Basic Laboratory Instrumentation PDF

Basic Laboratory Instrumentation  Spectrophotometry Spectrophotometry; is a technique used to measure the concentration of solutes in solution by measuring the amount of light absorbed by the solution in a cuvette placed in the spectrophotometer. Spectrophotometer; a machi...

Basic Laboratory Instrumentation  Spectrophotometry Spectrophotometry; is a technique used to measure the concentration of solutes in solution by measuring the amount of light absorbed by the solution in a cuvette placed in the spectrophotometer. Spectrophotometer; a machine that measure the amount of light or electromagnetic radiation (of certain frequency) transmitted or absorbed by the solution.  The absorption process In a solution, some molecules have ability to absorb light at a specific wavelength. The amount of energy absorbed depends on the amount of the substance present. The wavelength of the transmitted light combined to make up the new observed exit color detected by spectrophotometer. Transmitted energy expressed in terms of percent transmittance (%T): %T = Is /I0 x 100 If all light absorbed, the %T = 0, if no light absorbed at all then %T = 100. I = intensity of transmitted light & I0 = Intensity of incident light (original)  Beer-Lambert law Beer-Lambert law allows compounds to be quantified by their ability to absorb light that is relates directly to concentration. Beer’s law: Absorbance directly proportional to the concentration. Lambert’s law: Absorbance directly proportional to the length of the length of the light path through the sample. These two laws together forms the Beer-Lambert law, which states: A= abc where: A = absorbance (no unit), a = molar absorptivity constant (L/mole- cm), b = cell pathlength (cm), c = concentration of analyte (mol/L) I1 = intensity of transmitted light & I0 = Intensity of original light  Spectrophotometer Component Polychromatic light from light bulb composed of many different wavelength can be dispersed into separate wavelengths by refraction, by using prism or grating. Light Source: provides the light to be passed through the sample. Low pressure (vacuum) Tungsten Filament Deuterium Lamp: ultraviolet Light Tungsten Lamp: visible light (320-2500 nm) (160-375 nm)  Spectrophotometer Wavelength Selector (monochromator): used to select a given wavelength of light from the light source. Can be prism, reflection or diffraction grating or color filter Sample Cell: sample container of fixed length (b). Usually round or square cuvette made of material that does not absorb light in the wavelength range of interest Glass – visible region Quartz – ultraviolet Light Detector: measures the amount of light passing through the sample. Usually works by converting light signal into electrical signal, most common type is photomultiplier (PMP) tube.  Types of Spectrophotometers Single-Beam Instrument: sample and blank are alternatively measured in same sample chamber. Double-Beam Instrument: Continuously compares sample and blank. Automatically corrects for changes in electronic signal or light intensity of source  Turbidimetry and Nephelometry Light passing through a medium with dispersed particles either scattered or transmitted. In turbidimetry; the intensity of light transmitted is measured (measure cloudiness or turbidity of a solution). The photodetector is placed in direct line with the incident light (180° angle). The light source should emit a wavelength in the near ultraviolet range (290–410 nm). In nephelometry, the intensity of scattered light is measured. Light detector is oriented at an angles 30° or 90°. Both techniques commonly for immunoprecipitation or bacterial growth.  Fluorometry When molecules absorb light they reach a higher energy level or an excited state, when returns to ground state, loses the excess energy as heat or light emission (fluorescence). Each fluorescent compound has specific excitation wavelengths (often in the UV region) and emission wavelengths. The intensity of the fluorescence produced is directly proportional to the concentration of the fluorophore and the intensity of the excitation source. High-intensity radiation, most often in the UV range, is provided the excitation source, usually a mercury vapor, halogen, or xenon art lamp. Fluorescence assays are typically used to measure fluorescent tags or labels used in immunoassays and flowcytometry. Fluorometer or Spectrofluorometer  Chemiluminescence Chemiluminescence; is the emission of light as a result of chemical reaction. Certain organic compound, typically; luminol, react with oxidizing agent (H2O2) in the presence of catalyst (peroxidase enzyme) to produce and excited state product that while returning to ground state emit light in the shorter wavelength (425 nm) This techniques commonly used in some immunoassays, such as western blotting. Luminometer is the instrument that measures flashes of light produced by chemiluminescence. Luminometer  Atomic absorption spectrophotometry (AAS) AAS measures the absorption of a defined wavelength of light by ground-state atoms in a flame. Atomic absorption spectrophotometer  Atomic absorption spectrophotometry Most AAS systems are double-beam instruments AAS is accurate and sensitive; it serves as the reference method for some analytes, commonly used to measure metals in toxicology lab, including iron, lead, calcium, magnesium, zinc, and copper, in body fluids. The instrument cost, along with the technical expertise and time required for operation, limit the use of AAS in most clinical laboratories. Flameless atomic absorption uses a "graphite furnace" to atomize the sample; this technique is more sensitive than the flame methods.  Electrochemical measurements Quantitative determination based on electrochemistry is common in clinical laboratory second to photometric assays. Electrochemistry; study the relationship between electrical and chemical energy and interconversion between them, usually involves redox reactions (electron transfer reactions). Important terms: Oxidation; loss of electrons Reduction; gain of electrons Redox; Oxidation reduction reaction Oxidizing agent: element or compound that accepts an electron Reducing agent: element or compound that donates an electron  Reference Electrodes Standard Hydrogen Electrode Platinum metal immersed in H+ ion solution. Limitations: 1. Difficult to be used and to keep H2­gas at one atmosphere. 2. It needs periodical replating of Pt. sheet.  