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Light Sources Spectral Imaging Wavelength Selection Microscope Basics Optical Sectioning Fluorescent Proteins Spinning Disk Superresolution


The ZEISS Online Campus features interactive tutorials that have been developed to explore complex topics in all phases of optical microscopy and digital imaging. The tutorials are embedded within web pages that contain accompanying discussions addressing the subject phenomena and provide instructions for use and control of the interactive tutorials. Additional information is contained in review articles on selected topics.

Light Sources

Arc Lamp Instability - Illumination sources based on plasma discharge (arc lamps) require a considerable period after ignition to reach thermal equilibrium, a factor that can affect temporal, spatial, and spectral stability. This tutorial examines several of the origins of arc lamp instability, including wander, flare, and flutter.

Halogen Regenerative Cycle - In the halogen regenerative cycle, which operates in tungsten halogen incandescent lamps, vaporized tungsten reacts with hydrogen bromide to form gaseous halides that are subsequently re-deposited onto cooler areas of the filament rather than being slowly accumulated on the inner walls of the envelope. This interactive tutorial demonstrates how halogens combine with tungsten and oxygen to complete the halogen regenerative cycle in incandescent tungsten halogen lamps.

Coherence of Light - One of the important parameters of illumination sources is their coherence, which is somewhat related to brightness due to the fact that extremely bright light sources are more likely to be highly coherent. This tutorial examines how incoherent light emitted by an arc lamp can be passed through a slit and filter to increase coherence and narrow the wavelength band.

Elliptical Reflectors - Advanced light sources suitable for use in high-performance fluorescence microscopy couple metal halide arc lamps with elliptical collection mirrors and high-speed filter wheels for rapidly shifting the output wavelength. These sources also provide fiber optics or liquid light guides for coupling the output to the microscope optical train. This interactive tutorial explores how careful positioning of the arc with respect to elliptical reflector focal points is critical to the formation of a focused beam at the input of a liquid light guide.

Mercury Lamphouses - High pressure mercury plasma arc-discharge lamps are highly reliable, produce very high flux densities, and have historically been widely used in fluorescence microscopy. This interactive tutorial examines advanced mercury arc lamphouses that are capable of automatic bulb alignment and intensity control.

Light-Emitting Diode Operation - Among the most promising of emerging technologies for illumination in optical microscopy is the light-emitting diode (LED). These versatile semiconductor devices possess all of the desirable features that incandescent (tungsten-halogen) and arc lamps lack, and are now efficient enough to be powered by low-voltage batteries or relatively inexpensive switchable power supplies. This interactive tutorial explores how two dissimilar doped semiconductors can produce light when a voltage is applied to the junction region between the materials.

LED Illumination for Microscopy - Among the most promising of emerging technologies for illumination in optical microscopy is the light-emitting diode (LED). These versatile semiconductor devices possess all of the desirable features that incandescent (tungsten halogen) and arc lamps lack, and are now efficient enough to be powered by low-voltage batteries or relatively inexpensive switchable power supplies. The interactive tutorial featured in this section explores the ZEISS Colibri LED illumination system for widefield fluorescence microscopy.

Spectral Imaging

Additive Properties of Emission Spectra - This interactive tutorial explores how multiple spectra can be added to produce a composite emission spectrum similar to those encountered in spectral imaging of specimens labeled with multiple fluorophores.

Spectral Imaging with Linear Unmixing - Explore how mixed fluorophores having highly overlapping emission spectra can be separated into individual components using spectral imaging and linear unmixing techniques. This tutorial contains several examples with fluorophores emitting in the green and red spectral regions.

Emission Fingerprinting with Lambda Stacks - Use this tutorial to examine how lambda stacks can be used to extract information about individual spectral profiles in specimens labeled with highly overlapping fluorophores.

LSM 700 Light Pathways - The LSM 700 laser scanning confocal microscope from Carl Zeiss is designed for efficient separation of signals by efficient splitting of the emission using the variable secondary dichroic (VSD) beamsplitter to prevent crosstalk and enable spectral imaging as well as linear unmixing of highly overlapping fluorophores.

Spectral Imaging FRET with Biosensors - Spectral imaging of FRET biosensors using fluorescent proteins is an emerging technique for the analysis of events in cell biology. This tutorial explores the performance of a cameleon calcium biosensor and a caspase apoptosis indicator in spectral imaging.

