Flow Cytometry and Confocal Microscopy PDF

Flow cytometry and cell sorting. Confocal microscopy The text under the slides was written by Gábor Mocsár 2020 This instructional material was prepared for the biophysics lectures held by the...

Flow cytometry and cell sorting. Confocal microscopy The text under the slides was written by Gábor Mocsár 2020 This instructional material was prepared for the biophysics lectures held by the Department of Biophysics and Cell Biology Faculty of Medicine University of Debrecen Hungary https://biophys.med.unideb.hu 2 Wallace H. Coulter developed an impedance measurement method for the counting and sizing of particles. The technique was developed specifically to rapidly count blood cells by calculating changes in electrical conductance as cells suspended in a conductive fluid moved through a tiny orifice. Coulter is best known for its long line of developments in hematology research, and fundamentally changed hematology by enabling more precise and faster measurement of CBC (Complete Blood Count). 3 Flow cytometry is a fluorescence technique for determining the physical and chemical characteristics of a population of cells. During this process, a sample of cells is suspended in a fluid and injected into the flow cytometer instrument. Cell sorting is a process for purifying populations of cells, the sorting is based on the presence or absence of certain physical properties. Sorting can be done based on many properties, such as cell size, morphology, and protein expression. Separation of cells is based on droplet technology permitting recovery of subsets of cells for post-experimental use. 4 Modern flow cytometers are able to analyze several thousand particles per second in “real time” and can actively separate and isolate particles with specified optical properties at comparable speeds, if designed as cell sorters as well. Flow cytometry offers high- throughput, and the ability to analyze the fluorescence and light scattering properties of particles (cells) individually. 5 Flow cytometry uses the light scattered from cells or emitted from fluorescent dyes, and the multidimensional data can be used for multi-parametric analysis. Fluorescent labels are typically attached to an antibody that recognizes a target feature molecule at the cell surface or in the cell. A fluorophore is characterized by a specific peak excitation and emission wavelengths. There are many combination of available fluorescent dyes. Specific detection is based on specific excitation, corresponding to the peak in the absorption spectrum of the dye, and on specific detection, achieved by recording those photons, which are at around the peak of the emission spectrum of the fluorophore. This specific fluorescence detection is typically done by using optical filters (band pass, long pass or short pass) and dichroic mirrors. They are used to filter and move light to the detectors, such as photomultiplier tubes (PMTs). Hydrodynamic focusing is used in most cytometers for precise positioning of cells in a liquid jet to the laser beam. Cell sorting is a method to purify cell populations based on the presence or absence of specific fluorescent characteristics, the separation is done by electrostatic deflection of charged droplets in an electric field. Modern flow cytometers usually have multiple lasers and fluorescence detectors, allow multiple antibody labeling, and can more precisely identify a target population by their phenotypic markers, therefore multivariate data analysis capability is available. 6 A flow cytometer typically has three sub systems: Fluidics System – responsible for the delivery and focusing of cells to the laser beam. Optical System – responsible for the generation and collection of light signals. Electronics System – responsible for the conversion of light signals to a measurable electronic signal, and then measuring, amplifying, and digitizing that signal to be communicated to the computer. 7  Schematic diagram of a hydrodynamic system, from sheath focusing to fluorescent detection. Hydrodynamic focusing – the process by which cells are focused in the center of the laser beam one cell at a time. This is accomplished by forcing a large volume of liquid into a small volume in the flow cell. The liquid has two sources: the sample or core fluid containing the cells to be measured, and the sheath fluid responsible for preventing cells from colliding into the walls of the nozzle, which would damage them. Under optimal conditions (laminar flow) there is no mixing of the central fluid stream and the sheath fluid. When a cell suspension is run through the cytometer, sheath fluid is used to hydrodynamically focus the cell suspension through a small nozzle. The core stream of fluid takes the cells past the (blue) laser light one cell at a time. Light scattered from the cells or particles is detected as they go through the laser beam. Fluorescence detectors measure the fluorescence emitted from stained cells or particles. 