also been optimized for ultrashort pulses. These thin film polarizers have been designed to provide superior performance in ultrafast Ti: Sapphire regenerative amplifiers. While pulse lengths are relatively long in these amplifiers, pulse dispersion is still a concern if pulse width is to be maintained in the recompressed pulse. Multiple round trips in the amplifier have a multiplying effect on the dispersive characteristics of any optic in the cavity. For this reason, substantial effort has been made in designing and testing these polarizers for minimum pulse dispersion.

Once the second edition was safely off to the printer, the 110 larger world of micro-CT and micro-MRI and the smaller wo

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A polarizer is an optical component that is designed to filter, modify or analyze the polarization states of light. Polarizers can be integrated into optical systems to increase contrast, decrease glare or to measure changes in temperature, magnetic fields or gauge chemical reactions.

Light sheet fluorescence microscopy

The mechanism of polarization in a dichroic polarizer is selective absorption and transmission of incident radiation. Dichroic is the selective polarization absorption of the anistotropic polarizating material, also called diattenuation. Anisotropic means that a material exhibits the physical property that it has a different value when it is measured in different directions. Examples include oriented polymer molecules and stretched nanoparticles. Dichroic polarizers exhibit limited damage thresholds and environmental stability, with glass dichroic polarizers performing better than plastic dichroic polarizers in these areas. Dichroic polarizers are useful when very large apertures are needed for an application. They are also used for microscopy, imaging and display applications.

This research-level reference provides a review of the morphological techniques that have become a primary method of ana

Light sheet microscopyusing an Airy beam

k2: Minor transmittance or blocking efficiency is the transmission of linearly polarized light with the polarizer oriented for minimum transmission.

Defined relative to the plane of incidence of the light ray on a surface, there are two orthogonal linear polarization states that are important for reflection and transmission, p- and s-polarization. P-polarized light (from the German word parallel) has its electric field polarized parallel to the plane of incidence. S-polarized light (from the German word senkrecht) is perpendicular to the plane of incidence.

L Dyer · 2022 · 11 — As such, a decade after its introduction, light sheet fluorescence microscopy was named Method of the Year by Nature Methods for 2014.

Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution

These specialty optical fibers allow only one polarization state to propagate. The light introduced that has any other polarization direction will have significant optical loss and won’t be propagated through the fiber. Polarization fibers are designed to exhibit extreme birefringence, resulting in only light with the desired polarization direction being guided through the fiber, with all other polarization directions having very high losses. Polarization fibers offer several advantages over in-line polarizers, including lower insertion loss, higher extinction ratio, as well as no complicated component assemblies and packaging.

Confocalmicroscopy

Calcite linear polarizers use birefringence in crystalline materials to modify the polarization of incident light. The transmission of the desired polarization and the deviation of the remaining light is directly related to birefringent materials’ index of refraction, as well as the angles of the cut between the crystals. Crystalline polarizers usually are made up of two birefringent crystals cut and aligned at specific crystalline axes in order to attain a particular polarization behavior outcome. Crystalline polarizers offer a high optical purity, which is ideally suited for a wide variety of laser applications that require high damage thresholds with optimized extinction ratios. These polarizers feature high extinction ratios up to 100,000:1 and are contained in a mountable anodized aluminum housing. The polarizers include Glan-Laser Calcite Polarizers, Glan-Thompson Calcite Polarizers, Rotatable Glan-Thompson Calcite Polarizers and Wollaston Calcite Polarizing Prisms.

This calculator will calculate typical beam intensity min/max ranges for an ILT Light Measurement System you may be interested in purchasing.

H0: Open transmittance, (k12 + k22) / 2, is the transmittance of two polarizers oriented for maximum transmission in unpolarized incident light.

Polarization in fiber optics is a very important characteristic that can be utilized in any fiber optic measurements or systems. Fiber-Optic Polarization Control products include Manual Polarization controllers, Polarization Beam Combiners and Splitters, Fiber Optic In-line Polarizers, Fixed Ratio Porlarization Maintaining Couplers, Fiber Optic Faraday Rotator Mirrors and Fiber Optic Depolarizers.

Optically sectioned imaging by oblique planemicroscopy

Dec 16, 2021 — Light energy initiates the process of photosynthesis when pigments absorb the light. Organic pigments have a narrow range of energy levels that they can absorb.

Table of contents : What You Can Find in This essentialPrefaceContentsAbout the Author1 Introduction 1.1 Standard Microscopes 1.2 Laminae Lucis2 Beyond Resolution 2.1 The Alchemists and the Red Glass 2.2 Ultramicroscopic Particles3 Biological Photography 3.1 In der Beschränkung zeigt sich erst der Meister 3.2 Back to Unlimited Freedom 3.3 Versatile Illumination4 Glittering Particles and Colorful Molecules 4.1 Lightning Trap for Particles 4.2 Microscopic Fireflies5 OPFOS—Good Ideas and the Acronymic Suffering6 Recent Developments 6.1 A Look into Life 6.2 The Return of the Scan System 6.3 Self-Healing Radiation 6.4 A Little Over-Resolution After All 6.5 Two-Way Light Sheets 6.6 Rubber Lenses 6.7 Matrix—New Edition 6.8 The Vertical TurnWhat You Learned From This essentialReferences

A guide to light-sheetfluorescence microscopyfor multiscale imaging

Linear polarizers exhibit polarizing properties that are usually defined by a degree of polarization efficiency (P) and its extinction ratio (ρp), which can vary with wavelength and incident angle.

Structural Equation Modeling is a statistical method increasingly used in scientific studies in the fields of Social Sci

k1: Principal transmittance or insertion loss is the transmission of linearly polarized incident light with the polarizer oriented for maximum transmission.

Since light is an electromagnetic wave, its wave has an electric field, and this wave oscillates perpendicular to the direction of propagation. Unpolarized light has the direction of this electric field fluctuating randomly in time. Examples of unpolarized light include the sun’s light, halogen lights, LED spotlights and incandescent lightbulbs. Polarized light’s electric field has a well-defined direction. Laser light is the most familiar example of polarized light. There are three kinds of polarizations, depending on how the electric field is oriented:

LED Light Source - Light Source for Fiber optic Product.

Advances in Biological Science Research: A Practical Approach provides discussions on diverse research topics and method

Figure 7: In the rest frame of the moving camera (left,with the picture according to Figure 5), the direction of the light rays is different from that in ...

light sheetmicroscopy中文

Upon striking the sample, photons that match the energy gap of the molecules present (green light in this example) are absorbed, exciting the molecules. Other ...

Acceptance angle is the maximum deviation from the design incidence angle where the polarizer will still perform within its specifications. Angles of incidence of 0° or 45° or at Brewster’s angle is where most polarizers are optimally designed to work.

