Nikon MicroscopyU interactive Java and Flash tutorials are designed to simplify and explain complex topics in microscopy, optics, digital imaging, and photomicrography. Each tutorial explores an individual topic and takes advantage of the powerful Java technology to create interactive features that allow the visitor to change parameters and observe how this affects the phenomenon being studied. Use the links below to navigate to interactive Java tutorials of interest.

Microscope Alignment for Köhler Illumination - Perhaps one of the most misunderstood and often neglected concepts in optical microscopy is proper configuration of the microscope with regards to illumination, which is a critical parameter that must be fulfilled in order to achieve optimum performance. The intensity and wavelength spectrum of light emitted by the illumination source is of significant importance, but even more essential is that light emitted from various locations on the lamp filament be collected and focused at the plane of the condenser aperture diaphragm. This interactive tutorial reviews both the filament and condenser alignment procedures necessary to achieve Köhler illumination.

Eclipse L200 Microscope - Imagine you are dressed in a bunny suit and are about to examine newly fabricated chips under the microscope. Use this tutorial to explore how integrated circuit inspection microscopes are utilized to examine wafers in brightfield, darkfield, and differential interference contrast illumination.

Apodized Phase Contrast - In apodized phase contrast microscopy, halo attenuation and an increase in specimen contrast can be obtained by the utilization of selective amplitude filters located adjacent to the phase film in the phase plates built into the objective at the rear focal plane. These amplitude filters consist of neutral density filter thin films applied to the phase plate surrounding the phase film as illustrated in the tutorial window.

Specimen Contrast Enhancement with Apodized Phase Plates - Recent advances in objective phase ring configuration have resulted in a new technique termed apodized phase contrast, which allows structures of phase objects having large phase differences to be viewed and photographed with outstanding clarity and definition of detail.

Full-Frame CCD Operation - Full-frame charged coupled devices (CCDs) feature high-density pixel arrays capable of producing high quality digital images with the highest resolution currently available. This popular CCD architecture has been widely adopted due to the simple design, reliability, and ease of fabrication.

CCD Noise Sources - Noise sources vary in digital cameras. Photon noise, dark current, fixed pattern noise, and photo response nonuniformity are generated on the CCD itself, while reset noise, I/f noise, and quantization noise occur during amplification and conversion of the analog signal to a digital output.

Tube Lens Focal Length - With infinity-corrected optical systems, tube lengths between 200 and 250 millimeters are considered optimal. This is primarily because longer focal lengths will produce a smaller off-axis angle for diagonal light rays, leading to a reduction in system artifacts. Longer tube lengths also increase the flexibility of the system with regard to the design of accessory components.

Oblique Coherent Contrast Illumination - Featuring new objectives with substantially higher numerical apertures and an industry-leading zoom ratio of 15x, the Nikon SMZ1500 establishes a new standard in stereomicroscopy. This interactive Java tutorial explores the light path and images produced with Nikon's proprietary Oblique Coherent Contrast illumination system designed to optimize contrast in transmitted stereoscopic microscopy.

Toroidal Mirrors - The C-BD Diascopic Brightfield/Darkfield stand introduced with the Nikon SMZ-1500 uses a seven-sided toroidal mirror to substantially reduce stray light. Use this interactive Java tutorial to explore how mirror shape affects the amount of light entering the objective in darkfield stereoscopic microscopy.

Microscope Objectives: Immersion Oil and Refractive Index - The refractive index of the imaging medium is critical in determining the working numerical aperture of a microscope objective. A dramatic increase in numerical aperture is observed when the objective is designed to operate with an immersion medium such as oil, glycerin, or water between the front lens and the specimen cover glass. This tutorial explores how changes in the refractive index of the imaging medium can affect how light rays are captured by the objective.

Microscope Objectives: Numerical Aperture Light Cones - The light-gathering ability of a microscope objective is quantitatively 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. Higher values of numerical aperture allow increasingly oblique rays to enter the objective front lens, producing a more highly resolved image. This interactive tutorial demonstrates the change in numerical aperture light cones displayed by a microscope objective with corresponding changes in the angular aperture (and numerical aperture) of an objective.

Proximity-Focused Image Intensifiers - Image intensifiers were developed for military use to enhance our night vision and are often referred to as wafer tubes or proximity-focused intensifiers. They have a flat photocathode separated by a small gap on the input side of a micro-channel plate (MCP) electron multiplier and a phosphorescent output screen on the reverse side of the MCP. Use this interactive Java tutorial to explore how intensifier gain can be used to increase the output level of this device.

