Confocal Microscopy

Introduction

The major application of confocal microscopy in the biomedical sciences is for imaging either fixed or living tissues that have been labeled with one or more fluorescent probes. When these samples are imaged using a conventional fluorescence microscope, fluorescence in the specimen in horizontal planes outside of the region of interest interferes with resolution of structures in focus, especially for those specimens that are thicker than 2µm or so. The ability of the confocal microscope to eliminate the out-of-focus flare caused the explosion in its popularity.

Illumination in a confocal microscope is achieved by scanning one or more focused beams of light from a laser across the specimen. An image produced in this way is called an optical section to distinguish it from the more common physical section used by other microscopy techniques. Because it uses light, it is a noninvasive method of image collection as opposed to physical sectioning of fixed specimens. An important consequence of this is that with confocal microscopy the imaging of living specimens is made possible. Additionally, since multiple optical sections can be made at varying depths (“Z planes”), it is possible to construct a computerized 3-D image of the tissue.

The Research Resources Center presently has two confocal microscopes, a Zeiss LSM510 and a Zeiss PASCAL. They are being applied to both biological and materials samples.

The Evolution of Confocal Microscopy

Marvin Minsky created the basic design of the confocal microscope in the mid-1950s at Harvard University. His idea required that a laser be used as a light source instead of the mercury lamp used in a conventional epi-fluorescence microscope. An important design element was a “pinhole” set between the reflected light and a detector at the focused plane that reduced the signals from the out-of-focus planes.

In Minsky's original microscope the light source was stationary and the specimen itself was moved on a vibrating stage. This can eliminate lens defects but the movement of biological specimens can cause wobble and distortion and loss of resolution in the image. A real image was not formed in Minsky's original microscope - his contribution was to the major principles of confocal microscopy, i.e., the use of laser light to illuminate a thin plane of a relatively thick sample through a pinhole aperture. Several later major technological advances have made his design available to research by biologists. These advances include stable multiwavelength lasers for brighter point sources of light, more efficiently reflecting mirrors, sensitive low-noise photodetectors, fast microcomputers with image processing capabilities, elegant software solutions for analyzing the images, high-resolution video displays and digital printers, and brighter and more stable fluorescent probes.

Basic Principles and Components of the Confocal Laser Scanning Microscope

(Figure 1 - diagram on the right) Schematic display of the major parts of a confocal microscope. See text below for description.

Light source. Lasers are used as the light sources in confocal microscopes. All the energy of a laser is contained in a tightly coherent bundle that can be focused on an extremely small spot with limited diffraction by the objective. This spot is then scanned over the specimens resulting in an image with high resolution. Different laser sources produce light of different wavelengths, which can excite different fluorochromes thereby producing different colors. The variety of colors enable the investigator to study the interaction of as many as four macromolecules.

Probe. The fluorescent probe is the major target used in confocal microscopy. A fluorochrome is a chemical which, when exposed to a light of specific wavelength or a range of wavelengths, absorbs light energy and becomes excited, emitting light that appears in the image as a specific color. These colors, the fluorescent spectrum, vary, of course, with the emitted wavelength. Short wavelengths are perceived as blue or violet, longer wavelengths are detected as green, and orange and red represent the longest wavelengths. Wavelengths in the range of ultra violet (U.V.) and the infrared are not visible to the human eyes. After illumination by laser, the wavelength of emitted light of the probe is always longer than that of the illuminating light. For example blue light excites FITC, which emits green light. GFP and its variants emit light of different wavelengths (green, blue, cyan, yellow and red for GFP, BFP, CFP, YFP and RFP, respectively) and these have been established as a novel genetic reporter system expressed in living cells and animals.

Dichroics (dichroic mirror or dichroic beam splitters). This optical element always reflects light of lower wavelengths and transmits light of wavelengths higher than their rated value. Thus, a “Dichroic mirror FT510” reflects light with wavelength below 510nm and transmits wavelengths above 510nm. This characteristic permits multicolor detection.

Light path and pinhole. The laser beam (Fig 1) passes through a beam-expanding lens and reflects off the dichroic mirror to the sample, producing a diffraction-limited spot at the sample plane (b, lower part of image). The fluorescent emission focuses back through the objective lens and the dichroic mirror, through the focusing lens and pinhole aperture (a,b,c, upper part of image) to the photomultiplier detector. The smaller the pinhole is, the sharper the image. Each spot is called a pixel. Only in-focus light (b) arrives at the detector; all out-of-focus light (a,c) is eliminated. After scanning through X and Y directions, the optical section, composed of many pixels, is formed and stored in the computer imaging system.