Rolf Theodor Borlinghaus The Light-Sheet Microscopy Biological Structural Research in a Lateral View essentials Springer essentials Springer essentials provide up-to-date knowledge in a concentrated form. They aim to deliver the essence of what counts as "state-of-the-art" in the current academic discussion or in practice. With their quick, uncomplicated and comprehensible information, essentials provide: • an introduction to a current issue within your field of expertis • an introduction to a new topic of interest • an insight, in order to be able to join in the discussion on a particular topic Available in electronic and printed format, the books present expert knowledge from Springer specialist authors in a compact form. They are particularly suitable for use as eBooks on tablet PCs, eBook readers and smartphones. Springer essentials form modules of knowledge from the areas economics, social sciences and humanities, technology and natural sciences, as well as from medicine, psychology and health professions, written by renowned Springer-authors across many disciplines. More information about this subseries at http://www.springer.com/series/16761 Rolf Theodor Borlinghaus The Light-Sheet Microscopy Biological Structural Research in a Lateral View Rolf Theodor Borlinghaus Microscopia Palatina Sinsheim-Eschelbach, Germany ISSN 2197-6708 ISSN 2197-6716 (electronic) essentials ISSN 2731-3107 ISSN 2731-3115 (electronic) Springer essentials ISBN 978-3-658-32767-5 ISBN 978-3-658-32768-2 (eBook) https://doi.org/10.1007/978-3-658-32768-2 © Springer Fachmedien Wiesbaden GmbH, part of Springer Nature 2021 The translation was done with the help of artificial intelligence (machine translation by the service DeepL.com). A subsequent human revision was done primarily in terms of content. This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Responsible Editor: Stefanie Wolf This Springer imprint is published by the registered company Springer Fachmedien Wiesbaden GmbH part of Springer Nature. The registered company address is: Abraham-Lincoln-Str. 46, 65189 Wiesbaden, Germany What You Can Find in This essential • • • • An explanation of how ordinary and light-sheet microscopy differ What this technique has to do with alchemy How the technique with the light sheets was developed in photography Latest developments in the field of light-sheet microscopy v Preface Microscopes fascinate everyone. That’s probably because they provide insights that go beyond everyday experience. The same applies—albeit to a much lesser extent—to telescopes. And certainly the aesthetic gain also contributes to the fascination. A hearing aid does not work like this. It is always just an aid to compensate for defects, not a key that opens up new worlds of experience. In addition, microscopy has established itself as an extremely important tool in research and technology. Biology in particular would not be what it is today if there were no microscopes. Throughout history, there have always been phases in which it was thought that the end of the development of microscopic technology had to be reached. And then, with further developed methods, completely different dimensions opened up. And indeed, in the last 40 years light microscopy has been completely turned upside down by completely new methods. (You may want to reprimand the many superlatives, but they are really appropriate!) Nevertheless, old inventions still play a major role, and occasionally the synthesis of the current and the old results in something completely new. And seen in the light of day: Everything invented today is, of course, based on the foundation of the past. Light-sheet microscopy is not quite new—the first commercial device was already available 100 years ago. It was developed for colloid chemistry. After a long period of rest, it entered its successful phase in biology about 25 years ago. Nowadays, a prediction is usually expected as to how things will continue. This expectation remains unfulfilled. History is too unsteady to make serious predictions. Let us rather stay “on” and let us be surprised. This much can be predicted: there will still be surprises. vii viii Preface By the way: The colored illustrations are available for the readers of the b/w print work to understand as additional material for free download at www.spr inger.com on the product page of this book. Eschelbach (Germany) and La Serena (Chile) in October 2016 Rolf Theodor Borlinghaus Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1 Standard Microscopes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Laminae Lucis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 2 2 Beyond Resolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 The Alchemists and the Red Glass . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Ultramicroscopic Particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 5 6 3 Biological Photography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 In der Beschränkung zeigt sich erst der Meister . . . . . . . . . . . . . . . . 3.2 Back to Unlimited Freedom . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Versatile Illumination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 11 13 14 4 Glittering Particles and Colorful Molecules . . . . . . . . . . . . . . . . . . . . . . . 4.1 Lightning Trap for Particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Microscopic Fireflies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 17 19 5 OPFOS—Good Ideas and the Acronymic Suffering . . . . . . . . . . . . . . . 21 6 Recent Developments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1 A Look into Life . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 The Return of the Scan System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3 Self-Healing Radiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4 A Little Over-Resolution After All . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5 Two-Way Light Sheets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.6 Rubber Lenses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 25 26 28 29 30 31 ix x Contents 6.7 Matrix—New Edition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.8 The Vertical Turn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 32 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 About the Author Rolf Theodor Borlinghaus was awarded his doctorate in 1988 under Prof. Dr. Peter Läuger at the Institute of Biophysics at the University of Konstanz. He was working in Marketing and Product Management for two famous German optical companies for over 30 years and retired in 2020 for health reasons. He is also a freelance author, field botanist, and master in the art of living. xi 1 Introduction Summary What is the difference between ordinary microscopy and light-sheet microscopy? Before that the question arises: What is an ordinary microscope anyway? Usually, one assumes that it is a device with which objects can be viewed in a magnified form. Usually, the object is radiated through, so the light source is on the other side of the specimen in relation to the observer. In the past, when there were no electronic computers, game consoles or mobile phones, young people were occasionally given a microscope for Christmas. It was a black tube that was made stable by means of a foot. Underneath the tube was a movable mirror that allowed sunlight to be threaded into the tube. An object could be inserted between the tube and the mirror. At the upper end of the tube you could look into it. This is what a microscope looks like. Of course I also had such a device. But the quality of that microscope was so bad that it was not enough to ignite an enthusiasm for microscopy. So if you want to do something good for your children, give them a good microscope and do without electronic gadgets. Your children will be worth half a month’s salary after all? 1.1 Standard Microscopes The famous microscopes of Antonie van Leeuwenhoek around 1700 looked quite different and formally they were highly magnifying glasses. To magnify very strongly with a magnifying glass, the object (and also the observer) must be brought very close to the lens. For this reason alone it is easier to view the specimen in transmitted light. Nevertheless, reflected light microscopy, in which light falls on the specimen, is not a modern invention: Robert Hooke already described in detail a sophisticated device for incident light illumination in his famous book © Springer Fachmedien Wiesbaden GmbH, part of Springer Nature 2021 R. T. Borlinghaus, The Light-Sheet Microscopy, Springer essentials, https://doi.org/10.1007/978-3-658-32768-2_1 1 2 1 Introduction “Micrographia” from 1665 ([1]: Scheme 1, Fig. 5). In any case, a light-sheet microscope is not a transmitted light microscope and therefore not comparable to the junior microscope described above. The further development of microscopy initially very much promoted the transmitted light method. Small objects are usually thin and therefore rather transparent. And if they are too thick, they are cut into thin slices. This led to the development of microtomes. Between 1900 and 1980 microscopy was a fairly stable subject and there were only here and there mostly gradual improvements. Only fluorescence microscopy has taken off, after the coupling of fluorescent molecules with antibodies to make the appropriate antigens in tissue visible [2]. This method was later transferred to DNA fragments and thus made it possible to carry out the “in situ hybridization” [3], initially developed with radioactive substances, with fluorescence (FISH technique). These two methods really turned biology upside down. Since about 1980, new microscopy methods have been developed, which were especially aimed at improving the visibility of small details. This was achieved in two ways: On the one hand, the resolution itself was increased, which is now theoretically possible up to any level of accuracy (overview in [4]), and on the other hand, the blurred parts were suppressed as far as possible, which is what confocal microscopy does (overview in [5]). Light-sheet microscopy itself is initially not a method for improving optical resolution. Like confocal microscopy, it is a method in which optical sections are generated which can then be reconstructed to form a coherent image. It is also possible to limit the observation to only one plane, but without disturbing unsharpness from other planes. Often images from the same plane are also taken as a time series—in this way one obtains a film in which one can observe structural changes, such as in the development of embryos. Spatio-temporal changes in the concentration of metabolites or ions can also be observed in this way. 1.2 Laminae Lucis So what’s the deal with these ominous sheets of light? If you belong to the somewhat older generation, you may remember romantic moments when you sit in a barn whose plank façade is not completely sealed, but here and there a narrow gap allows sunlight to pass through. In the past, the builders of such barns were excellent pragmatists and not so much freedom-averse perfectionists. It was usually very dusty in such barns, and the incident sunlight transformed the otherwise rather gloomy scenery into a fairy-tale building with many different and surprising light effects. The sunlight was scattered by the many millions of dust particles 1.2 Laminae Lucis 3 Fig. 1.1 Sunlight falls through a metal grille into a dusty barn. The dust particles scatter the light in all directions and can therefore be seen from outside the beam path (dark field). Apart from the geometry, this is already “light-sheet photography.” and the volume penetrated by the radiation appeared as a bright, luminous structure (Fig. 1.1). Such impressions have become rare in our modern dustless and sterile boring world. If you are lucky enough to live in an area that has not been devastated by land consolidation, you can observe similar effects in fog in the wild. To be able to see this phenomenon, the observer must be outside the beam of light, that is, in darkness. The observed region itself is just as dark, only the scattering particles stand out against the dark background. In microscopy, such methods are therefore called “dark field microscopy” (which has nothing to do with the grape variety “Dunkelfelder”). 