Birefringent Crystals in Polarized Light - This interactive tutorial explores how birefringent anisotropic crystals interact with polarized light in an optical microscope. The specimen is a virtual tetragonal crystal having an optical axis oriented parallel to the long axis of the crystal. Light entering the crystal from the polarizer travels perpendicular to the optical (long) axis of the crystal.

Polarizer Rotation and Specimen Birefringence - When a birefringent material is placed between crossed polarizers in an optical microscope, light incident upon the material is split into two component beams whose amplitude and intensity vary depending upon the orientation angle between the polarizer and permitted vibration directions of the material. This tutorial explores the effects of polarizer rotation on specimen birefringence as observed in a polarized light microscope.

Photomask Reticule Operation - Practice adjustment of the photomask reticule mounted in a focusing eyepiece using this interactive tutorial. Slider controls allow the visitor to focus the specimen and adjust illumination intensity. In addition, the reticule crosshairs can be focused, and the illumination color changed with a set of radio buttons.

Stereomicroscopy Fluorescence - The illuminator for epi-fluorescence on a stereomicroscope functions in a manner that is similar to those employed on more complex compound microscopes. Typically, the fluorescence illuminator consists of a xenon or mercury arc lamp contained in an external lamphouse that is attached to the microscope via an intermediate tube (or vertical illuminator) positioned between the microscope zoom body and observation tubes. This interactive tutorial explores internal optical pathways of the Nikon SMZ1500 stereomicroscope equipped with an epi-illumination intermediate tube and lamphouse.

Focus and Alignment of Mercury and Xenon Arc Lamps - Mercury and xenon arc lamps are now widely utilized as illumination sources for a large number of investigations in widefield fluorescence microscopy. Visitors can gain practice aligning and focusing the arc lamp in a Mercury or Xenon Burner with this interactive tutorial, which simulates how the lamp is adjusted in a fluorescence microscope.

Reflected Confocal Microscopy: Integrated Circuit Inspection - Examine individual layers on the surface of integrated circuits with this interactive tutorial. Photomicrographs for the tutorial were derived from a Nikon Optiphot C200 reflected light confocal microscope.

Laser Scanning Confocal Microscopy - (approximately a 30 second download on 28.8K modems) Several methods have been developed to overcome the poor contrast inherent with imaging thick specimens in a conventional microscope. Specimens having a moderate degree of thickness (5 to 15 microns) will produce dramatically improved images with either confocal or deconvolution techniques. The thickest specimens (20 microns and above) will suffer from a tremendous amount of extraneous light in out-of-focus regions, and are probably best-imaged using confocal techniques. This tutorial explores imaging specimens through serial z-axis optical sections utilizing a virtual confocal microscope.

ACT-1 Main Window - Upon initalization, the ACT-1 software main window appears on the computer monitor. This window presents a variety of on-screen elements including menu bars and a Windows-style tool bar that facilitates capture and correction of digital images. There are two image display areas, a larger window appearing on the left portion and a smaller window positioned on the right. The tutorial operates in a manner that is similar to the native Nikon software. Currently, working modules include the thumbnail area, and all portions of the Live window.

Live Settings Panel Operation - Utilize this interactive Java tutorial to explore the various features available in the Nikon DXM 1200 ACT-1 software Live settings panel. Among the options included in the tutorial are exposure time, focus, the focus mark, sensitivity, and the color levels function. Tutorial controls are identical to those found in the native software.

White Balance Calibration - Explore calibration of the DXM 1200 imaging control software for white balance to alleviate color shifts and unwanted hues in digital photomicrography with this interactive tutorial. Either a single point or a rectangular marquee-selected area on the image can be specified for measuring the white balance.

Image Rotation - Live and captured images displayed in the Nikon DXM 1200 ACT-1 control software image windows can be rotated and flipped by a menu found in the Live settings panel. This interactive tutorial explores the appearance and orientation of flipped images. Control of the applet features is identical to that found in the native Nikon software.

Retouching Digital Images - Images captured by the Nikon DXM 1200 digital camera system can be conveniently processed by a retouching menu available with the ACT-1 control software. Correction factors include image brightness, contrast, gamma, sharpness, hue, saturation, intensity, rotation, color balance, white/black balance, and cropping. This interactive tutorial explores how images can be corrected using the ACT-1 Retouch Image window.

Option Settings Panel - The Option settings panel in the DXM 1200 ACT-1 control software can be utilized to configure various functions related to image capture. Among the variables controlled by this panel are image resolution, auto printing, automatic image enhancement, noise reduction, and color mode.