The confocal microscope provides high-resolution images in the axial dimension by minimizing blur from out-of-plane fluorescence. For example, the optical sections collected by confocal microscopy (Fig 2) shows clearly the distribution of Gsa protein from diffuse pattern to perinuclear pattern after antidepressant treatment.

Figure 2. The left image is of rat glioma cells treated with antidepressant and the right image is a control, non-treated cell. The primary antibody is against Gs alpha and the secondary antibody is an Oregon Green labeled goat anti-rabbit antibody. Images from the LSM510 by Dr. Robert J. Donati. (larger image 820x400)

Dr. Robert J. Donati, Postdoctoral Research Associate, Dr. Mark Rasenick's laboratory, Department of Physiology and Biophysics.

Images in Fig 3 were obtained from pollen powder, which has strong autofluorescence with different wavelengths that are detected with triple-wavelength plus DIC mode. The right side uses a conventional fluorescence microscope and is characterized by a somewhat blurred picture, whereas the left side images came from a confocal image with the thickness less than 1µm showing considerable detail within structures.

Figure 3. Image of pollen powder (autofluorescent colors) (larger image 743x400)

In summary, the basic principle of the confocal microscope is that the laser beam is focused on a region (spot) in the specimen and detection is confined to the same region. An aperture consisting of a pinhole in front of the detector eliminates light from out-of focus elements. The term “confocal” means that the light of excitation and the light of emission are at the same focus.

Capabilities of the Confocal Microscope

Certain features of the confocal process that allow the investigator to image specimens in a way never before possible are discussed here.

• The optical section is the basic image unit of confocal microscopy. Because of the availability of multi-line lasers, current systems have the capability of exciting as many as four fluorochromes which makes it possible to show numerous components of a specimen simultaneously in separated or combined images. It also provides a non-confocal conventional transmitted light-image called DIC (differential interference contrast) together with confocal fluorescence imaging. Using the three-channel function, it is possible to detect three different proteins or nucleic acids with different probes that are present in separated or in different combinations (Fig 4).

  Figure 4. Images of mouse cardiomyocytes stained with RACK (RITC), PKC (FITC) and nuclei (DAPI). The images were obtained by Dr. David L. Geenen. (larger image 1200x400)

  Dr. David L. Geenen, Research Associate Professor of Medicine (Cardiology).

  The LSM510 offers five channels (blue, green, red, dark red and DIC). Because the difference between the red and dark red is not obvious, a pseudocolor - for example, yellow - can be used as a substitute for dark red, as shown in Fig 5.

  

  Figure 5. Autofluorescence of pollen powder was imaged by the LSM510 confocal scope with red (a), blue (c), green (d), dark red (e - displayed as false color yellow for clarity), plus a transmitting channel (DIC) (b). Image (f) is the combination of the five images from (a) to (e). (larger image 386x257)

  
  

• A Z-stack is a sequence of optical sections collected at different levels from a specimen using computer controlled stepping in the Z (vertical) direction by preset distances (Fig 6). From the Z-stack, the various patterns of staining at different levels of the specimen can be observed. The Z-stack of optical sections can be processed relatively easily into a 3D representation of the specimen.

  Figure 6. Confocal Z-series images of rat utricle stained with Calretinin (green-FITC), Peripherin (red-Texas red) and nuclei of hair cell (blue-DAPI). From the work of Dr. Anna Lysakowski and medical student Scott Guth. (larger image 401x300)

  Dr. Anna Lysakowski (right) Department of Anatomy and Cell Biology and medical student Scott Guth.

  
  

• The specimen can be optically sectioned orthogonally by scanning a single line at different Z depths in X or Y direction. From these data, an XZ or YZ profile is produced from a stack of Z sections (Fig 7). It allows the user to observe the structure of tissues or cells from different directions.

  

  Figure 7. Orthogonal section from Fig 6. XZ, YZ and XY views are revealed in one image. From Anna Lysakowski, Department of Anatomy and Cell Biology. (larger image 400x400)

  
  

• A series of optical sections collected with a time-lapse program can be used to trace the movement of certain molecules in live tissue or cells. For example, the release of certain dyes from synaptic vesicles by a stimulation ion can be recorded at preset time intervals (Fig 8). Figure 9 shows a series of images showing the movement of PKC from cytoplasm to cell membrane under the induction of PMA.

  

  Figure 8. The RRC’s LSM510 confocal microscope is helping Dr. Aixa Alfonso and Dr. Andrea Holgado de Brigueda of the Department of Biological Sciences and the Laboratory of Integrative Neuroscience explore the mechanism of synaptic vesicle biogenesis and recycling at C elegans neuronal terminals. Images of FM4-64 loaded synaptic vesicles before (a) and after (b) chemical depolarization are shown. The images were collected by using the LSM510 with the time series software. Analysis and calculation of flourescence from the time series images (c) were obtained using NIH image software.