Fluorescent Protein FRET Biosensors - Spectral imaging has been very useful for the examination of fluorescent protein biosensors to determine the presence or absence of FRET in response to a biological stimulus.

3-Channel QUASAR Detection Unit - The ZEISS QUASAR photomultiplier detection technology is based on a filter-free system that guides the desired wavelength range to the target detector using adjustable optical wedges and slider light stops.

34-Channel QUASAR Detection Unit - Employing a special 32-channel photomultiplier, the ZEISS multichannel QUASAR detection unit is ideal for enhancing lambda stack acquisition speed for live-cell imaging experiments.

Wavelength Selection

Matching Filter Sets with Microscope Light Sources - In fluorescence microscopy, the excitation light is generally passed through a bandpass interference filter to select a specific band of wavelengths that are used to illuminate the fluorophore. Depending upon the filter characteristics and the light source, the amount of light available for excitation can vary by a wide margin. This interactive tutorial is designed to enable the visitor to choose between various ZEISS filter sets and common microscope illumination sources to determine the optimum combination for a specific application.

Filter Wheel Wavelength Selection - One of the most useful and cost-effective configurations to achieve wavelength selection in fluorescence microscopy involves placing a polychroic mirror in a standard fluorescence filter optical block and using separate filter wheels under computer control to rotate the proper excitation and emission filters into the optical pathway when necessary. This interactive tutorial examines wavelength switching with an aftermarket filter wheel coupled to an external metal halide lamphouse.

High Speed Wavelength Switching - Temporal investigation of events in living cells requires the ability to capture successive images on a wide spectrum of timescales, often spanning the range of microseconds to minutes. The Sutter Lambda DG-4 device featured in this tutorial is a complete interference filter-based xenon-powered illumination system that exhibits switching speeds of less than 2 milliseconds.

Microscope Basics

Optical Pathways in the Transmitted Light Microscope - The design of an optical microscope must ensure that the light rays are organized and precisely guided through the instrument. This interactive tutorial explores the function of the field and condenser aperture diaphragms of a transmitted light microscope.

Microscope Alignment for Köhler Illumination - Illumination of the specimen is the most important variable in achieving high-quality images in microscopy and critical photomicrography or digital imaging. This interactive tutorial explores how to establish Köhler illumination on a transmitted light microscope.

Objective Specifications - Microscope objectives are precision optical systems that feature a wide range of magnifications, numerical aperture, immersion media, specialized contrast applications, and other properties. This interactive tutorial examines the specifications found on typical objectives.

The Concept of Magnification - A simple microscope or magnifying glass (lens) produces an image of the specimen upon which the microscope or magnifying glass is focused. This interactive tutorial explores how a simple magnifying lens operates to create a virtual image of the specimen on the retina of the human eye.

Microscope Conjugate Planes - The conjugate planes critical for establishing proper illumination in the microscope are examined in this interactive tutorial. Four conjugate planes can be brought simultaneously into focus: the field diaphragm, the specimen plane, the intermediate image plane (where the reticule is positioned), and the human eye.

Fixed Tube Length Microscope Conjugate Field Planes - The geometrical relationship between image planes in the traditional fixed tube length (usually 160 millimeters) optical microscope is explored in this tutorial. In most of the imaging steps in the microscope optical train, the image is real and inverted, but a virtual image is also produced in one of the image planes.

Infinity Corrected Microscope Conjugate Field Planes - A majority of modern research microscopes are equipped with infinity-corrected objectives that no longer project the intermediate image directly into the intermediate image plane. Light emerging from these objectives is instead focused to infinity, and a second lens, known as a tube lens, forms the image at its focal plane.

Infinity Optical System Basics - Infinity-corrected microscope optical systems are designed to enable the insertion of auxiliary optical devices into the optical pathway between the objective and eyepieces without introducing spherical aberration, requiring focus corrections, or creating other image problems.

Field Iris Diaphragm Function - When the microscope is properly configured for Köhler illumination, the field diaphragm is imaged in the same conjugate plane as the specimen, and in fact, all of the image-forming conjugate planes are simultaneously imaged into each other and can collectively be observed while examining a specimen in the eyepieces.

Numerical Aperture and Light Cone Geometry - The light-gathering ability of a microscope objective is expressed in terms of the numerical aperture, which is a measure of the number of highly diffracted image-forming light rays captured by the objective. This interactive tutorial explores the effect of numerical aperture on light cone geometry.