2 Beyond Resolution Summary Although light-sheet microscopy is a very current topic—it was voted “method of the year” in 2014 [9]—and although some would like to call the method “modern,” the idea is already comparatively old: Over 100 years. As already noted, the method is not one of those that can improve optical resolution across the diffraction-limited barrier. Nevertheless it was invented to make the invisible visible. A contradiction? 2.1 The Alchemists and the Red Glass The question began around the year 1680, when a busy glass alchemist had made an exciting invention in Berlin. Johann Kunkel had succeeded in producing red glass [6]. This was a result of alchemistic research, and—as usual in alchemy—it consumed a lot of gold. Typically human, there was also a dispute about priorities—and thus about money, influence, and power. Before Kunkel, JC Orschall [7] already described recipes how to make red glass. The alchemists of that time had in mind to produce gold from other substances. It was a common hypothesis that by “inoculating” with a suitable substance (the philosopher’s stone) any material could be converted zymologically into gold. As a plausible example, a dough was used which, by adding a small amount of sourdough, would be transformed into sourdough in its entirety. As no one had any idea about bacteria and yeasts and their effects, this was a perfectly reasonable, logical, and scientifically sound thesis. Only younger charlatans have used alchemy as a stepping stone to get money out of the pockets of other immature people with all kinds of questionable signs and mysterious sounding names. The many obscure remedies that they can buy for expensive money on the Internet are the heirs of this strategy today. Science © Springer Fachmedien Wiesbaden GmbH, part of Springer Nature 2021 R. T. Borlinghaus, The Light-Sheet Microscopy, Springer essentials, https://doi.org/10.1007/978-3-658-32768-2_2 5 6 2 Beyond Resolution itself has evolved and today the conversion of other elements into gold is a topic of nuclear physics and the effort is many orders of magnitude higher than the return—it is not worth it. This above mentioned red glass, usually called “gold ruby glass,” is a suspension of very small grains of gold in a solid glass matrix, a colloid. If the size of these particles is less than the diffraction-limited Abbe resolution [8], which in practice is about 200 nm, then the particles cannot be seen under a normal microscope, especially their size cannot be measured. Even if a particle can no longer be resolved, it still causes a disturbance because the light waves are “bent” a little even on very small particles. Sunlight is scattered by oxygen molecules, so we see the sky in blue, at least during daytime on a clear day. The color is due to the fact that this form of scattering is much more effective for blue light than for longer wavelengths. The small particles of metallic gold are not only good scattering objects, but they also absorb electromagnetic energy. After all, the outer electrons in a metal are not firmly bound to an atom, but can move relatively freely as an “electron cloud”—this is what makes the metal what it is. In the small grains of gold, these electron clouds can now be made to vibrate against the atomic nuclei, in much the same way that soup sloshes back and forth in a plate when you knock it from the side. The oscillation frequency cannot be arbitrary, but follows quantum mechanical rules. The particle can therefore only absorb photons of certain energy. Through the absorption the electron gas receives an energy quantum, which is called plasmon. Since the energy of the quantum determines the color of the light, a suspension of very small gold particles appears colored. If particles of about 30 nm are used, light in the blue-green region of the spectrum is absorbed and then scattered in all spatial directions. Therefore, the suspension—here the gold ruby glass—appears deep red in transmitted light (Fig. 2.1). 2.2 Ultramicroscopic Particles The desire was now to estimate the size of these particles. Since it is below the resolution limit, they are not visible in a normal microscope. Today it might be possible to use contrast-enhancing methods with cameras to make the disturbances visible in a transmitted light image. The transmitted light image shows only a very weak modulation—not visible to the eye—almost the entire range of brightness values remains empty. With contrast enhancement, this empty part of the gray area is cut off, in the simplest case this is done by electronically shifting the zero line. The remaining small modulation of brightness is 2.2 Ultramicroscopic Particles 7 Fig. 2.1 Gold ruby glass with the typical red coloration in transmitted light of a desk lamp. The red coloration comes from the absorption of short wavelengths. Red light is not absorbed, so in transmitted light only the red part of the white light is visible. Origin of the glass: Farbglashütte Reichenbach GmbH; Reichenbach/OL. Mechanical processing: Optical training workshops, Leica in Wetzlar then amplified so extremely that the diffraction figures generated by the particles become visible. Of course, the background is very much disturbed by blurred image parts of other layers, so that hardly usable images can be generated. The analogous contrasting method, “video microscopy” [10] led in the 1950s to the proof of the spindle apparatus during cell division and was for a few years the “hit” in biological microscopy. However, the answer to the question of how large the particles in gold ruby glass might be came as early as 1903 [11]. The colloid researcher Richard Zsigmondy and the physicist Henry Siedentopf developed a device at Carl Zeiss in Jena that was supposed to make these gold particles visible. Since the contrast is too small in transmitted light and too much blurred information in reflected light prevents the identification of the particles, they used a dark field method. In dark field, the specimen is illuminated at an angle that is outside the cone for observation, Fig. 2.2 left. The extreme case of such a dark field illumination is a right angle to the optical axis of the microscope, Fig. 2.2 right. Thus the principle of light-sheet microscopy is actually already described. To create such an effect in the gold ruby glass, Siedentopf and Zsigmondy used a light source whose radiation could be limited in height and width by a slit; the size could be adjusted with micrometer screws. This slit was then imaged into 8 2 Beyond Resolution Fig. 2.2 Left: Dark field. If the illumination (thick, blue arrow) is at an angle that does not fall within the aperture angle of the objective lens (spanned by the two finer, red arrows), the background (the field) remains dark for the observer. Right: The extreme case is an illumination perpendicular to the optical axis of the microscope. This is the case in light-sheet microscopy the glass to be examined. As a result, a thin layer within the glass at an angle of 90 degrees to the optical axis is illuminated (Fig. 2.3). The scattering at the gold particles then leads to bright spots against a black background in the observing D L Od H P Oc S T Fig. 2.3 Gap ultramicroscope by Henry Siedentopf (schematic). The light source L, here the sun, is imaged onto an adjustable slit S by means of a heliostat H using a telescope T. This slit is then transferred to the specimen P by means of an objective lens OC . There a thin layer, adjustable in height and width, is strongly illuminated. Small particles (here: Small gray circular area) scatter the light also perpendicular to the illumination axis and can be transferred to the detector D with an additional objective lens OD 2.2 Ultramicroscopic Particles 9 Fig. 2.4 Stars in a glass. The piece of gold ruby glass from Fig. 2.1 in the light-sheet microscope (left) and in the confocal microscope (right). The difference is only to be found in the magnification scale and the size of the diffraction discs, both of which are determined by the optics used. A section of the starry sky offers exactly the same impression, because here too only the diffraction patterns of distant objects are visible, which cannot be resolved for us here, so the sources appear quasi point-like part of the microscope—if everything is carefully adjusted and the density of the particles is not too high (Fig. 2.4 left). The authors called the particles that are not visible in an ordinary microscope “ultramicrons,” from Latin “ultra” to English “beyond,” that is, beyond the light microscopic resolution limit. “Microns” would then be particles that can be seen and measured in a microscope. Today, the term “nanoparticle” is used for ultramicrons, which is by no means more helpful, from Latin “nanus” the “dwarf” and Latin “particula,” which means “particle.” The microscope received the sweeping name “slit ultramicroscope.” The unsharp parts can also be eliminated in a confocal microscope. The result is shown in Fig. 2.4 on the right. Both methods produce optical sections and deliver very similar images. So, to sum up: The slit ultramicroscope had the following characteristics: 1. The illumination is perpendicular to the optical axis of the microscope (maximum dark field) 2. The geometry of the illumination is selected by a slit diaphragm in such a way that only the thinnest possible layer in the preparation is illuminated (light sheet). 