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.
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.
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 enhancng lambda stack acquisition speed for live-cell imaging experiments.
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.
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.
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. Information pertaining to the specifications of microscope objectives are inscribed on the decorative barrel by the manufacturer. This interactive tutorial examines the specification found on typical objectives.
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.
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 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.