Airy Disk Formation - When an image is formed in the focused image plane of an optical microscope, every point in the specimen is represented by an Airy diffraction pattern having a finite spread. This interactive tutorial explores the origin of Airy diffraction patterns formed by the rear aperture of the microscope objective and observed at the intermediate image plane.

Spatial Frequency and Image Resolution - When a line grating is imaged in the microscope, a series of conoscopic images representing the condenser iris opening can be seen at the objective rear focal plane. This tutorial explores the relationship between the distance separating these iris opening images and the periodic spacing (spatial frequency) of lines in the grating.

Conoscopic Images of Periodic Structures - This tutorial explores the reciprocal relationship between line spacings in a periodic grid (simulating a specimen) and the separation of the conoscopic image at the objective aperture plane. When the line grating has broad periodic spacings, several images of the condenser iris aperture appear in the objective rear focal plane.

Numerical Aperture and Image Resolution - The image formed by an objective at the intermediate image plane of a microscope is a diffraction pattern produced by spherical waves exiting the rear aperture and converging on the focal point. This tutorial explores the effects of objective numerical aperture on the size of Airy disk patterns.

Fundamental Aspects of Airy Disk Patterns - This tutorial explores how Airy disk pattern size changes with objective numerical aperture and the wavelength of illumination. It also simulates the close approach of two Airy patterns as they approach the Rayleigh criterion for determining the ability to resolve two closely spaced objects in the microscope.

Oil Immersion and Refractive Index - One way of increasing the optical resolving power of the microscope is to use immersion liquids between the front lens of the objective and the cover slip. This tutorial explores how changes in the refractive index of the imaging medium can affect how light rays are captured by the objective, which has an arbitrarily fixed angular aperture of 65 degrees.

Condenser Numerical Aperture - The size and numerical aperture of the light cone emitted by a substage condenser is determined by adjustment of the aperture diaphragm. This interactive tutorial examines how changing the aperture iris diaphragm opening size alters the size and angle of the light cone.

Condenser Aperture Diaphragm Function - The size and numerical aperture of the light cone produced by the condenser is determined by adjustment of the aperture diaphragm. Appropriate use of the adjustable aperture iris diaphragm (incorporated into the condenser or just below it) is of significant importance in securing correct illumination, contrast, and depth of field.

Condenser Light Cones - It is critical that the condenser light cone be properly adjusted to optimize the intensity and angle of light entering the objective front lens. Each time the objective is changed, a corresponding adjustment must be performed on the condenser to provide the proper light cone to match the numerical aperture of the new objective.

Coverslip Thickness Correction - High magnification dry objectives are often subject to aberration artifacts due to variations in cover glass thickness and dispersion. This tutorial demonstrates how internal lens elements in such an objective may be adjusted to correct for these fluctuations.

Focus Depth and Spherical Aberration - Explore the three-dimensional aspects of spherical aberration that is generated when imaging deep into specimens using the meridional section of a point spread function with this interactive tutorial. Spherical aberration is a significant problem when imaging specimens in aqueous media.

Inverted Microscope Lightpaths - Microscopes featuring an inverted-style frame are designed primarily for live-cell imaging applications and are capable of producing fluorescence illumination through an episcopic and optical pathway. This interactive tutorial explores illumination pathways in the Zeiss Axio Observer research-level inverted tissue culture microscope.

Reflected Light Microscope Optical Pathways - Reflected light microscopy is often referred to as incident light, epi-illumination, or metallurgical microscopy, and is the method of choice for fluorescence and for imaging specimens that remain opaque even when ground to a thickness of 30 micrometers.

Optical Sectioning Microscopy

Optical Sectioning Microscopy - Traditional widefield fluorescence microscopy produces images of thick specimens that often contain a high level of background signal, which dramatically obscures specimen detail and reduces contrast. To obtain crisp and sharp images, optical sections can be generated using either computational (deconvolution) or structured illumination techniques. This interactive tutorial explores the basic concept of optical sectioning using an animated cell model.

Structured Illumination Microscopy: ZEISS ApoTome Basics - Optical sections through thick specimens can be obtained in widefield fluorescence microscopy using structured illumination, as has been implemented in the ApoTome auxiliary device manufactured by ZEISS. This tutorial examines the necessary optical elements to equip a widefield microscope for structured illumination and presents typical image stacks obtained with the ApoTome.