10 2 Beyond Resolution 3. By adjusting the position of the preparation in the z-axis (“focusing”), different planes in the substrate can be examined. The success of this device was that the contrast could be increased by several orders of magnitude. Contrast is the ratio of signal (what I want to see) to background (what I don’t care about). The background in the dark field microscope is black. And the light sheet construction ensures that even blurred background information disappears from other layers. In order to be able to examine liquid suspensions of colloidal gold, a device was later developed which made it possible to bring different solutions one after the other directly under the objective without much preparation effort. This was achieved by means of a chamber whose supply line could be charged with the different solutions. This was an early perfusion device, which is now indispensable in the microscopy of living cells or tissues. And how can the size of the particles be determined if they can be detected but not measured? The authors have used the two most obvious methods for this purpose. First, they were able to calculate the volume in which the particles become visible, that is, the thickness of the light sheet multiplied by the area of the illuminated field of view. How much gold was present in the glasses or suspensions was precisely specified during production and was therefore known. With this information it is also possible to calculate what mass of gold must be present in the illuminated volume. Now one only needs to divide this mass by the counted quantity of particles, et voilà—one has the average mass of the particles, which leads to the diameter of the particles using the specific weight of gold. Instead of counting the particles, you can also measure their distances from each other, from which the number in the volume can be estimated. Both methods lead to similar results. However, one must always bear in mind that the size of such particles is distributed around a mean value. The shape of the distribution cannot be deduced from such measurements at first. After all, particle sizes down to 6 nm could be detected back then—smaller than 1/30 of the optical resolution! At this point, another instrument should be mentioned which was also developed in cooperation with Carl Zeiss and is very similar to the slit ultramicroscope. The light-section method of Gustav Schmaltz [18] also produces a light sheet by means of a slit. This was used to illuminate surfaces of workpieces made of metal or ceramics. The reflected light has the shape of the profile of the surface and could be recorded by a camera. In this way, geometries can be measured and defects in such objects can be detected. The term “light-section” is still in use today in technical applications, but has not been introduced in biological microscopy. 3 Biological Photography Summary From a completely different angle, there were developments in the middle of the last century that basically led in the same direction. Surely you have often regretted that in photographs, part of the image is always out of focus. This is especially noticeable in close-ups and when you have to work with a small aperture because of the light conditions. The reason for this is the limited depth of field: The image is only really sharp in one plane. Before and after that, we let something pass as “sharp” if we cannot resolve the blur caused by our limited vision. In everyday life, our smart brain blocks out everything that is not sharply imaged on the retina— seeing is therefore a highly subjective process. It is only when we take a photograph that we notice the disappointing quality of the image—the optics cannot be cheated. Although cameras for modest use, such as mobile phones, are already beginning to incorporate cheating in the form of obligatory image processing. Such a device— or more precisely: The manufacturer—deprives you of your sovereignty over the interpretation of what you see and externally determines your perception. Be alert! 3.1 In der Beschränkung zeigt sich erst der Meister1 The blurred camera images thus result from the fact that information not only falls from the focal plane onto the film (or chip), but also image components from the blurred planes in front of and behind it. A confocal microscope cuts off these parts and thus provides optical sections [5]. But at the expense of time: The image must 1“ the art is how to set the limits” Goethe, JW from 1815: The Sonnet. © Springer Fachmedien Wiesbaden GmbH, part of Springer Nature 2021 R. T. Borlinghaus, The Light-Sheet Microscopy, Springer essentials, https://doi.org/10.1007/978-3-658-32768-2_3 11 12 3 Biological Photography be built up sequentially point by point. Only one single point is illuminated at a time. More restrictions cannot be imposed on the image formation process. In classical microscopy, an attempt is made to outwit the problem by cleverly restricting the object: A thin section, such as can be made with a microtome, will only provide information from this thin skin of specimen. And if the section is thinner than the depth of field in the microscope with the optics just used, then there will be no blurred parts in the image. This is the reason why so much effort is put into cutting techniques and embedding between two flat glass surfaces. In reflected light microscopy, similar results are obtained by elaborate polishing of the surfaces of opaque objects such as minerals or metals. Scientific photography has gone one step further to achieve “extreme depth of field in microscopy” [12]. The basic idea was: Instead of laboriously processing the specimen, it is also possible to illuminate only a small part of the specimen. And if you do it skillfully, only such an area which is also sharply imaged by the camera. How can this be done? In principle the answer has already been given by Siedentopf and Zsigmondy. Whether the work from 1903 was on the table when the “extreme focal depth” microscope was developed can probably no longer be reconstructed. The two methods nevertheless look very similar. The starting point for the microscope with extreme depth of focus is also illumination through the narrowest possible slit (Fig. 3.1). Such an illumination slit was created here by spraying a thin film of black paint on a glass and then drawing a very thin line in this film with a razor blade. With suitable optics, this slit can be imaged back into the specimen. As with the slit ultramicroscope, the photograph is taken at right angles to the illumination. The illuminated layer must be thinner than the depth of field of the observation optics, here the microscope objective lens. A distinction is made between “depth of field” and “depth of focus” [13]. Depth of field refers to the depth of field in object space, that is, the area from the specimen that we perceive as sharp. On the image side of the camera lens is the sensor, today a semiconductor element, only a few years ago a film emulsion. This sensor must now also be located within the sharpness range of the image produced by the optics, that is, within the depth of focus. This question was even more important then than today, when films were still being used, which always had certain unevennesses that had to be smaller than this image-side depth of focus. With an illumination that is only limited within the depth of field, the image consequently does not contain any blurred parts, since everything that is blurred remains in the darkness. The result is an optical section, whereby the 3.2 Back to Unlimited Freedom 13 C O P I S L Fig. 3.1 Design of the microscope with extreme depth of focus (according to [12]). A light source L illuminates a thin slit S. This slit is imaged into the specimen P with illumination optics I and generates a light sheet along the illumination axis. (The illumination is yellow.) A camera C is used for observation, and its objective lens O sharply images a region of the specimen. This area is the depth of field, indicated by the outer, red curved lines and is perpendicular to the observation axis. The light sheet is thus embedded in the depth of field of the camera. Consequently, only sharp image portions are recorded (yellow, thicker line section on the surface of the specimen). To image the entire object, the specimen can be moved continuously along the observation axis (double arrow). An image is produced whose depth of field is defined by the traversing range, which theoretically can be as large as desired section is already produced on the illumination side—in contrast to the confocal microscope. 3.2 Back to Unlimited Freedom This procedure was developed initially for mineralogy. There, for example, the three-dimensional structure of crystals grown on substrates is to be investigated. Since the crystals are mainly found in more or less perpendicular orientation to the substrate, a problem arises in detecting the reflected light. One can imagine the crystals like the skyscrapers of a big city. The illumination might be created by a lighthouse, which at a certain floor height illuminates a beam of light in a circle around the buildings. Although this is not intended for the urban area and the inhabitants would rightly complain to the port authority, for the moment we will leave this human factor out of consideration. In this example, if an observer in a helicopter over the city looks down on the buildings, he will not see much: 14 3 Biological Photography Not much of the light will be reflected perpendicular to the lighting. His situation will be better if he is above the lighthouse and then looks at the city from a different angle. Now he will see the strip of light scurrying across the buildings. Instead of creating this viewing angle by changing the design of the microscope, the authors have simply tilted the city by one angle. The simple solution was to tilt the specimen on a support by 45 degrees against the axis of the microscope. Nevertheless, the result is not satisfactory: You can only see the reflections from the height of the lighthouse. Everything that is above or below is—as desired—in the dark. If you now take a picture with the camera, you can only see the structures of, for example, the seventh floor. This is the principle of an optical cut. The wish is, of course, to be able to see the buildings from the basement to the roof. But for this purpose you only need to change the z-position of the microscope table. In doing so, the position of the sharply imaged layer in relation to the illuminated layer remains constant, only the structure selection in the preparation changes. The original text reads as follows: “The symbolic worm is really the elevating mechanism for the stage of an ancient Ernst (Leitz) Wetzlar microscope No. 274703 …”2 . The adjustment of the specimen was thus achieved by means of the z-drive present in the “antique” Leitz microscope. The drive knob was connected to a lever mechanism, which in turn could be moved by a motor. In this way it was possible to move the entire specimen through the light sheet “in one go.” If during this time the camera was set in the “B-position,” that is, to continuous exposure, a complete image of the crystal structures was recorded on the film. Today’s devices consistently first record a stack of discrete optical sections, which are then combined again to form a projection image. Care must be taken to ensure that the distances between the images are sufficiently small to ensure that they are smaller than the focus range. This, of course, results in a large amount of primary image data. The advantage is that projections in any direction can be calculated from the primary data. Although this was not possible with the method described above, this device was suitable for taking an image with extreme depth of field directly at high speed and without image stacks. 