  

  Figure 9. PKC epsilon/EGFP c-terminal fusion protein in Babta AM treated HEK cells in response to MPA stimulation. PMA induced translocation of PKC isoform occurs from cytoplasm to the membrane. These images were collected from the LSM510 with time-lapse software by Dr. Wonhwa Cho and his graduate student John Rafter, Department of Chemistry. (larger image 1200x192)

  
  

  Dr. Andrea Holgado de Brigueda, Postdoctoral Research Associate, Department of Biological Sciences.

  John Rafter, graduate student in the laboratory of Dr. Wonhwa Cho, Department of Chemistry.

  
  

• Time-lapse sequences of Z-series can also be collected from living preparations using the Zeiss LSM510 to produce four-dimensional (4D) data sets.

• Unstained preparations or immunogold or silver labeled samples can also be viewed with confocal microscopy using reflected light imaging.

• A topography function can extract, process, present and measure the surface textures from a 3D image stack. This is particularly interesting with respect to materials applications.

• The microscope can also create non-confocal conventional transmitted light-images called DIC (differential interference contrast). It is often informative to collect a transmitted image of a specimen and to merge such DIC with one or more confocal fluorescence images of labeled cells together with confocal fluorescence imaging.

• Co-localization of molecules produced within living or fixed samples is one of the important applications of confocal microscopy (Fig 10).

  Figure 10. Neuroblastoma N-2A cells were double stained by RITC-labeled monoclonal anti-(-tubulin and FITC-labeled polyclonal rabbit anti G(( antibody were scanned by the LSM510 confocal microscope with multitracking scan. Colocalization areas are displayed with false color yellow in (a) and (b) and are merged in (c). These images were obtained by Dr. Tulika Sarma of Dr. Mark Rasenick’s laboratory, Department of Physiology and Biophysics. (larger image 418x149)

  Dr. Tulika Sarma, Postoral Research Associate in Dr. Mark Rasenick's laboratory in the Department of Physiology and Biophysics.

  
  

• FRET (fluorescence resonance energy transfer) is a quantum mechanical phenomenon that occurs between a fluorescence donor and a fluorescence acceptor in close proximity (100Å of separation) if the emission spectrum of the donor overlaps with the excitation spectrum of the acceptor. This is a very powerful tool in the study of molecular interaction within living organisms. Currently, the optimal donor-acceptor pair consists of optimized CFP and YFP variants, respectively. In order to make use of this method, the wavelengths of laser and specific filters have to be set properly. This technique is used in genomics for identification of single-nucleotide polymorphisms, etc.

• FRAP (fluorescence recovery after photo bleaching) is a technique which uses a short pulse of intense laser light to irreversibly destroy (photo bleach) fluorescence in a small micron-size area. Recovery of fluorescence into the area being photobleached occurs as a result of diffusion exchange between bleached and unbleached fluorescent molecules. New GFP fusion proteins are excellent reagents for applying in photobleaching studies.

Related Technologies

Microscopy technology continues to improve at a rapid rate. Two recent advances include multiphoton imaging and deconvolution microscopy.

Multi-photon microscopy uses a scanning system that is identical to that of the laser scanning confocal but without the confocal feature (i.e., without the pinhole). Because the laser excites the fluorochrome only at the point of focus, a pinhole is not necessary. Using this method, photobleaching is reduced, which makes the technology more amenable to imaging living tissues. The method also has certain limitations: For a given fluorochome, the spatial resolution using multiphoton imaging is slightly lower than that obtained using confocal imaging. Furthermore, if there exists a UV chromophore in the sample that absorbs at the excitation wavelengths, then there is a possibility of thermal damage to the specimen. The major obstacle to the wider use of the technique at present is the expense of the instrumentation and the need for frequent careful adjustments of the laser.

Deconvolution is a technique that uses a computer to calculate and remove the out-of-focus information from a fluorescence image. Like the confocal scope, it can image Z scans through the tissue sample.

Technological improvements such as these, including the combination of confocal and multiphoton microscopy and improved probes will provide a powerful means of imaging the spatial distribution and behavior of macromolecules in cells.