8 A fluid nozzle is a device in a flowing system that is designed for controlling characteristics of a fluid flow. The nozzle in a flow cytometer is a fluid chamber to which the sample fluid (containing cells) is driven to flow through the center of the laser beam. The fluid nozzle is one of the main components of a hydrodynamic focusing system. The size of the nozzle and tubes are such that the flow is laminar provided that appropriate pressure is applied. The aperture is circular with a diameter of 50-200 μm, the nozzle size should not be smaller than the diameter of the cells in the flowing system. 9 As shown in the image, blue laser light scattered from the cells is detected as they go through the laser beam. Detection of the scattered laser light can be used to measure volume (by forward scatter, FSC) and morphological complexity (by side scatter, SSC) of cells or other particles, even those that are non-fluorescent. 10 Although the illumination source of modern flow cytometers is either a lamp or a laser, lasers are used in most cases nowadays. Both sources have advantages and disadvantages and selection is based on the desired application. Lasers provide monochromatic, high intensity excitation, but their wavelengths are limited by the properties of the lasing material. Arc lamps have two major types, mercury vapor and xenon lamps. These light sources have the advantage of not requiring any specialized electronics or cooling which makes them much cheaper than lasers. Xe lamps and mercury arc-lamps have several emission maxima, which is advantageous especially when they match the absorption maxima of fluorescent probes. Mercury vapor lamps have a strong emission maximum at 365 nm, which enables its use for the excitation of most DNA probes. The spectrum of xenon lamps is continuous in a wide range which makes it very suitable for the excitation of several florescent probes. The use of these lamps requires special optical configuration and their focusing is more difficult than that of lasers. 11 Typically, the flow cytometers are equipped with lasers (Light Amplification by Stimulated Emission of Radiation) as primary excitation light source. The laser light is coherent, laser output wavelength has narrow bandwidth, and therefore the wavelength of the excitation can be specified with high precision. The coherence property of lasers ensures that the photon density at the illumination point is high enough to allow us to detect the scattered laser (FCS, SSC) or emitted fluorescence intensities. The emitted light of other light sources, such as arc lamps or LEDs (light emitting diodes), which are frequently used light sources in fluorescence microscopy, is not coherent, the output light travels in all directions from the source, and special optics are needed to focus it onto a measurement point. 12 Laser lines commonly used in flow cytometry, and absorption/emission spectra of fluorescent dyes are shown in the figure. A wide range of fluorophores can be used as labels in flow cytometry (from UV to Far Red), each fluorophore has a characteristic peak excitation and emission wavelength. Modern flow cytometers usually have multiple lasers and fluorescence detectors, allowing labeling with multiple antibodies and multidimensional cellular data analysis. 13 Schematic optical configuration of a flow cytometer with a single laser beam Schematic diagram of a flow cytometer, showing hydrodynamic focusing of the fluid sheath with a nozzle, a laser (blue), laser beam focusing optics, emission mirror and filters, photomultiplier tubes (PMTs), signal processing unit. Reminder: A flow cytometer typically has three sub systems: Fluidics System – responsible for the delivery and focusing of cells to the laser beam Optical System – responsible for the generation and collection of light signals Electronics System – responsible for the conversion of light signals to a measurable electronic signal, and then measuring, amplifying, and digitizing that signal to be communicated to the computer. 14  Schematic optical configuration of a flow cytometer with two laser beams. (Components for cell sorting are also included, discussed later.) As shown in the image, the flow cytometer uses blue laser light for generating and detecting the scattered laser light (forward scatter and 90° side scatter), for measuring the volume and morphological complexity of cells. A detector in front of the light beam measures forward scatter light signals (FSC) and detectors to the side measure side scatter light signals (SSC). Orange laser light excites specifically the fluorescent dyes that are conjugated to antibodies, causing the dyes to emit light at specified wavelengths. Fluorescence sensors, called photomultiplier tubes (PMTs), measure the fluorescence signal intensity emitted from stained cells. 