3.3 Versatile Illumination However, there is one flaw in the process: The shadows cast by the lateral lighting still leave some things in the dark. In a futuristic manhunt with lighthouse 2 The microscope with the abovementioned number left the Wetzlar factory on February 8, 1930 (many thanks to Aarne Liebich, Hessian Economic Archive in Darmstadt). 3.3 Versatile illumination 15 and helicopter in the metropolis, the light-shy gangsters still have the opportunity to hide in the dark corners between the houses. How can this be remedied? Obviously one must illuminate the shadows. The easiest way to do this is to add more light sources, which throw light sheets from different directions onto the interesting objects. This is achieved by arranging several light sources with the thin slit described above around the specimen. Such a device was first developed for macro photography [14], because the spatial conditions are more favorable there. In principle, however, there is no real difference between “microscopy” and “macroscopy,” the boundaries are fluid, the optics are also scalable with respect to diffraction limitation and, at most, mechanics and optics become increasingly complex with smaller fields and thinner light sheets. A large-format solution was therefore the use of two or three slide projectors (older readers will remember these devices), each equipped with an optical slit instead of a slide [14]. Obviously, biology in the 1980s was not yet so benevolently blessed with financial resources, so that instructions for making such a slit were also provided: Two razor blades are glued to a glass slide frame at the smallest possible distance from each other so that a gap between the blades is centered on the glass as horizontally as possible … The commercially available projectors were then arranged around the object so that the light blades overlapped exactly. This requires some patience, which is necessary to keep the exact z-position in the depth of field of the camera as well as to adjust the different light sources in exactly one plane. The light sheets must all be arranged without any tilting errors. The same author produced a version that was less complicated to adjust and certainly more suitable for daily practice [17]. The light sheet is created here by arranging two flat plates parallel to each other at a very small distance. The distance went down to 0.3 mm. This would correspond to a microscopy method with a numerical aperture of 0.04, as typically found at objective magnifications of 1 ×···2 ×. Illumination is then performed with filament bulbs, with the filament adjusted parallel to the gap between the two plates. In the middle of the two plates there is a larger opening through which the specimen is pushed during exposure. The plates act as a light “tunnel,” which corresponds to quite effective apertures and produces well collimated light (as long as the distance is not too small). You can then arrange as many light sources around the object as you like, at any angle and even in different colors, without any major adjustment effort. In particular, this arrangement is stable over a long period of time, for example until the next morning after work (Fig. 3.2). 16 3 Biological Photography F O L2 L3 P1 d L1 P2 S z Fig. 3.2 Simplified device for taking images with extreme depth of field (modified according to [17]). Two plane-parallel plates P1 and P2 at distance d are illuminated by light sources L1 … Ln in such a way that a light sheet is created in the opening of the plates. The subject S is moved through this light sheet in z-direction and the scattered, reflected or emitted light is imaged through an objective lens O onto the detector F If you make an effort and implement a few control units for sample movement and image acquisition, this “simplified unit” already meets more than the requirements in illumination and data acquisition for what you would call a modern light-sheet microscope: • • • • The light sheet is thinner than the depth of field of the objective lens. 3-D grid option (z-stacking). Instant 3-D projection image in a single shot. Any number of simultaneous lighting sources. Freely adjustable in angles and colors. • Multi-parameter recordings in many channels with sequential recording. • Specimen movement in z and rotatable in any angle. • Time series recording of living objects or of parameters changing in time. 4 Glittering Particles and Colorful Molecules Summary No, this is not about particles from particle physics. They’re really too small for our purposes. Rather, we use scattering on small objects, on crumbs of the most varied composition, to make flow conditions in liquids or gases visible. In biological applications, small biological objects, such as cells or cell compartments, are often “decorated” with a luminous marker, which can then also be used for such flow measurements. So far, we have generated light sheets by imaging a slit created in various ways into the object under investigation. But there are more possibilities. And one does not have to limit oneself to reflecting or scattering objects. Especially in biology, other contrasting techniques are common. The most famous and currently most successful is fluorescence. 4.1 Lightning Trap for Particles 1 Light-section arrangements were already used for flow measurements almost 50 years ago [16]. Instead of a slit, however, a different solution is used. Optical lenses have the property to concentrate light into a very small spot. If the lens is homogeneously illuminated, the spot is a diffraction pattern, the Airy pattern ([5], Sect. 1.3.2). Now it is possible to produce lenses that focus only in one of the two spatial directions and leave the other direction unaffected. A plano-convex “cylindrical lens” reminds of a groundsill, as used in road traffic for educational 1 In fact, a light-sectioning method based on the illustrated gap for flow measurement was already presented 80 years ago and documented with examples ([18], p. 81). © Springer Fachmedien Wiesbaden GmbH, part of Springer Nature 2021 R. T. Borlinghaus, The Light-Sheet Microscopy, Springer essentials, https://doi.org/10.1007/978-3-658-32768-2_4 17 18 4 Glittering Particles and Colorful Molecules G SL FL f d Fig. 4.1 Light sheet by expanding a laser beam with a glass rod. A light beam SL (here for the sake of simplicity with square profile of edge length d) is collected in a glass rod G in a focal line f. Behind f, the beam expands to form a light sheet FL of thickness d. Behind the focal line, the intensity of the light decreases linearly in the direction of propagation of the light purposes in speed control. The simplest form of a cylindrical lens is a glass rod (Fig. 4.1). If a thin beam is shone on a glass rod, for example with a laser, the laser light is focused into a line parallel to the axis of the glass rod. In the direction of the axis, however, the geometry of the beam remains the same. Behind this focal line, the light expands in a plane perpendicular to the axis of the rod, thus creating a sheet of light that is always as thick as the diameter of the laser beam. In the resulting light sheet, particles in suspension will scatter the light, such as dust particles in a barn. A camera set up perpendicular to the light sheet can then record the particle distribution at any time. If you take pictures fast enough one after the other, you can measure the trajectories of individual particles and from these, you can measure the speed of the particles at any point of the light sheet without interfering with the flow process. In principle, such a system performs the same as the slit ultramicroscope of 1903, but was developed for these dynamic measurements. The particles are much larger, and therefore the optical effort is quite low. Modern measuring methods also use laser pulses emitted in rapid 4.2 Microscopic Fireflies 19 succession, so that the same particle is imaged twice or more on one image. The distance between these particle images can then be used to conveniently calculate the velocity and from this the flow velocity and direction of the gas or liquid under investigation. This method (Particle Image Velocimentry, PIV) is routinely used today in fluid mechanics. 4.2 Microscopic Fireflies The light blades that are generated here make particles visible that are located inside the thin disk. These can be gold particles in glass or plastic beads in water, steam droplets or smoke particles in the air. All these particles are initially scattering objects, which means that light of the same color is simply directed in different directions. At a distance, this can be compared to a reflecting surface. The essential principle is the dark field method: The illumination beam and the observation cone are not coaxial but intersect, usually at an angle of 90 degrees. Fluorescence is a modern contrast method that is also very well suited for dark field applications. Light as an electromagnetic wave can interact with matter in that the oscillating electric field interacts with the positive and negative charges of atoms and molecules. The energy schemes for scattering and fluorescence are shown in Fig. 4.2, where the lengths of the arrows correlate with the energy. Without irradiation energy, the molecule is in the ground state G, which still has various (thermal) sub-states. In the case of fluorescence (on the right of the picture), a photon is first absorbed, that is, the energy of the photon is absorbed by the electronic system of the molecule. The molecule changes to a higher, excited state A, which is also split into different thermal sub-states. Very quickly after absorption, the molecule then releases a small part of the energy as heat to the environment. It remains in the excited state for a relatively long time and will then emit the remaining (smaller) energy again as light particles. The emitted photon with the lower energy therefore has a longer wavelength than the one that is irradiated. It is “redder” than the exciting photon. Fluorescence microscopy makes use of this fact by allowing the excitation and emission of light to be separated according to color in the beam path. The comparatively long time is usually a few nanoseconds and is described by the “fluorescence lifetime.” In the case of scattering (left in the picture), the energy of the