Structured Illumination Microscopy: ZEISS ApoTome Operation - The basic concept behind the ZEISS ApoTome is the use of an evenly spaced grid in the aperture plane to serve as a mask through which the specimen is illuminated. The grid is inserted into the light path of the microscope and uses the epi-illuminator lens system to project a shadow of the grid lines into sharp focus, superimposed on the specimen, in the objective focal plane.

Optical Sectioning with Structured Illumination - Among the numerous advantages of structured illumination microscopy is the ability to produce crisp and distinct optical sections having a thickness that coincides with the objective resolution. This interactive tutorial explores optical sectioning with the ZEISS ApoTome.

VivaTome Basics - In aperture correlation microscopy, the final image is calculated in three steps: image extraction and mirroring followed by registration of both images and, finally, the actual calculation of the optical section itself. In the registration step, distortions as mapped in a previous calibration step are corrected between the two imaging beam paths.

VivaTome Optical Train - In the ZEISS VivaTome, a rotating disk having a defined grating pattern is located in one of the microscope conjugate image planes. Excitation light is directed through this disk, and the transparent regions on the disk are placed very close together so that approximately 50-percent transmission efficiency through the disk is achieved.

Optical Sectioning with Aperture Correlation - Aperture correlation microscopy combines the light efficiency of structured illumination with the acquisition speed of a spinning disk confocal instrument. This interactive tutorial simulates a virtual aperture correlation microscope.

Comparison of Confocal and Widefield Microscopy - Laser scanning confocal microscopy is capable of producing the highest out-of-focus discrimination of all routine optical sectioning techniques. This interactive tutorial explores optical sectioning with confocal microscopy and compares these sections to the results obtained with widefield fluorescence.

Fluorescent Proteins

Enhanced Green Fluorescent Protein (EGFP) Chromophore Formation - Still the "gold standard" in fluorescent protein technology, the enhanced version of GFP features a chromophore based on a para-hydroxybenzylidene substituted imidazolinone.

DsRed Fluorescent Protein Chromophore Formation - The chromophore of the first reported red fluorescent protein extends conjugation into the polypeptide backbone to generate fluorescence in the longer wavelength regions.

zsYellow Fluorescent Protein Chromophore Formation - The ZsYellow fluorescent protein chromophore features a novel three-ring system and peptide backbone cleavage due to the substitution of lysine for serine as the first amino acid residue in the chromophore tripeptide.

mKusabira Orange Fluorescent Protein Chromophore Formation - The final step in mKO chromophore maturations involves the formation of a novel five-member thiazole ring system when the Cys65 hydroxyl moiety attacks the carbonyl of Phe64 and cyclizes.

mOrange Fluorescent Protein Chromophore Formation - In a manner similar to mKusabira Orange, mOrange chromophore maturation involves the formation of a novel five-member oxazole (rather than a thiazole) ring system.

eqFP611 Chromophore Formation - A planar trans motif is found in the chromophore of the red fluorescent protein eqFP611, isolated from a sea anemone, and displays one of the largest Stokes shifts and red-shifted emission wavelength profiles of any naturally occurring fluorescent protein.

HcRed Fluorescent Protein Chromophore Formation - Although HcRed shares only approximately 21 percent amino acid sequence homology with GFP, enough critical amino acid motifs are conserved to form a very stable three-dimensional beta-barrel structure.

Kaede Fluorescent Protein Chromophore Formation - Upon illumination of the green species with ultraviolet light, the Kaede chromophore undergoes polypeptide chain cleavage between His62 and Phe61 to generate red fluorescence.

Kindling Fluorescent Protein (KFP1) Chromophore Formation - Investigations into the mechanism of kindling fluorescent protein photoswitching suggest that a cis-trans isomerization of the hydroxybenzilidine chromophore moiety is a key event in the switching process.

PA-GFP Chromophore Photoactivation - By replacing the threonine at position 203 with a histidine residue in wild-type GFP, researchers produced a variant having negligible absorbance in the region between 450 and 550 nanometers, thus dramatically enhancing contrast.

Dronpa Fluorescent Protein Chromophore Photoswitching - The most prominent and well-studied photoswitchable fluorescent protein is named Dronpa (named after a fusion of the Ninja term for vanishing and photoactivation), which is a monomeric variant derived from a stony coral tetramer.