15 Flow cytometers use fluorescence (FL-) channels, i.e. suitably designed optical paths, to detect light emitted from one specific dye. Separation of channels is achieved by using various optical elements selectively passing light of a small range of colors while reflecting other colors. These optical elements are listed below: Long pass (LP) filters allow transmission of photons above a specified wavelength. Dichroic filters/mirrors (such as dichroic LP mirrors) are positioned at a 45° angle to the light beam, and they typically reflect light below a certain wavelength, and allow light to pass above this wavelength. 16 As shown in the image, blue laser light is scattered from the cell as it goes through the laser beam. Light scattered at low angles is termed forward scatter (FSC) or forward angle light scatter (FALS) light, and it is collected along the same axis as the laser beam by a detector. Detection of the forward scattered laser light can be used to determine the volume of cells. 17 The measurement of forward scatter signal allows the discrimination of cells by their size, because larger cells refract more light than smaller cells. Therefore FSC intensity is proportional to the diameter of the cell. Application: by measurement of the FCS signal of a blood sample it is possible to distinguish between larger monocytes and smaller lymphocytes. 18 As shown in the image, blue light is scattered from the cells as they go through the laser beam. Side scattered light (SSC) is light is refracted by cells and it travels in a direction perpendicular to its original path (typically measured at a 90° angle to the excitation line). It provides information about the granularity and internal complexity of cells. Cells with a low granularity will produce less side scattered light, while cells with higher internal complexity will result in a higher side scatter signal. 19 20 21 7 The figure shows the direction in which the three major types of signals are measured in flow cytometry. Both fluorescence and side scatter are measured at a direction perpendicular to the excitation line. Forward angle light scatter is detected in a direction almost parallel to the excitation line. As shown in the image, red light is scattered from the cells as they go through the laser beam. As cells are translucent, many photons will pass through the cytoplasm. If a photon strikes an organelle (ER, nucleus, etc), the photon will be reflected at a larger angle than those generated by the forward scatter phenomenon. The more organelles/bits inside the cytoplasm, the more photons will be scattered, therefore side scatter signal is proportional to the internal complexity of cells. 22 As mentioned earlier, flow cytometers use separate fluorescence (FL-) channels to detect light emitted from one specific dye. Separation of channels from multiple dyes is achieved by using various optical elements to direct photons of the correct wavelength to each sensor (PMT):  Band pass (BP) filters allow transmission of photons that have wavelengths within a narrow range.  Short pass (SP) filters allow transmission of photons below a specified wavelength.  Long pass (LP) filters allow transmission of photons above a specified wavelength.  Dichroic filters/mirrors (such as dichroic LP mirrors) are positioned at a 45° angle to the light beam. In long pass dichroic filter, photons above a specific wavelength are transmitted straight ahead, whilst photons below the specific wavelength are reflected at a 90° angle. Short pass dichroic filters behave in the opposite way, i.e. they reflect above a certain wavelength while allowing photons below this wavelength to go across. 23 The flow cytometers use separate optical channels and detectors to detect emitted fluorescent or scattered laser light. The light sensitive detectors are either photomultiplier tubes (PMTs) or photodiodes (e.g.: avalanche photodiode, APD). PMTs are the most commonly used light detectors, see next slide. 24  Schematic optical configuration of a flow cytometer, and main components of a photomultiplier tube As shown in top-left the image, the cells, as they go through the laser beam, will scatter laser light, and their fluorescent labels will emit photons, then the optical elements will direct the photons to the dedicated sensor. The intensity measurement of the emitted fluorescent or scattered laser light intensities is typically achieved by photomultiplier tubes, as light sensitive sensors. Main components and the working principle of photomultiplier tubes is shown in top-right image. Photomultiplier tubes (PMT) are light sensitive detectors, and the amplitude of their output signal is proportional to the intensity of the incident light. The incoming photon is ejecting a few primary electrons from the photocathode by the photoeffect. These primary electrons move toward the first dynode because they are accelerated by the electric field. After striking the first dynode, two, three or more electrons are emitted, and these electrons are in turn accelerated toward the second dynode, then even more electrons are emitted etc, Finally, the multiple dynode stages multiply the photoelectron produced by incident light 108-fold. The last stage is called the anode, which is reached by a large number of electrons resulting in a sharp current pulse. The current pulse is converted to voltage peak that is easily detectable with fast signal processing devices. The photomultiplier tubes (PMTs) communicate the signal to the computer. 