illumination photons does not match a distance of the available states. In the example, the energy is too low and the wavelength is therefore too long. In the case of an interaction, the system now assumes a so-called “virtual state,” but the energy 20 4 Glittering Particles and Colorful Molecules Fig. 4.2 Scattering and fluorescence in the term diagram. Scattering (left): An illuminated object is initially in the ground state G. If the energy of the incoming photons (In) does not match a higher energy state, the system assumes a virtual state V from which the photon is immediately released (Scatter). The energy is unchanged (and thus the color of the scattered light), but the direction may have changed. Fluorescence (right): If the illuminating photon can put the electronic system of the object into an excited state A, the photon is absorbed (Absorption). It falls very quickly into the lower oscillation state (dotted arrow). After a characteristic time, the energy is released again as light (Emission), and the system returns to one of the oscillation states of the ground state. Here, the energy of the emitted photon is lower, the wavelength is longer, the color “redder.” cannot be absorbed, the quantum mechanical selection rules prohibit this. Consequently, the photon will have the same energy during its further journey as it had at the beginning. However, the direction can be different, which is why scattered light can also be recorded outside the beam direction. Biological preparations can now be stained in a variety of ways with fluorescent molecules that specifically mark desired structures. This started with accidental discoveries in histological staining, gained considerable momentum through specific labelling using antibodies or DNA fragments and has now culminated in the use of fluorescent proteins that can be activated in living organisms at almost any location and in any context through genetic manipulation. And all this in almost any color. Structural research can thus investigate the interaction of different cell components. By staining living specimens, we can now look directly into the mysterious machinery of life processes, so to speak—in the sense of the word. In the following, some steps in the development of light sheet microscopy for application in biology are illuminated with fluorescent dyes. Of course, this can only be done very incompletely in this medium; and because the fermentation process has, so to speak, only just begun to really take off, one must expect exuberant innovations as soon as they are published. 5 OPFOS—Good Ideas and the Acronymic Suffering Summary The development of the first device for light-sheet microscopy on fluorescence-dyed objects in biology had an optical background: If you want to record a thick specimen in all three dimensions, you usually take a “stack of images.” One starts at the surface and then focuses incrementally into the specimen to the other side, taking a “flat” image after each step. In a confocal microscope you can use optical sections, the thickness of which is limited by diffraction. However, there is a problem: The light distribution is not the same in all three spatial directions. A point is transformed into an ellipsoid of rotation when illuminated. The same applies to the sensory part of the light path. As a result, the lateral resolution is—in the best case—twice as high as the axial resolution (see e.g., [5]). The smaller the aperture of the objective, the greater the difference and longer the ellipsoid. In addition, in the case of a cylindrical figure, a smaller part of the cylindrical surface at the top and bottom contributes to the signal than at the sides. The latter appear brighter when the surface is colored— for example of membranous structures—because more area is immersed in the point spread function (PSF) (see Fig. 5.1). To circumvent this problem, Voie et al. [19] have used light sheet microscopy for fluorescence. The specimen, the cochlea of guinea pigs, is a cylindrical object in the first approximation. In order to generate the light sheet, a cylindrical lens was also used here, but in a different form than described above for the particle measurements (Fig. 5.2). A laser beam was first widened so that its diameter was within the desired dimension of the light sheet. This beam is then focused with a cylindrical lens of longer focal length than a glass rod. The focal length is chosen so that the “focal line” mentioned in Fig. 4.1 becomes a “focal surface.” Strictly speaking it is still a line, but the changes in thickness along the beam axis are not as steep. Now, if a © Springer Fachmedien Wiesbaden GmbH, part of Springer Nature 2021 R. T. Borlinghaus, The Light-Sheet Microscopy, Springer essentials, https://doi.org/10.1007/978-3-658-32768-2_5 21 22 5 OPFOS—Good Ideas and the Acronymic Suffering Fig. 5.1 Anisotropy in microscopy. The point spread function PSF cuts more area from a horizontally lying cylinder (Cyl, here only a cross section is shown) at the lateral flanks and the pixel is therefore brighter than at the polar flanks. The resolution, inversely proportional to the expansion d of the PSF is axially worse (dz is larger) than laterally (dxy is smaller) Fig. 5.2 Light sheet microscope for biological objects with fluorescence staining. The light (beam from left to right in green) from a laser (Laser) is expanded with a beam expander and focused by means of a cylindrical lens (LCyl ). In the focus area, the object (Ob) is mounted rotatably (dr). The emission (from bottom to top in red) is recorded by a camera (Camera) perpendicular to the illumination certain tolerance is allowed, this area around the focus line can be considered as √ a light sheet. It has been agreed that a deviation from 2 from the thinnest point will be considered tolerable. This arrangement thus produces a light sheet whose intensity does not depend on the position along the beam axis; perpendicular to it, the intensity is distributed as determined by the beam profile of the laser. However, the thickness of the light sheet is determined by the optics, it is approximately λ/NA, where λ is the wavelength and NA the aperture in the focusing direction. 5 OPFOS—Good Ideas and the Acronymic Suffering 23 This procedure is particularly important for microscopy, as the aim is to produce thin sheets with high apertures. The cochlea is now irradiated by the thin sheet of light “from left to right.” The fluorescent dye used is excited in the illuminated region. The camera, whose depth of field is greater than the thickness of the light sheet (see Fig. 3.1), records the image. In contrast to confocal microscopy, fluorescence is only excited in the layer that is then also recorded by the camera. This preserves the dye and the preparation. It should be noted, however, that the illumination intensity naturally decreases from left to right, as the dyes absorb light and opaque components and scattering particles reduce the excitation light. Thus, the image is much brighter on the side on which the light enters than on the opposite side (depending on the individual situation in the preparation). Of course, the emitted light from the focal plane must still penetrate the material above before it reaches the detector; here too, signal is lost and the image quality is reduced. In “ordinary” microscopy, the disturbing effects are noticeable in that the object on the surface initially looks very bright and sharp, but as soon as you focus in depth, the signal becomes weaker and the image appears blurred. The advantage of the light-sheet method is therefore not a better penetration or a better image quality with otherwise the same optical conditions, but the fact that only that part of the object is irradiated from which signal is also picked up— this leads to less fading of the fluorochromes. The second advantage is that the recording can be done in parallel, as the whole two-dimensional image is recorded in one single exposure and not scanned pointwise. This increases the acquisition speed considerably—this is particularly helpful when examining living specimens. But we are still in the Cartesian coordinates of the light sheet and the camera field. The abovementioned cochlea was now not shifted linearly in the z-direction, as is the case with normal focusing, but was mounted so that it could be rotated incrementally around its axis. An image was recorded after each angular adjustment. The image series is not a stack but a rotation sequence, which fits well to the polar coordinates of the object. The three-dimensional structure could then be reconstructed from these series [20]—with the same resolution and brightness (with regard to PSF) in all areas. The method also offers a solution to the above described problem of intensity decrease along the illumination: If the specimen is rotated by 360° during serial exposure, two series of complementary images are obtained over 180° each, which are illuminated from the first side and from the opposite side. If one combines such images, one can at least partially compensate the losses. In the common microscope, a comparable solution would be that after having taken a stack of 24 5 OPFOS—Good Ideas and the Acronymic Suffering images, one turns the preparation once like a steak in a pan and then records a second stack. The procedure was called “orthogonal-plane fluorescence optical sectioning” by the authors and was christened with the acronym “OPFOS.” In the 1980s, it became fashionable to assign a new name and a corresponding acronym to each modification of a process. The flood of such abbreviations quickly grew into a tsunami that still continues today. Perhaps because as an author you have the opportunity to use a short artificial word to direct the searchers in the Internet machines to your work? Or is it—similar to graffiti tags—only used for territorial marking? In any case, in the following we will use as few such acronyms as possible to keep the presentation understandable and civilized. 6 Recent Developments Summary Light-sheet microscopy is always a variant of the slit ultramicroscope developed by Siedentopf. Essential is the lateral illumination of a small, selected volume, usually in perpendicular orientation to the observation axis. In addition to applications in fluid mechanics, surface inspection and macroscopic imaging of biological objects, the microscopic use of light sheets in biology has experienced a particularly rapid upswing. The first useful images quickly led to the first improvements and modifications. Whether the technology is now saturated and progress will remain rather gradual, or whether there will be dramatic new developments is, in my opinion, impossible to predict. After all, prophetic predictions are often only quoted because they later proved to be correct. Those that did not come true are often forgotten, even if they come from the same author. This is due to the love of people to tinker with great examples and follow them. To truly enjoy freedom, one must grow up and let open questions stand as open questions. So instead of stirring in the coffee grounds, let’s describe some of the methods and variations on the topic that have been published since the turn of the millennium. 