DN100 Camera Control Unit Simulation - When the DN100 Camera Control Unit (CCU) is powered on, the display window appears on the computer monitor. The display window presents a color image captured at 1.3 megapixel resolution that is refreshed at a rate of 15 frames per second. An array of on-screen control windows that facilitate the capture and correction of digital images is accessible from the main display window. In addition, the front panel of the CCU contains a number of indicators and buttons that can be used to monitor the CCU and gain access to its image processing and networking capabilities.

DN100 Web Browser Simulation - When the Nikon DN100 web browser interface initializes, three virtual rooms are available to the operator from the entry page. In one of these rooms, a single microscope image with a remote control panel appears in the web browser window. The remote controls provided by the instrument allow a remote microscopist to access many of the features of the camera control unit, including exposure settings, image processing functions, and the ability to capture and download live images in Bitmap or JPEG format. Other noteworthy features include an electronic zoom and pan capability, as well as an annotation function that allows the operator to superimpose a hand-drawn diagram on the image window.

Astigmatism - Astigmatism aberrations are similar to comatic aberrations, however these artifacts are not as sensitive to aperture size and depend more strongly on the oblique angle of the light beam. The aberration is manifested by the off-axis image of a specimen point appearing as a line or ellipse instead of a point. Depending on the angle of the off-axis rays entering the lens, the line image may be oriented in either of two different directions, tangentially (meridionally) or sagittally (equatorially). The intensity ratio of the unit image will diminish, with definition, detail, and contrast being lost as the distance from the center is increased.

Chromatic Aberration - Chromatic aberrations are wavelength-dependent artifacts that occur because the refractive index of every optical glass formulation varies with wavelength. When white light passes through a simple or complex lens system, the component wavelengths are refracted according to their frequency. In most glasses, the refractive index is greater for shorter (blue) wavelengths and changes at a more rapid rate as the wavelength is decreased.

Field Curvature - A simple lens focuses image points from an extended flattened object, such as a specimen on a microscope slide, onto a spherical surface resembling a curved bowl. The nominal curvature of this surface is the reciprocal of the lens radius and is referred to as the Petzval Curvature of the lens. Curvature of field in optical microscopy is a common and annoying aberration that is familiar to most experienced microscopists.

Geometrical Distortion - Distortion is an aberration commonly seen in stereoscopic microscopy, which is manifested by changes in the shape of an image rather than the sharpness or color spectrum. The two most prevalent types of distortion, positive and negative (often termed pincushion and barrel, respectively), can often be present in very sharp images that are otherwise corrected for spherical, chromatic, comatic, and astigmatic aberrations. In this case, the true geometry of an object is no longer maintained in the image.

Coverslip Correction Collars - High magnification objectives designed to be used with air as the immersion medium between the front lens and the cover glass are prone to aberration artifacts due to variations in cover glass thickness and dispersion. This tutorial demonstrates how internal lens elements in a high numerical aperture dry objective may be adjusted to correct for these fluctuations.

Geometrical Construction of Ray Diagrams - A popular method of representing a train of propagating light waves involves the application of geometrical optics to determine the size and location of images formed by a lens or multi-lens system. This tutorial explores how two representative light rays can establish the parameters of an imaging scenario.

Perfect Lens Characteristics - The simplest imaging element in an optical microscope is a perfect lens, which is an ideally corrected glass element that is free of aberration and focuses light onto a single point. This tutorial explores how light waves propagate through and are focused by a perfect lens.

Perfect Two-Lens System Characteristics - During investigations of a point source of light that does not lie in the focal plane of a lens, it is often convenient to represent a perfect lens as a system composed of two individual lens elements. This tutorial explores off-axis oblique light rays passing through such a system.

Projection and Viewing Eyepieces - The eyepiece (or ocular) is designed to project either a real or virtual image, depending upon the relationship between the intermediate image plane and the internal eyepiece field diaphragm. Explore how eyepieces can be coupled to the human eye or a camera system to produce images generated by the microscope objective.

Condenser Image Planes - In a microscope optical system, the lamp filament is imaged in the focal plane of the condenser aperture diaphragm when the microscope is configured to operate under conditions of Köhler illumination. This tutorial explores the relationship between image planes relevant to the field and condenser diaphragms and how aperture size affects ray trace pathways.

Microscope Conjugate Field Planes - In a microscope optical system, the lamp filament is imaged in the focal plane of the condenser aperture diaphragm when the microscope is configured to operate under conditions of Köhler illumination. This tutorial explores the relationship between image planes relevant to the field and condenser diaphragms and how aperture size affects ray trace pathways.