Indicator Electrodes An electrode that response to the analyte in the solution. Glass membrane electrodes and ion-selective electrodes (ISEs) are common examples. Common Indicator Electrodes Used in pH meters For different electrolytes like Na, Cl, K and Ca Microscopy  Introduction The microscope magnifies the image of very small objects thus making them visible to the human eye. Microscopes are used in various sections of clinical labs to: Evaluate stained blood smears and tissue sections Perform cell counts Examine urine sediment Observe cellular reactions Interpret smears containing microorganisms A simple microscope can resolve below 1 micrometer (μm) A compound microscope can resolve down to about 0.2 μm. simple microscope compound microscope  Types of Microscopes Microscopes can be divided into the following 3 categories; Light microscopes: Dark-Field microscope Phase-contrast microscope Epi-fluorescence microscope Polarizing and differential interference Bright-filed microscope: Confocal laser scanning microscopes Electron microscopes Transmission election microscope (TEM) Scanning electron microscope (SEM) Clinical microscopes fall into the classification of light microscopes. Compound Microscope Parts  Parts of the Microscope Ocular lenses: The oculars, or eyepieces, located at the top of the microscope, are attached to a barrel or tube connected to the Microscope arm through which the object is viewed, Ocular lenses contains a magnifying lens. The usual magnification is 10 times (10x), but 15x, 20x also available. Objective lenses: Most microscopes are equipped with at least three objectives or magnifying lenses and these are available in a variety of magnifications. The scanning lens, magnification (4x) The low-power objective lens, magnification (10x) The high-power objective lens, magnification (40x) The oil-immersion objective lens, magnification (100x) Objective  Parts of the Microscope Condenser: The condenser focuses or directs the available light into the objective. Diaphragm: The iris diaphragm is located in the condenser unit It regulates the amount of light that strikes the object being viewed. Coarse and Fine focus adjustments: The coarse adjustment is used to focus with the low- power objective only. The fine adjustment is used to give a sharper image after the object is brought into view with the coarse adjustment  Illumination source of Microscopes Various types of illumination sources are used in different microscopes. Light microscopes use visible light, ultraviolet light, or lasers. High-resolution microscopes called confocal microscopes use focused laser beams of very narrow wavelength to illuminate specimens. Electron microscope uses electron beam to illuminate specimens instead of light or laser.  Magnification in Light Microscope Magnificcation; is the enlargment of the image Magnificaiton of a microscope is given by: Mmicroscope = Mobjective X Meyepiece Generally microscope has the following magnifications; Total Objective Ocular Term magnificatio lens lens n Scanning 4x 10x 40x Low power 10x 10x 100x High power 40x 10x 400x Oil 100x 10x 1000  Phase contrast Microscope With phase-contrast microscopy, the background (field) appears gray and the specimen is Cells bright. Used to view unstained samples of cells, and transparent materials such as urine sediments. Also commonly used for Urine Casts performing white blood cell and platelet counts using the hemocytometer. Blood Fun cells gi  Dark-Field Microscope In the DF microscope, the light in the center of the condenser is blocked to cause light to hit the specimen at a high angle, scattering the light rays. The image obtained is a bright specimen against a dark field Unstained cells, particularly Cells living or moving cells such as sperm or parasites, are easily seen using dark field microscope. Syphilis Sperms  Bright-field Microscope Used to view stained specimens, such as stained blood smears or stained bacterial smears. Stained Parathyroid tissue Stained blood Stained smear Bacteria Plasmodium  Differential Interference Contrast (DIC) Microscopes Microscopes that introduces contrast to images of specimens which have little or no contrast by using a polarized light beam. Used for unstained specimens and can produce three- dimensional images. Reveal information about the structure and composition of substances and can be especially useful in identifying urine crystals. Provide excellent resolution and contrast and make it possible to view cell membranes and compartments within unstained cells, such as cell nuclei.  Fluorescence microscope Use ultraviolet (UV) light to illuminate the specimen stained with fluorescent dye. A fluor or fluorescent dye is a substance that absorbs short- wavelength (exciting) light and emits longer wavelength (emitting) light. Used to identify microorganisms such as mycobacteria, and to detect the presence of antibodies in certain diseases such as syphilis and lupus erythematosus. Fluorescence microscope Im  Confocal Laser Scanning Microscopes (CLSM) Sometimes called confocal microscopes, are one of the most powerful light microscopes. Laser beam is used as light source for illumination. These microscopes can be used to examine fluorescently stained specimens but also have other applications. Specimens can be viewed and scanned to create a series of images that are captured digitally to give 3d structure.  Electron Microscope Specimens are visualized by focusing an electron beam on the specimen rather than light waves or illumination with a light source. Electron microscopes provide much greater magnification (up to 1,000,000x) and resolving power than light microscopes, for observation of fine structural details. There are two types of electron microscopes ⎼ Transmission electron microscope (TEM) ⎼ Scanning electron microscope(SEM)  Transmission electron microscope (TEM) Transmission electron microscopy (TEM) is a high-resolution imaging technique in which a beam of electrons passes through a thin layer of sample to produce an image. Corona Virus Transmission electron microscope Cell mitochondria under  Scanning electron microscope(SEM) A scanning electron microscope (SEM) is a type of electron microscope that produces images of a sample by scanning the surface with a focused beam of electrons. Dividing liver cancer cell Color enhanced SEM image of blood cells Human Bacteriophage  Transmission (TEM) and Scanning (SEM) Electron Microscopes and images Comparison SEM TEM Showed some bacteria on a surface. The interior structure of a single bacterium.