6.1 A Look into Life As mentioned above, light-sheet microscopy has two distinct advantages for living objects: High frame rates and low fading (and thus low production of toxins from bleached dyes). Therefore, this topic was taken up and worked on in the group around E. Stelzer [21]. An essential aspect in the examination of living objects is the preparation. The classical method of “somehow” placing objects on slides and then covering them with a cover glass so that they can be examined with corrected © Springer Fachmedien Wiesbaden GmbH, part of Springer Nature 2021 R. T. Borlinghaus, The Light-Sheet Microscopy, Springer essentials, https://doi.org/10.1007/978-3-658-32768-2_6 25 26 6 Recent Developments optics in an ordinary microscope obviously causes difficulties for the living object [22]. Typical living objects in current biological research are frog eggs, nematodes, larval stages of fruit flies, seedlings of an otherwise rather inconspicuous weed, the thale cress, and many more. These creatures are not found in nature on microscope slides (apart from a few fenestrophilous mosses and algae) and prefer to be able to interact with the environment in freedom rather than having a cover glass put over them. Earthly creatures are not flatlanders. One solution to the problem of three-dimensional growth is to embed the objects in soft agar. This is a gelatinous mass obtained from red algae. Those who enjoy slimming cures may know agar-agar as a laxative filler that is indigestible to humans. In a soft agar, water, nutrients and minerals can easily diffuse and the living being embedded in it does not have to starve to death, but can develop comparatively comfortably. The supply of the necessary substances, temperature control and lighting (for plants) can be ensured for weeks by computer-controlled machinery. This made it possible to study developmental processes directly in the living organism. By means of fluorescent proteins, which the genetically manipulated object itself can produce at almost any location, individual developmental stages, switching on and off of gene expression, interactions between different molecules, but also simply changes in shape can be followed over hours and days. Special recording techniques also allow large-surface overview displays, for example of entire living brains at very high spatial resolution and over longer periods of time [24]. 6.2 The Return of the Scan System One advantage of light-sheet microscopy is the higher recording rate, because a camera records a complete image at once—in contrast to a laser scanning method, where the object is scanned point-by-point. With a cylindrical lens it is possible to create a static light sheet, but the disadvantage remains that this light sheet is only as homogeneous as the beam profile of the laser. Lasers usually have a Gaussian profile. Therefore, a considerable part of the profile has to be cut off if one wants to create more or less homogeneous conditions. In addition, the use of cylindrical optics does not always allow for the desired thin light sheets. The best results are obtained with conventional optics that focus point-shaped and are circular. Here, as is well known, the focus is not a line but a point (cum grano salis). And one can now define a line in the vicinity of this point that is sufficiently uniform in thickness, which can then be regarded as a “thread of light” (Fig. 6.1). 6.2 The Return of the Scan System 27 LR MR X Y z x y Fig. 6.1 Scanned light sheet. Laser light (beam from below, green arrow) is guided by a scanning mirror MR into raster optics LR . This optics creates an area around the focal point which can be called a light thread. When the light thread in the specimen is moved in the xy-plane by the scanning process, the entire field is illuminated. The recording time of the camera must be synchronized with this raster process or be much longer than a raster process These considerations, combined with the well-known laser scanning technology, easily result in a method in which the light sheet is generated by scanning a light thread in the plane of observation [25]. While the light thread crosses the field of view, a camera records the emission of the fluorescence. Since the thread is continuously guided across the field, there are no gaps in the image. Scanning mirrors are usually made up of a torsion motor with a mirror attached to the axis. A torsion motor is basically an electric motor in which the rotor axis is firmly connected to the stator on one side, so the rotor cannot really rotate but can only twist. Rapid reversal of voltage then results in an equally rapid rotation of the mirror alternately in both directions. Such motors are widely used, for example in the barcode scanners at the supermarket checkout, but also in confocal laser scanning microscopy. Raster frequencies of up to several thousand periods per second are not unusual. This method therefore also allows image acquisition rates in the kilohertz range. In a confocal point-raster method, two such mirror functions are used for the two dimensions in the plane. In the kilohertz range, only lines can be generated there. Therefore, images consisting of several hundred lines can only be generated several hundred times slower. Scanning optics is a well-established technology in light microscopy, and very thin light sheets of high quality and good homogeneity can be produced here. If one already uses a scanning method for the xy-plane, it is of course obvious to adjust the position of this plane also in the z-axis via a scanning mirror in order to generate an image stack. This would have the advantage that the specimen itself does not have to be moved, which is often large, heavy and sensitive to vibrations. 28 6 Recent Developments However, in the case of axial scanning, the depth of field of the camera must also be adjusted, for example, by mechanically adjusting the microscope objective. 6.3 Self-Healing Radiation For most of us, “healing” is first of all a surprising predicate for radiation. After all, the contaminated area around Chernobyl is highly damaging to health and the “Gorleben shall live” sticker on the parka is supposed to protect us from radiation. Ionizing radiation is probably not quite so unconditionally deadly after all [26], and of course this refers to electromagnetic radiation of low energy anyway—for example the warming and life-giving sunlight. And what damage that needs to be healed can such a beam suffer? We imagine an ordinary “laser beam” to emit light with a certain cross section and then maintain this cross section. In the same way that a well-made twisted yarn has the same cross section from start to finish. Light rays, no matter how well made they are, do not have a constant cross section due to diffraction phenomena. And the light thread described above for the raster process is not a ray at all, as we imagine it, but a long-drawn-out point spread function. The cross section is at best an Airy pattern at the focus position (and only there). The term of a light beam used in geometrical optics has no cross section at all and is only a constructive aid. Now opticians have asked themselves the question whether there might not be a beam profile that does not change even over any distance of propagation. And indeed such a thing was found [27] and named “Bessel beam.” Unfortunately, a real Bessel beam exists only in theory, but it is possible to generate an approximate Bessel beam over a certain distance [28] and thus to rasterize a light sheet. While a Gaussian beam shows the profile of a Gaussian curve, a Bessel beam has a profile of concentric rings, with a thread-like core in the middle. In practice, the proportions of these components can be varied, and the inner component can be made so small that the light sheet is ultimately thinner than the classically focused light. Unpleasantly, however, a large part of the energy is found in the outer shells, which again limits the advantage. Nevertheless, the inner filament is much better preserved than in the classic case, even in objects with many obstacles: Even behind a small disturbing object, this inner filament forms again after a short distance. Hence the term “self-healing.” The advantage of a light sheet of Bessel beam is therefore thinner optical sections and better penetration. 6.4 A Little Over-Resolution After All 6.4 29 A Little Over-Resolution After All In the introduction it was mentioned that light-sheet microscopy is not a method to exceed the classical resolution limit, although combinations of light-sheet microscopy and super-resolution techniques will of course lead to better resolution [29]. However, the concept of quasi-Bessel-beam illumination from the previous section, in combination with other techniques (which are not super-resolution techniques themselves), still allows resolutions beyond the diffraction limit—at least as far as the thickness of the optical section is concerned (Fig. 6.2). A possible combination partner is two-photon fluorescence microscopy. The basic idea behind two-photon excitation [31] is the splitting of the excitation photon in the fluorescence process. If, for example, excitation with a blue photon of 480 nm is possible, then this can also be done with two photons of the wavelength 960 nm, which would then be infrared radiation. Since two photons have to react with the fluorescence molecule, the concentration dependence is a quadratic one, as chemical kinetics teaches (for repetition it is recommended [30]). In a Bessel beam this leads to the fact that fluorescence is excited only in the inner filament, while in the outer sheaths the intensity is not sufficient to produce noticeable excitation states. Thus, the disturbing emission from the outer areas is suppressed and the layer thickness is less than in classical excitation. Fig. 6.2 Effect of two-photon excitation in a light beam. Left for a Gaussian profile, right for a Bessel profile. While in single-photon excitation (a and b, in blue) a Bessel beam also produces fluorescence in the outer envelopes, this is suppressed in two-photon excitation (c and d, in red), although larger wavelengths are used there, which proportionally reduce the resolution a b c d 30 6 Recent Developments Fig. 6.3 Isotropic resolution by crossed use of two identically designed light sheets. Details in the text 6.5 Two-Way Light Sheets As already mentioned, the point spread function is much longer axially than radially. In order to improve the resolution by using two identical PSFs crossed, a concept was introduced which uses the original microscope only as a luxurious specimen holder and puts the light-sheet method over it [33]1 . Here, two identical microscope objectives are used to create two light sheets that intersect at an angle of 90°. They are each at 45° to the optical axis of the original microscope. Each objective is linked to an apparatus which allows it to function both as illumination with a light sheet and as observation with a camera. Light source and camera path are separated by a beam splitter. During recording, a light sheet is moved through the specimen from top to bottom from one side by means of a scanning mirror, while the emission is recorded on the other side. To synchronize the depth of field with the position of the light sheet, the second objective is focused by a piezo element. Since both sides are symmetrically constructed, after the first pass, illumination and observation can be changed and a second stack recorded (Fig. 6.3). Afterward, the entire data is summarized and ‘muddled up’ with a few sophisticated algorithms. The result is a three-dimensional data set with a uniform (isotropic) resolution in all directions in the range of the light sheet used. 1A light-section method, in which illumination and observation were also arranged in a vshape over the sample and the original microscope was completely omitted, was already described at the beginning of the last century as a “surface tester that can be placed on top”: ([18], p. 77). 