25  Schematic diagram about the generation of a voltage pulse in the PMTs  When no fluorescing cells pass through the laser beam, no photons are scattered and emitted, therefore no signal is detected.  As a fluorescently labeled cell passes through the laser beam, they are excited by the laser beam, fluorescent photons are emitted and so the intensity of the voltage measured with the PMT increases.  After the cell passes through the laser beam, it creates a peak of photon emission over time detected by the PMT.  As the cells are completing their path through the laser beam, they leave a pulse of voltage detected by the electronic system. The current is then converted to a voltage pulse, referred as an event.  The total pulse height, width and area can be determined with dedicated fast signal processing unit. 26  The discriminating function of the signal processing electronics. A discriminator is circuit that can be adjusted to accept or reject voltage signals of different characteristics (such as pulse height or width) measured by PMTs. Therefore, only voltage signals that are above a preset threshold will be collected. The discriminator is set to only a single value. If this threshold is not reached, then the signal is not detected. 27 The measured light intensities in flow cytometry are highly variable, therefore usage of special signal amplifying and conversion units are needed. The amplification system can be linear or logarithmic. Amplifiers should meet very specific criteria i.e. they should be linear in a wide range and with a relatively small noise. In addition to linear amplifiers there are also logarithmic amplifiers, which are able to cover a wide range of intensities by logarithmically transforming the signals (this is especially useful in the quantitative determination of surface molecules). The amplified signals that are collected from forward- scattered light (FSC) and side-scattered light (SSC) as well as dye-specific fluorescence photoelectron-multipliers are transformed to numbers by analog-digital converters (ADC) that can be processed and visualized by a computer. Top-left image: SSC-H vs FSC-H scatter plot shown with Linear- Linear (Lin-Lin) axes scales; each dot represents a single cell; SSC-H: height of the side scatter channel voltage signal events; FSC-H: height of the forward scatter channel voltage signal events. Top-right image: PE-CD4 vs FITC-CD3 scatter plot shown with Logarithm- Logarithm (Log-Log) axes scales; each dot represents a single cell; PE-CD4: height of voltage signal measured in PE channel; FITC-CD3: height of voltage signal measured in FITC channel; (CD4: a cell surface marker, a glycoprotein found on the surface of immune cells such as T cells; CD3: a cell surface marker, a protein complex is involved in activating T cells.) 28 The signals derived from the PMTs show time dependence corresponding to the flow of cells into and out from the laser beam. The amplified signals that are collected from photoelectron- multipliers are transformed to numbers by analog-digital converters (ADC). Multidimensional signal processing analyses enables the processing of the height (amplitude) and width of the signals as well as the area (integral of the signal) under each peak. Modern flow cytometers usually have multiple lasers and fluorescence detectors allowing multiple antibody labeling. By labeling cells with multiple antibodies researchers can more precisely identify a target population by its phenotypic markers, therefore multivariate data analysis capability is available. 29 The 'acquisition' is the process of collecting signal data from (fluorescently labelled) samples using the flow cytometer. During the acquisition, the computer is displaying the measured parameters (e.g.: SSC-H, FSC-H, PE-H, FITC-H etc.) on pre-defined scatter plots or on histograms in real time. All parameters that are above a preset threshold value will be collected and stored, referred to as an (true) event. 30  In the above data-table, part of a list mode data is shown Flow cytometry data can be acquired and stored in so-called “list mode” files. In this form of data, the various parameters of each individual cell are stored together: the rows of the file correspond to the individual cells (event), whilst the columns represent the different parameters. This excerpt of an exemplary list-mode file stores data of three cells. Four parameters were recorded from each cell. The names of these parameters are shown in the top row of the table. Two of the parameters are light scatter parameters (FS – forward scatter, SS – side scatter), while the other two parameters are fluorescence parameters (FITC, PE – names of two fluorophores). In this exemplary list-mode file the forward scatter and side- scatter values of the first cell are 50 and 100, respectively. The FITC and PE intensities of the same cell are 80 and 90, respectively. 