Light Microscopy

Light microscopy covers a wide range of techniques including phase contrast microscopy, interference microscopy, polarization microscopy, fluorescent microscopy.  It can be used to investigate the shape, structure and composition of materials.   Phase contrast microscopy is widely used to study the structure of living cells because, with this technique, internal structures can be observed without killing and staining the cell.  In addition, motion pictures of dividing cells or moving cells can be made using phase contrast microscopy.  The interference microscope involves passing two separate beams of light through the specimen. With the appropriate instrument, the mass of material per unit area of the specimen can be determined, and contour mapping of small objects is possible.  Crystalline or fibrous elements, both of which are characterized by an orderly or layered molecular structure, are studied with a polarizing microscope; the polarizing microscope has been particularly useful in studying the detailed structure of bone.   In fluorescence microscopy, the images seen are molecules of fluorescent dyes added to cells that attach to specific cellular components. Appropriate filters are required to insure that only the light of the correct  wavelength contributes to the image. Fluorescent antibodies have been used to locate specific kinds of proteins and other materials in certain cells of a tissue or in certain regions of a cell. The antibodies can be prepared for example by injecting into a rabbit an antigen (e.g., the protein myosin), which stimulates white blood cells called lymphocytes to synthesize antibodies that react specifically with the antigen. After the antibodies are isolated and purified, a fluorescent dye, fluorescein, becomes attached to them by a chemical reaction. If the fluorescent antibodies are spread over a tissue, they attach specifically to the molecules that stimulated their formation (myosin). The fluorescence microscope reveals the sites containing the antigen-antibody complex as bright luminescent areas in a dark background.

In a confocal imaging system a single point of excitation light (or sometimes a group of points or a slit) is scanned across the specimen.  The point is a diffraction limited spot on the specimen and is produced either by imaging an illuminated aperture situated in a conjugate focal plane to the specimen or, more usually, by focusing a parallel laser beam. With only a single point illuminated, the illumination intensity rapidly falls off above and below the plane of focus as the beam converges and diverges, thus reducing excitation of fluorescence for interfering objects situated out of the focal plane being examined. Fluorescent light (i.e. signal) passes back through the dichroic reflector and then passes through a pinhole aperture situated in a conjugate focal plane to the specimen. Any light emanating from regions away from the vicinity of the illuminated point will be blocked by the aperture, thus providing yet further attenuation of out-of focus interference.  Light passing through the image pinhole is detected by a photodetector.  Usually a computer is used to control the sequential scanning of the sample and to assemble the image for display onto a video monitor. Most confocal microscopes are implemented as imaging systems that couple to a conventional microscope.  The resolution obtained by a confocal microscope can be better by a factor of up to 1.4 than the resolution obtained with the microscope operated conventionally.  A confocal microscope can also be used in reflection mode and still exhibit the same out-of-focus rejection performance. This application is often used to image the surface of specimens.

A useful primer can be found at:  http://micro.magnet.fsu.edu/primer/index.html 

For more information please consult an ITG staff member or follow other links on the ITG web pages.

Related instruments:

Dissecting light microscope

Stereology workstation

Laser scanning confocal microscope

Fluorescent light microscope

Related Forums:

Leica SP2 single and multi-photon confocal microscopes.

The Internet Atlas of Histology.

Introduction to the Beckman Microscopy Suite.

StereoInvestigator: The Fuss About Using Stereology.

Automated Quantitation of Labeled Cells: The MCID System in Action.

Quantitative Microscopy Using Fluctuation Spectroscopy.

Using Optical Tweezers to Manipulate Cells.

Neurolucida and Stereoinvestigator: Two systems for Analysis of Morphology of Biological Structures.

A Web Atlas of Cellular Structures using Light and Confocal Microscopy.

An Introduction to the Light Microscope in the ITG.

Related Technical Reports:

Cell Biological Applications of Fluorescence Microscopy.

A Web Atlas of Cellular Structures Using Light and Confocal Microscopy. 

Related projects:

Web Atlas of Cellular Structures

Electron Microscopy

An electron microscope is an electron accelerator that focuses an electron beam with the aid of electromagnetic lenses. The accelerating voltage is typically in the range of 60-400 keV.  Lenses focus the electron beam and magnify the image after the electrons pass through the specimen. The lenses and the specimen stage are mounted in a vertical, lead-lined cylindrical column that allows the interior to be maintained under vacuum. The vacuum is needed so that the electrons do not collide with air molecules and get knocked off course before they reach the specimen. A good vacuum is required (10^-7 torr) since the mean free path of an electron is 125 cm in a vacuum of 10^-4 torr.

In the scanning electron microscope (SEM), a beam of accelerated electrons is used to image surface features of specimens. The surface topography of a specimen is generated by the electrons reflected (backscattered) or given off (secondary electrons) by the specimen struck by the electron beam. This is accomplished by focusing a narrow, intense beam of electrons to form a very small spot of illumination (between 2Å & 200Å diameter) on the specimen. This fine spot is then moved sideways by deflecting the beam so that a very narrow ribbon of specimen, whose width corresponds to the diameter of the spot, is traversed by the electron probe spot.  Scanning electron microscopy can be used to image the topography the sample or to determine the local composition, crystal structure and orientation, and electrical and optical properties of the sample.

For more information please consult an ITG staff member or follow other links on the ITG web pages.

Related instruments:

Transmission electron microscope.

Environmental scanning electron microscope.

Related Forums:

Microscopic studies of Equine Osteoarthritis.

Pseudo-color Stereograms from the ESEM.