RECENT RRC NEWS Flow Cytometry The RRC Flow Cytometry Service (FCS) has purchased a BECTON-DICKINSON LSR bench top analyzer. The instrument has been installed and will be fully operational soon. It has an air cooled Helium-Cadmium UV laser, as well as the usual 488nm air-cooled Argon laser. It is ideal for calcium measurements using INDO-1, live cell DNA analysis using Hoechst reagents, and detection/analysis of cells that express various blue fluorescent proteins such as CYAN. The new instrument uses software (Cell Quest) that many users of the Becton-Dickinson FACScalibur® are familiar with. Investigators who demonstrate proficiency in operating this instrument will be able to use it without assistance from RRC personnel. The Coulter EPICS 753 has been decommissioned to provide space for the new instrument. As a result, in the near future the lab will not be able to sort particles/cells stained with UV excitable probes, or to analyze/sort particles stained with probes that have to be excited by laser sources different from 488nm, 633nm or UV wavelengths. The RRC is pleased to welcome Ms. Pinal Patel full time to the FCS. She will help train users in instrument operation and will provide service on the RRC flow cytometers/cell sorters. Pictured: Dr. Karen Hagen (right), Director of Flow Cytometry, and Pinal Patel, Research Specialist in the Health Sciences and new assistant to Karen in the flow lab. Electron Microscopy There have been several improvements and changes in the RRC Electron Microscopy Service (EMS): New SEM for EMS-west Pictured: Linda Juarez, RRC EM technician, practices using the new Hitachi S-3000N Variable Pressure Scanning Electron Microscope (VPSEM). The new scope provides investigators with new capability and power. The JEOL JSM-35C was finally switched off after twenty-two years of service to the UIC research community. During the following week it was removed and the room cleaned and repainted for the arrival of a new SEM. The new microscope is a Hitachi S-3000N Variable Pressure Scanning Electron Microscope (VPSEM), and opens up a new area of application for the EMS, that of variable pressure imaging. EMS users were allowed access less than two months after the JSM-35C was switched off. In conventional high vacuum mode the S-3000N outperforms the old JSM-35C. The imaging resolution at 25kV is 3.5nm compared with 7nm. The microscope also has a unique dual bias gun design, which increases the beam current by a factor of five at low accelerating voltages (less than 5kV). This allows good low-voltage imaging of surface detail on specimens, and the ability to image some non-conducting specimens without charging. If low voltage mode does not work for your non-conducting specimens, variable pressure (VP) mode will! In the VP mode the specimen chamber is operated at a pressure of 1-270Pa by leaking air into the chamber. Charging is the result of the build up of negatively charged electrons in a specimen unable to conduct them away. The poor vacuum in VP mode leads to the generation of positively charged ions, a result of the interaction of the electron beam with gas molecules, which can neutralize the negative charge on the specimen. Specimens that have proved difficult to image in conventional SEM even when coated, such as teeth, are easily imaged in VP mode without coating. The ability to look at all dry specimens with no preparation is the biggest advantage of the new microscope. In VP mode the conventional secondary electron detector (SED) cannot be used and a backscattered detector is used instead. Our S-3000N also has an Environmental SED which works by measuring the positive specimen current built up in the specimen in poor vacuums. Images can only be collected at slow scan speeds but contain the same information as a conventional SED image. The microscope also has a user friendly Oxford Inca EDX system with a light element detector. One user has already tested this down to boron! In VP mode, the beam spreading caused by the gas degrades spatial resolution. As the pressure rises, an electron skirt forms around the beam that can be several millimeters in diameter. However, good spatial resolution analysis is possible in high vacuum mode and spatially resolved analysis in VP mode can be carried out by X-ray mapping, which averages out the skirt. New EDX Systems for EMS-east Thermo Noran has supplied two new EDX systems for the JEM-2010F and JEM-3010 along with a new 40mm2 detector for the JEM-2010F. The new systems are Vantage PC based systems using similar software to the previous Voyager UNIX system. The existing EDX detector from the JEM-2010F was transferred to the JEM-3010. Analysis of areas with a 5nm probe is possible on the JEM-3010 and down to 0.5nm in the JEM-2010F. The Tracor Northern EDX system on the JEOL JXA-733 Microprobe has also been upgraded using a CRB award to Earth and Environmental Sciences. The new system, which uses the existing EDX spectrometer, is based on an EDAX EDAM III system and is integrated with the Advanced Microbeam WDX system installed in 1998. New in Specimen Preparation In EMS-east, the Interface Physics Group has purchased a new Fishione 1010 Low Angle Ion Mill with Liquid Nitrogen specimen cooling. Special holders allow milling down to 0 degrees (compared to 8 degrees on the earlier Fishione 3000). We will also shortly receive a new rotary slurry cutter from South Bay Technology which will be used to cut 3mm specimens from a wide range of materials. In EMS-west, the renovation of the new specimen preparation lab and sectioning room is now complete with the arrival of a Leica UCT microtome. The new microtome is identical to the one in EMS-east and brings our capabilities for ultrathin sectioning up to date. The existing Sorval MT-6000 will be retained for thick sectioning.

Wed Mar 20 16:11:00 CST 2002