31  The distribution of FITC (fluorescein isothiocyanate) fluorescence in a cell population represented with a one-parameter frequency histogram The single-parameter frequency histogram is a simplest way of representing the distribution of numerical data. Histograms display a single measurement (scattered or fluorescence light intensity) parameter on the x-axis and the number of events (cell count) on the y-axis. The distribution of the measured data can be characterized by statistical parameters of the data, e.g.: average, standard deviation, skewness, coefficient of variation etc. Histograms cannot display the correlation between two parameters, but they can visualize a parameter from multiple cell-populations in the same graph, e.g.: in the top right figure, two histograms from different cell population were presented in the same graph. 32 Top left image, dot-plot or scatter plot: The traditional form of visualizing a two-parameter data set is known as a “dot plot”. In the dot-plot, each point corresponds to one cell. The two axes measure two different parameters (x axis: 90° light scatter (side scatter) signal; y axis: forward scatter signal) in a way that the coordinate values of the cells are proportional to the parameters measured. Top right image, density dot plot: Two parameter data of each point can be encoded by colorful visualization. Density plots display two parameters as a frequency distribution. However, a single dot corresponds to a single cell in a dot plot, the color of a single dot in a density plot decodes the number of cells falling into a given area in the plot. Type of plot is useful for displaying subpopulations characterized by a very high density of events. 33 Gates are boundaries placed around cell populations that have common features like scatter or marker expression to quantify and study these populations. Cells are first gated on the basis of their scatter properties, top-left dot-plot: x axis: 90° light scatter (side scatter signal); y axis: forward scatter signal. The black polygon located on the figure is a primary gate, i.e. the cells within the polygon are selected for further analysis. This is achieved by restricting the plotting of data to cells found in the gate. Bottom-left histogram: the frequency histogram of the FITC intensity is only shown for cells in the gated population defined on the FCS-SSC dot-plot. Bottom-middle dot-plot: fluorescence intensity distribution in the two-parameter dot plot is visualized only for the population gated on the FCS-SSC dot-plot. (x axis: FITC intensity; y axis: PE intensity). Cells can be further subdivided based on the intensity of surface markers (red and purple colors). Back-gating is a method to confirm the correctness of a gating pattern. The population that has been identified by a particular gate is gated again on entirely different parameters. To confirm the identity of these populations with gating strategies, they can be back-gated along FSC and SSC parameters. The bottom-right dot plot on the right confirms these populations on the basis of their scatter properties. 34 Cell sorting is flow cytometric method to purify cell populations from a sample based on the presence or absence of specific parameters. Precise droplet forming devices combined with fast signal processing electronics are needed to achieve the best purity of the sorted cells. If cells are collected under sterile conditions, these sorted cells can be further cultured and manipulated. 35 The sorting process starts when a sample is injected into a stream of sheath fluid that passes through the vibrating nozzle, the disturbance in the stream causes it to break into a droplet containing ideally only one cell. 36 After the fluid passes through the vibrating nozzle, the disturbance in the stream causes it to break into a droplet containing ideally only one cell. A charging collar (or ring) is placed at the point where the stream breaks into separated droplets. The charging of the droplets is based on the presence or absence of specific parameters, e.g.: dim or bright fluorescence intensity (black or red cells) detected from the cell present in the droplet. Then the charged droplets (with cells) pass through electrostatic deflection plates that divert them into collection tubes based upon their charge: unlabeled cells go straight on without deflection into the waste and red cells are collected in the collection tubes. 