Infinity Microscope Conjugate Field Planes - The geometrical relationship between image planes in the optical microscope configured for infinity correction with a tube lens is explored in this tutorial. In such a microscope, magnification of the intermediate image is determined by the ratio of the focal lengths of the tube lens and objective lens.

Interactive Flash Tutorial Index - Built with current Flash programming technology, these tutorials combine three-dimensional graphics and images with interactive animations that explore a variety of concepts. Similar to Java applets, Flash tutorials are controlled by slider bars, pull-down menus, and radio buttons.

Bias Retardation Effects on Specimen Contrast - The introduction of bias retardation in differential interference contrast (DIC) microscopy renders the specimen image in pseudo three-dimensional relief where regions of increasing optical path length (sloping phase gradients) appear much brighter (or darker), and those exhibiting decreasing path length appear in reverse. This interactive tutorial explores the effects of varying bias retardation on contrast as a function of thickness for a wide spectrum of semi-transparent specimens.

Nomarski Prism Action in Polarized Light - When a Nomarski or modified Wollaston compound differential interference contrast (DIC) prism is sandwiched between two crossed polarizers and examined with light transmitted through both polarizers and the prism, a pattern of parallel interference fringes with a predominant central black band (fringe) can be observed. This interactive tutorial explores how varying prism wedge geometry, utilized for different objective numerical apertures, affects the interference pattern observed between crossed polarizers.

DIC Microscope Component Alignment - The proper adjustment and alignment of differential interference contrast (DIC) optical components is critical to imaging performance, so it is imperative that the microscopist recognize misalignments and component mismatches, and take the necessary steps to correct these errors. This interactive tutorial, hosted on the Nikon MicroscopyU website, examines conoscopic and orthoscopic viewfields in a DIC microscope under a variety of configurational motifs, and discusses many of the important aspects recommended for satisfactory microscope alignment.

Comparison of Phase Contrast and DIC Microscopy - The most fundamental distinction between differential interference contrast (DIC) and phase contrast microscopy is the optical basis upon which images are formed by the complementary techniques. Specimens examined by these contrast-enhancing methods produce images that are often quite different in appearance and character when objectively compared. This MicroscopyU interactive tutorial explores many of the similarities and differences exhibited between images captured with phase contrast and DIC microscopy.

DIC Microscopy with de Sénarmont Compensators - Although in traditional designs, differential interference contrast (DIC) microscopes introduce bias retardation into the matched condenser and objective Nomarski (or Wollaston) prisms by translating one of the prisms across the optical axis, the same effect can also be achieved through the use of a simple de Sénarmont compensator with fixed Nomarski prisms. This MicroscopyU interactive tutorial examines the relationship between wavefronts emerging from a de Sénarmont compensator and how they can be controlled to produce positive and negative bias retardation (contrast) effects in a DIC microscope.

Wavefront Relationships in de Sénarmont and Nomarski DIC - In differential interference contrast (DIC) microscopy, the spatial relationship and phase difference between ordinary and extraordinary wavefronts is governed either by the position of the objective prism (Nomarski DIC) or the relationship between the polarizer and a thin quartz retardation plate in a de Sénarmont design. This interactive tutorial explores the similarities and differences between the wavefront relationship in the two microscope configurations.

The de Sénarmont DIC Microscope Optical Train - Although traditional differential interference contrast (DIC) optical systems introduce bias retardation into the wavefront field by translation of the objective Nomarski prism, the same effect can be achieved through the application of a fixed Nomarski (or Wollaston) prism system and a simple de Sénarmont compensator consisting of a quarter-wavelength retardation plate in conjunction with either the polarizer or analyzer. This interactive tutorial explores the wavefront relationship in a de Sénarmont DIC microscope optical train as the polarizer is rotated with respect to the fast axis of the retardation plate.

Optical Sectioning with de Sénarmont DIC Microscopy - The ability to image a specimen in de Sénarmont DIC microscopy with large condenser and objective numerical apertures enables the creation of remarkably shallow optical sections from a focused image plane. Without the disturbance of halos and distracting intensity fluctuations from bright regions in lateral planes removed the focal point, the technique yields sharp images that are neatly sliced from a complex three-dimensional phase specimen. This property is often utilized to obtain crisp optical sections of cellular outlines in complex tissues with minimal interference from structures above and below the focal plane.

Wavefront Relationships in Reflected Light DIC Microscopy - In reflected light differential interference contrast (DIC) microscopy, the spatial relationship and phase difference between ordinary and extraordinary wavefronts passing through the optical system is governed either by the position of the objective prism (Nomarski DIC) or the orientational relationship between the polarizer and a thin quartz retardation plate in a de Sénarmont design. This interactive tutorial explores the similarities and differences between the wavefront relationships in the two microscope configurations.