Photoconversion of Kaede/Eos Highlighters - Unlike photoactivatable fluorescent proteins, Kaede and Eos are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions for optical highlighting.

Excited-State Proton Transfer - When excited with ultraviolet light, the tyrosine residue in the neutral chromophore of wild-type GFP becomes a strong acid and transfers a proton through a novel hydrogen bond network in a process known as excited-state proton transfer.

Spinning Disk

Spinning Disk Fundamentals - Explore how light passes through the pinholes on a spinning disk microscope to produce multiple excitation beams that are swept across the specimen as the disk spins. The Nipkow disk is located in a conjugate image plane and scans with approximately 1000 individual light beams.

Yokogawa Spinning Disk - The most advanced design in spinning disk instruments was engineered by Yokogawa Electric Corporation of Japan and implemented in a series of increasingly complex disk scanning units. This tutorial examines the operating principles of the Yokogawa scanning units.

Pinhole Crosstalk in Spinning Disk Microscopy - Axial resolution in spinning disk microscopy is largely defined by the size of the pinhole or slit and the separation distances between these apertures. This tutorial demonstrates how fluorescence removed from the focal plane can generate pinhole crosstalk.

Microlens Arrays in Spinning Disk Microscopy - The amount of light transmitted through the Nipkow disk in spinning disk microscopy is determined by the diameter of the pinhole or slit and the distance between these apertures. This tutorial explores how the amount of light passed through a disk can be increased by using microlens arrays on the upper disk in a two-disk system.

Camera Exposure and Disk Speed in Spinning Disk Microscopy - In spinning disk microscopy using a Yokogawa scan head, the camera exposure times are dependent upon the intensity of fluorescence emission gathered by the specimen and vary widely from one sample to another, and the disk rotation speed must be carefully adjusted to match the camera exposure time.

Superresolution Microscopy

Photoactivation Localization Microscopy (PALM) - Photoactivated localization microscopy (PALM) is a superresolution technique that dramatically improves the spatial resolution of the optical microscope by at least an order of magnitude (featuring 10 to 20 nanometer resolution), which enables the investigation of biological processes at close to the molecular scale.

Stimulated Emission Depletion (STED) Microscopy - Superresolution microscopy using stimulated emission depletion (STED) creates sub-diffraction limit features by altering the effective point spread function of the excitation beam using a second laser that suppresses fluorescence emission from fluorophores located away from the center of excitation.

Superresolution Structured Illumination Microscopy (SR-SIM) - SR-SIM is capable of achieving a lateral resolution of 50 to 60 nanometers and an axial resolution ranging from 150 to 300 nanometers. The technique relies on superimposing different grid orientations on the specimen to generate raw images, which are reconstructed into high resolution derivatives.

The RESOLFT Concept - The theoretical foundation necessary for achieving resolution beneath the diffraction barrier, which is actually composed of a family of physical concepts, was first advanced by Stefan Hell and associates with their introduction of the idea of reversible saturable (or switchable) optical fluorescence transitions (RESOLFT).

The PALM Concept - Photoactivated localization microscopy (PALM) relies on the stochastic activation of fluorescence to intermittently photoswitch individual photoactivatable molecules to a bright state, which are then imaged and photobleached. Thus, very closely spaced molecules that reside in the same diffraction-limited volume are temporally separated.

The Stimulated Emission Depletion (STED) Concept - Point-spread engineering techniques designed to circumvent the diffraction barrier all rely on a time-sequential readout of fluorescent probe photoswitching. The first technique successfully applied to superresolution biological imaging of fixed cells was the RESOLFT method named stimulated emission depletion (STED).

Depletion Lasers in STED Microscopy - In STED microscopy, the specimen is illuminated by two synchronized ultrafast co-linear sources consisting of an excitation laser pulse followed by a red-shifted depletion laser pulse that is referred to as the STED beam. Pulsed lasers are used to produce radially symmetric depletion zones.

Saturated Structured Illumination Microscopy - Saturated structured illumination microscopy is a superresolution technique where non-linearity arises from saturation of the excited state. SSIM and related methodology can readily be implemented on a widefield microscope with a single laser system and standard fluorophores.

Superresolution Microscopy with STED - STED takes advantage of the RESOLFT concept by significantly modifying the shape of the excitation point-spread function by manipulating the phase, pulse width, and intensity of the excitation and depletion lasers. This interactive tutorial explores how images are constructed using STED microscopy.