37 The sheet fluid passes through the electrostatic deflection plates 38 The numerous proteins expressed on the surface of cells are characteristic of the cell type and can indicate the functional state of the cells e.g. active vs inactive. The determination of the presence of a surface antigen combined with relative proportion from the whole cell population is of major importance in clinical research as well as in routine diagnostics. Typically, monoclonal antibodies labelled with fluorescent probes make it possible to study surface antigens. In the example above, two markers were used:  Antibody binding to the cell surface CD4 molecules - labelled with red dye. CD4 (cluster of differentiation 4) is a glycoprotein found on the surface of immune cells such as T helper cells, monocytes, macrophages, and dendritic cells.  Antibody binding to the cell surface CD2 molecules - labelled with blue dye. CD2 (cluster of differentiation 2) is a cell adhesion molecule found on the surface of T cells and natural killer (NK) cells. Immunophenotyping is the analysis of heterogeneous populations of cells for the purpose of identifying the presence and proportions of the various populations of interest. With immunophenotype analyses, the presence or absence of CD4 and CD2 surface markers can help to identify the different subpopulation and activation states of T cells. 39  color dot-plot: FITC-CD8 vs PE-CD4 During the course of immunofluorescent phenotyping, one-dimensional –usually logarithmic – distributions are used, but during the simultaneous labelling of different antigens, two- dimensional visualization of data is applied. Quadrant analysis: This type of analysis can be used when two different categories of cells exist for two parameters:  cells with high CD4 expression -> high FITC-CD4 intensity referred as CD4+  cells with low CD4 expression -> low FITC-CD4 intensity: CD4-  cells with high CD8 expression -> high PE-CD8 intensity: CD8+  cells with low CD4 expression -> low PE-CD8 intensity: CD8- Overall, there are four mutually exclusive categories and each cell in the population can be uniquely placed into one. This type of relative proportional analysis is frequently used in immunophenotyping, when cells are categorized for the presence or absence of a cell surface protein marker based on labeling with specific antibodies. Categories/quadrants and the corresponding relative cell number proportions from the given example: 40 1 quadrant: CD8- and CD4+ cells (also called CD4 single positive cells), 45% 2 quadrant: CD8+ and CD4+ cells (also called double positive cells), 2% 3 quadrant: CD8- and CD4- cells (also called double negative cells), 27% 4 quadrant: CD8+ and CD4- cells (also called CD8 single positive cells), 26% Data check: 45+2+27+26=100 % 41  The DNA content of cells changes during the course of the cell-cycle. DNA-distribution can be used to determine the proportion of cells in a given phase of the cell-cycle. The figure depicts a hypothetical DNA-distribution schematically representing the peaks corresponding to various phases of the cell cycle. As DNA is doubling during S-phase, cells are found with a DNA content ranging between 2N and 4N. A histogram of DNA content against cell numbers gives the normal DNA distribution profile for a proliferating cell population. The frequency histogram can provide information about percentage of cells in the major phases of the cell cycle. 42  The DNA content of cells during normal cell-cycle. Cell cycle analysis by DNA content measurement is a method that most frequently employs flow cytometry to distinguish cells in different phases of the cell cycle. Before the analysis, the cells are usually permeabilized and treated with a fluorescent dye that stains DNA quantitatively, such as propidium iodide (PI). Cell cycle anomalies can be revealed by the analyses of the DNA content frequency histogram. 43  The DNA-distribution of a cell population drawn from a rapidly growing tumor tissue The DNA distribution that departs from the normal pattern is representative of the aneuploid nature of the tumor; the first peak of the histogram corresponds to the 2N DNA content whereas the second corresponds to the aneuploid DNA content (the so-called DNA index corresponding to this peak is 1.21). The peak that gives the highest DNA-fluorescence (small red peak at around intensity value 800) belongs to the cells of the aneuploid clone being in the G2/M phase. Aneuploidy is the presence of an abnormal number of chromosomes in a cell. About 68% of human solid tumors are aneuploid. The aneuploid nature of a cancer cell has an important prognostic value and useful in following the course of therapy. 