Introduction to the Beckman Microscopy Suite.

Extraction of Microbial 16S rRNA Gene Sequences From Hot Spring Travertine.

Microstructure and Stress Evolution during Drying of Binary Colloidal Systems.

New Technology for the ESEM-FEG in the Beckman Microscopy Suite.

Automated Data Collection for Cryo Electron Microscopy.

Energy-Dispersive Spectroscopy Using the Environmental Scanning Electron Microscope.

Computational Methods for Three-Dimensional Reconstruction of Macromolecular Complexes.

An Interactive Remote Microscopy Application for WWW-Based Control of an Environmental Scanning Electron Microscope (ESEM).

ESEM: An Introduction and Discussion to the Technology and its Applications.

Studying the Structure of Materials using Electron Microscopy, Diffraction and Modeling.

Leginon: Automated Acquisition of 1000 Electron Micrographs a Day.

Transmission Electron Microscopy: What Information can be Obtained from the Ultrathin Section?

JavaScope: Control of a Transmission Electron Microscope from a Web Browser.

Related Technical Reports:

An Interactive User Interface for Automated Acquisition of Transmission Electron Micrographs.

An Automated System For Maintaining Liquid Nitrogen Levels In The Gatan Cryostage.

An Integrated System For Transmission Electron Microscopy.

Bugscope: The Second Year of a Sustainable Remote Microscope Project for K-12 Education Outreach.

Evenly Dispersed Gold Beads on TEM Grids. 

Improving the Positional Accuracy of the Goniometer on the Philips CM200 TEM.

Automated Acquisition of Cryo Electron Micrographs Using Leginon.

Bugscope: Sustainable Internet Access to a ESEM for the K-12 Classroom.

Nondestructive Imaging of Pin-Mounted Museum Specimens of Insects Using the Environmental Scanning Electron Microscope (ESEM).

irma: An Interactive Remote Microscopy Application for WWW-Based Control of an Environmental Scanning Electron Microscope (ESEM).

Improving the Positional Accuracy of the Goniometer on the Philips CM SERIES TEM.

JavaScope: A Web-Based TEM Control Interface.  

Leginon: A System for Fully Automated Acquisition of 1000 Electron Micrographs a Day.  

Preparation of Catalase Crystals. 

TEM Length Calibrations.

Related projects:

Remote Microscopy

Automated Microscopy

Bugscope

Scanning Probe Microscopy

Scanning-tunneling microscopy (STM) can image surfaces of conducting materials with atomic-scale resolution. It uses an atomically-sharp metal tip that is brought very close to the surface. When the tip and sample are connected with a voltage source, a small tunneling current flows between the tip and sample surface. This current can be measured, and the magnitude depends on the distance between the tip and the surface. As the tip is moved laterally across the surface, a feedback mechanism moves the tip up and down to maintain a constant tunneling current. Rastering the tip across the surface therefore produces a topographic map of the surface.

Atomic-force microscopy is similar to scanning-tunneling microscopy (STM) in that it can image surfaces at atomic-scale resolution. The difference between AFM and STM is that AFM does not require that the sample be an electrically conducting material. Like STM it uses an atomically-sharp tip that is brought very close to the surface. The tip will feel a chemical attraction or repulsion and will move up or down on its supporting cantilever. The key to the sensitivity of AFM is in monitoring the movement of the tip. A common means of monitoring the tip movement is to use a laser beam that is reflected or diffracted by the tip or cantilever. Up or down movement of the tip is then detected by changes in the laser beam position. As in STM, rastering the tip across the surface produces a topographic map of the surface with atomic resolution.

Light at optical wavelengths, 500 nm for example, cannot be focused to spots much smaller than approximately one micrometer due to diffraction. This diffraction limit prevents optical imaging beyond the micrometer scale. Near-field scanning optical microscopy (NSOM) uses a light source of less than 100-nm diameter to beat the diffraction limit of conventional optical microscopy. The main concept of NSOM is to place the sample very close to the light source, i.e., in the near field, so that the imaging resolution is determined by the diameter of the light source.   The near-field light source is made from a glass capillary that is heated with a CO₂ laser and pulled to an atomically-sharp tip. The outside of the capillary is coated with silver for reflectivity except at the very end of the tip. Laser light is focused into the glass capillary and propagates to the tip by internal reflection (as in optical fibers). A small amount of the light leaks out of the tip via the evanescent wave. The near-field source is brought very close to a sample, and the sample is rastered back and forth to produce an image. NFOM can use either absorption or fluorescence as the optical signal.

For more information please consult an ITG staff member or follow other links on the ITG web pages.

Instruments in the suite that fall into the general category of scanning probe microscopy include:

Please follow the hyperlink to learn more about these instruments.

Related instruments:

Atomic force microscope

Near field scanning optical microscope

Related Forums:

Silicon-Based Molecular Nanotechnology.

Introduction to the Beckman Microscopy Suite.

Probing Polymer Subsurface Structure Using Tapping Mode Atomic Force Microscopy.

Surface plasmon resonance microscopy with a near field scanning optical microscope.

Measuring Surface Adhesion and Stiffness on the Nanometer Scale Using Pulsed-Force Microscopy.

Near Field Scanning Optical Microscopy in the Microscopy Suite.

Magnetic and Electronic Force Microscopy using the AFM.

Introduction to Atomic Force Microscopy.

Related Technical Reports:

Imaging Electrochemical Controlled Chemical Gradients Using Pulsed Force Mode Atomic Force Microscopy.

Imaging Ultrathin Organic Films on the Nanometer Level Using Surface Plasmons.