Optical Sectioning in Reflected Light DIC - The ability to capitalize on large objective numerical aperture values in reflected light DIC microscopy enables the creation of optical sections from a focused image that are remarkably shallow. Without the confusing and distracting intensity fluctuations from bright regions occurring in optical planes removed from the focal point, the technique yields sharp images that are neatly sliced from a complex three-dimensional opaque specimen having significant surface relief. This property is often employed to obtain crisp optical sections of individual features on the surface of integrated circuits, as explored in the interactive tutorial, with minimal interference from obscuring structures above and below the focal plane.

Phase Contrast Microscope Alignment - Careful alignment of the phase contrast microscope is essential to produce the maximum contrast effect without introducing artifacts that degrade specimen appearance. This interactive tutorial examines variations in how specimens appear through the eyepieces (at different magnifications) when the condenser annulus is shifted into and out of alignment with the phase plate in the objective.

Optical Pathways in the Phase Contrast Microscope - The most important parameter in the design of a phase contrast microscope is to isolate the surround and diffracted light waves emerging from the specimen so that they occupy different locations in the diffraction plane at the rear aperture of the objective. This interactive tutorial explores light pathways through a phase contrast microscope and dissects the incident electromagnetic wave into surround (S), diffracted (D), and resultant (particle; P) components.

Phase Plate Configuration Effects on Specimen Contrast - The transmission and retardation properties of surround (undiffracted) light passing through the phase plate annulus in phase contrast microscopy can significantly affect the overall specimen contrast observed in the microscope. This interactive tutorial explores contrast variations induced by altering phase plate absorption and retardation characteristics.

Positive and Negative Phase Contrast - Depending upon the configuration and properties of the phase ring positioned in the objective rear focal plane, specimens can be observed either in positive or negative phase contrast. This interactive tutorial explores relationships between the surround (S), diffracted (D), and resulting particle (P) waves in brightfield as well as positive and negative phase contrast microscopy. In addition, phase plate geometry and representative specimen images are also presented.

Specimen Optical Path Length Variations - Phase contrast microscopy interprets differences in specimen optical path length as fluctuations in light intensity, which are readily observed as variations in contrast through the microscope. This interactive tutorial explores the effects of refractive index and thickness changes on the apparent overall optical path length, and demonstrates how two specimens can have different combinations of these variables but still display the same path length.

Interaction of Light Waves with Phase Specimens - Upon encountering a phase specimen, an incident illumination wavefront is deformed according to the geometry, refractive index, and thickness of the specimen. This interactive tutorial examines the variety of deformations observed in wavefront shape as specimens having differing characteristics are illuminated with a planar beam of light.

Shade-Off and Halo Phase Contrast Artifacts - Two very common effects observed in phase contrast images are the characteristic shade-off and halo patterns in which the observed intensity does not directly correspond to the optical path difference (refractive index and thickness values) between the specimen and the surrounding medium. This interactive tutorial demonstrates shade-off artifacts in positive and negative phase contrast microscopy.

Apodized Phase Contrast - In apodized phase contrast microscopy, halo attenuation and an increase in specimen contrast can be obtained by the utilization of selective amplitude filters located adjacent to the phase film in the phase plates built into the objective at the rear focal plane. These amplitude filters consist of neutral density filter thin films applied to the phase plate surrounding the phase film as illustrated in the tutorial window.

Apodized Phase Plates and Specimen Contrast - Recent advances in objective phase ring configuration have resulted in a new technique termed apodized phase contrast, which allows structures of phase objects having large phase differences to be viewed and photographed with outstanding clarity and definition of detail.

Fluorescence Resonance Energy Transfer with Fluorescent Proteins - Fluorescent proteins are increasingly being applied as non-invasive probes in living cells due to their ability to be genetically fused to proteins of interest for investigations of localization, transport, and dynamics. In addition, the spectral properties of fluorescent proteins are ideal for measuring the potential for intracellular molecular interactions using the technique of Förster (or fluorescence) resonance energy transfer (FRET) microscopy. Because energy transfer is limited to distances of less than 10 nanometers, the detection of FRET provides valuable information about the spatial relationships of fusion proteins on a sub-resolution scale. This interactive tutorial explores various combinations of fluorescent proteins as potential FRET partners and provides information about critical resonance energy transfer parameters, as well as suggestions for microscope optical filter and light source configuration.