6.7 Matrix—New Edition 31 Such a device is independent of the actual microscope, it allows examining classically prepared specimens again and is also suitable for fast images of living objects. 6.6 Rubber Lenses In the 1970s, the term rubber lens was generally known and used for pancreatic optics, today the super cool sounding word “zoom” is used for it. This is a pity, because the very latest technology is indeed very similar to a real rubber lens—as is the oldest optical device of all: The eye lens, which is now literally a rubber lens. Modern rubber lenses use liquids that are made to change the refractive power of the actual lens through clever procedures. This can be done continuously and quickly. Speed is particularly important here, because even the displacement of a comparatively heavy, highly corrected and highly apertured lens is rather sluggish compared to the possible recording rates and the desired speeds. Classical vario lenses, in which lenses and lens systems are shifted against each other by mechanical actuators, are of course even slower. Modern rubber lenses have therefore already been used for light-sheet microscopy [34]. 6.7 Matrix—New Edition Although fluorescence has become a very effective and indispensable tool for studying all kinds of structures and metabolic changes, it has an Achilles’ heel: The fluorochromes bleach out. When the molecules are elevated into the excited state, this means that the electronic system has been changed and contains more energy. But the electronic system also makes up the chemical bonds. So it is easy to see that an excited system is much more likely to make chemistry than a system in the ground state. This chemistry is often a decay chemistry. Not only does the fluorescence molecule disappear and the sample become darker, but often reactive fragments are formed that are unhealthy for living material—often fatal. These bleaching processes are not only dependent on the total energy introduced, but also on the dose [35]. The same amount of light in small packages causes less damage than if it is dumped in a single large carload. As a continuation of the Bessel beam concept, beam patterns were introduced for lighting that resemble pearl necklaces instead of threads: Small light droplets 32 6 Recent Developments along the illumination axis [36]. This should reduce fading at high resolution and speed. However, the associated optical setup is complex and the desired beam shape is quickly destroyed in the specimen by the irregular distribution of differently refractive materials. Therefore, the penetration depth is certainly very limited. 6.8 The Vertical Turn A good way of generating light sheets is the beam scanning principle (Sect. 6.2). Well-established microscopes using this principle are confocal laser scanning microscopes. This is the first ingredient. The basic difference between ordinary microscopy and light-sheet microscopy is the orientation of the illumination axis to the observation axis. In ordinary microscopy they coincide (0° or 180°), in light-sheet microscopy they are perpendicular to each other. To convert an ordinary microscope into a light-sheet microscope, the illumination must therefore be bent by 90°. The optical element with which beams are usually bent is a mirror. This is the second ingredient. Using these two ingredients, Leica in Mannheim developed a device that is both a confocal laser scanning microscope and a light-sheet microscope [37]. The principle was given the sweeping name “vertical turn.” In fact, a bend in the optics becomes a blessed thing here. In a confocal point-scanning microscope, an image is generated in incident light just like in a conventional fluorescence microscope. The image plane is oriented perpendicular to the optical axis, just as one would expect in a regular microscope. In an ordinary fluorescence microscope, the entire field of view is illuminated and viewed at once, that is, all pixels are viewed simultaneously—a parallel imaging method. In a scanning microscope, only one single point is illuminated and observed at a time: A serial imaging process. This point must be traversed line by line in the image plane from top left to bottom right, as was the case in old television sets of the previous century. How long it takes to record a line depends of course on the speed of the scanning mirror, the typical value of 1 kHz has already been mentioned. The frame rate therefore depends on this raster frequency and on the number of lines with which an image is to be recorded. The line feed is guaranteed by a second scanning mirror. The number of lines per image can be set as desired and should be adapted to the optical resolution in a high-quality image. The smallest format contains only a single line, which is recorded in quick succession, the second mirror remains in rest position. The resulting data can be written into a two-dimensional image, which now does not 6.8 The Vertical Turn 33 have the dimensions x and y, but the dimensions x and t. In biology, such a recording is called a kymogram and is used for very fast processes in living specimens. The illuminated partial volume is a surface as described in Sect. 6.2, basically a sheet of light—but we are still looking at the edge of the sheet (Fig. 6.4 left). Now we mount a mirror at the height of the focus position and aim at this mirror with the second scanning mirror as a new rest position. The mirror is arranged so that the light is deflected exactly horizontally. The scanning movement thus illuminates a sheet of light in the focal plane, whose emission can be recorded by a camera (Fig. 6.4 right). Since only one scanning mirror is active in the lightsheet mode, the second one can be used to deflect the light onto the deflection mirror. At first it does not matter in which direction the light irradiates the sample. It is also possible, for example, to attach a second mirror and illuminate the sample from the opposite side. In this way, two images are obtained which can be fused to reduce the absorption artifacts. In extreme cases, instead of a plane L2 x x S1 y S2 y L1 Fig. 6.4 Vertical turn principle. Left: Confocal raster mode. Excitation light (beam from below in green) is guided through a microscope objective L1 onto the sample and scanned in x and y points (green points). The emission light (red arrow pointing downward) is collected by L1 and fed to the detector (not shown). Right: Light-sheet mode. Excitation light (beam from below in green) is guided through L1 to the mirror S1 and thus horizontally in the focal plane (dotted line) into the sample. The sample is scanned line by line. The emission light (red arrow pointing upward) is collected by L2 and fed to the detector (not shown). Alternatively, the mirror S2 can be used to illuminate the sample from the opposite side 34 6 Recent Developments mirror, a section of the conical mantle can be used as a mirror and a rotating line can be created in the focal plane by controlling both scanning mirrors. Since the light sheet is formed from the focus of the illuminating lens, when switching between these two methods, this lens must be adjusted so that the illumination is at the correct position. The difference is the distance from the deflecting mirror to the center of the field of view. The advantage of such a concept is that confocal and light-sheet microscopy are combined in one device. This is not only a question of cost, but also allows the combination of these two methods in the same experiment [38]. In confocal mode, for example, a selected region could be exposed to light in order to switch on photoactivatable fluorescent proteins or to use related techniques. After the exposure, further events can then be followed in the light-sheet mode with high time resolution. What You Learned From This essential • • • • How a light-sheet microscope works. What you can use light sheets for. How to use them to detect the invisible. Which current innovations in this technology are important. © Springer Fachmedien Wiesbaden GmbH, part of Springer Nature 2021 R. T. Borlinghaus, The Light-Sheet Microscopy, Springer essentials, https://doi.org/10.1007/978-3-658-32768-2 35 References 1. Hooke R (1665) Micrographia or some physiological description of minute bodies made by magnifying glasses with obeservations and inquiries thereupon. Royal Society, London 2. Coons AH, Creech HJ, Jones RN (1941) Immunological properties of an antibody containing a fluorescent group. Proc Soc Exp Biol Med 47:200–202 3. Pardue ML, Gall JG (1969) Molecular hybridization of radioactive DNA to the DNA of cytological preparations. Proc Natl Acad Sci USA 64(2):600–604 4. Borlinghaus RT (2016) Unbegrenzte Lichtmikroskopie. Über Auflösung und SuperHochauflösung und die Frage, ob man Moleküle sehen kann. Springer, Wiesbaden 5. Borlinghaus RT (2016) Konfokale Mikroskopie in Weiß. Optische Schnitte in allen Farben. Springer, Berlin 6. 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The material that is used in the manufacture of a polarizer and the actual polarizer design combine to determine the laser damage threshold. Birefringent polarizers have the highest laser damage threshold.  Beamsplitters, which are two optics cemented together, will have low laser induced damage threshold and air-spaced birefringent polarizers have high laser induced damage threshold.

This book contains all the necessary information and advice for anyone wishing to obtain electron micrographs showing th

BALL MOUNT WITH 1/4-INCH SCREW. Angle your camera independently of Ring Light.

Coordination compounds have been well-known for their wide variety of applications for over a century, as well as enhanc