44 45 Abbe diffraction limit: Ernst Abbe found in 1873 that light with wavelength λ, traveling in a medium with refractive index n and converging to a spot with half-angle θ will have a minimum resolvable  distance of d that can be calculated with the formula: d  If two objects points are farther 2 n sin away from each other than the minimum resolvable distance (middle figure on the left), their images will not overlap. If, however, the two object points are closer than the minimum resolvable distance (rightmost blue figure), the images of the two objects will overlap preventing their discrimination. Abbe limit is around d = 200 nm for commercial light microscopes, which is small compared to the size of cells (1 μm to 100 μm), but large compared to viruses and proteins. In order to increase the resolution, shorter wavelengths can be used such as in electron microscopy. The two formulas on the right describe the de Broglie wavelength () of a particle with a momentum of mv, and the wavelength corresponding to electrons accelerated through a voltage of V. 46  Differences between a fluorescence microscope and a confocal microscope A fluorescence microscope excites a wide column of the specimen section, which causes emission of fluorescence from both inside and outside the focal plane (focal plane: black horizontal line). Therefore, out-of-focus fluorescence contributes to the recorded image (both blue and purple light rays reach the camera). In confocal microscopy, a small pinhole placed next to the PMT. The pinhole will exclude out of focus light (both blue and purple light rays are blocked out by the pinhole), allowing only fluorescence originating from the focal plane to be collected. Additionally, a simple fluorescence microscopes is less complex than a confocal microscope. A simple fluorescence microscope is usually equipped with an arc lamp combined with a digital camera and filter sets only. In a confocal system, there are laser units, a confocal ‘scan head’ and computer(s) for controlling multiple parameters in the system as well as image processing. 47  The confocal principle in fluorescence laser scanning microscopy The term “confocal” refers to both illumination and detection, i.e. detection of fluorescence is restricted to the same spot where illumination light is focused. In the illustrated schematic optical configuration, coherent light emitted by the laser system passes through a pinhole, which is overlapping with the detection volume of the second pinhole positioned in front of the detector. 48  Main components of the ‘scan head’ of a commercial confocal laser scanning microscope The laser focus in the specimen is scanned in a raster pattern, this mechanism requires a synchronization between the horizontal and vertical movement of the laser spot. Coordination of the movement of the laser spot, typically achieved by using two mirrors, one scanning along the x-axis and the other on the y-axis, produces the rectilinear raster scan. 49  A single-beam laser scanning confocal microscope The sampling spot must be moved through the specimen and the resulting signal collected and stored for each pixel. The scan head controls the excitation and detection of the photon signal required to construct the confocal fluorescence image. The components of a typical commercial scan head generally include one or more laser inputs, fluorescence filter sets, a raster scanning mechanism, one or more pinhole apertures, and detectors (usually photomultiplier tubes, PMTs) for multiple fluorescence wavelength detections. 50 Main components of a confocal microscope:  microscope, equipped the scan head and sample holder stage  fluorescence filters  laser unit 51  Images of a conventional fluorescence and confocal microscope system The advantage of a confocal microscope is demonstrated by these two images. Out of focus light makes images produced by conventional fluorescence microscopes blurred. On the other hand, a confocal microscope only collects light emitted by a single plane of the specimen making the images of thick specimens much sharper than with a conventional fluorescence microscope. The pinhole excludes out of focus light from image formation, allowing only fluorescence originating from focal plane to be collected. Magnifications are 250 and 300 respectively. 52 Images of a conventional fluorescence and confocal microscope system 53 Images of a conventional fluorescence and confocal microscope system 54 Images of a conventional fluorescence and confocal microscope system 55  Images of a conventional fluorescence and a confocal microscope system Fluorescence image on the right: A conventional fluorescence microscope excites a wide column of the specimen section, and collects the emission of fluorescence from both inside and outside the focal plane. Therefore, out-of-focus fluorescence contributes significantly to the recorded image. Fluorescence image on the left: the pinhole excludes out-of-focus light from image formation, allowing only fluorescence originating from the focal plane to be collected: no photons detected from the focal plane, a dark image is recorded. 56 Capturing multiple two-dimensional images at different depths in a sample enables the reconstruction of three-dimensional structures (a process known as optical sectioning) within a sample. The image on the left shows an axial (top) and a lateral view (bottom) of a single hamster ovary cell. The image was reconstructed from optical sections of actin-stained specimen (confocal fluorescence). VoxBlast Image courtesy of Doctors Ian S. Harper, Yuping Yuan, and Shaun Jackson of Monash University, Australia. (see Journal of Biological Chemistry, 274: 36241, 1999). 57 58