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 machine that m...

# 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): $%T = \frac{I}{I_o} \times 100$ - If all light absorbed, the %T = 0, if no light absorbed at all then %T = 100. - $I$ = intensity of transmitted light & $I_o$ = 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) $I_1$= intensity of transmitted light & $I_o$= Intensity of original light ## 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, isprovided 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. ## 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. ## Atomic absorption spectrophotometry (AAS) - AAS measures the absorption of a defined wavelength of light by ground-state atoms in a flame. ### 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 H₂ gas 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. ## 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. ### 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, 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-immersjon objective lens, magnification (100x) ##### 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: $M_{microscope}$ = $M_{objective}$ X $M_{eyepiece}$ - Generally microscope has the following magnifications; | Term | Objective lens | Ocular lens | Total magnification | |---|---|---|---| | Scanning | 4x | 10x | 40x | | Low power | 10x | 10x | 100x | | High power | 40x | 10x | 400x | | Oil immersion | 100x | 10x | 1000 | ### Phase contrast Microscope - With phase-contrast microscopy, the background (field) appears gray and the specimen is bright. - Used to view unstained samples of cells, and transparent materials such as urine sediments. - Also commonly used for performing white blood cell and platelet counts using the hemocytometer. ### 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 living or moving cells such as sperm or parasites, are easily seen using dark field microscope. ### Bright-field Microscope - Used to view stained specimens, such as stained blood smears or stained bacterial smears. ### 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. ### 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. #### 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.