Spectrophotometry and Microscopy Quiz

Basic Laboratory Instrumentation

• Basic laboratory instrumentation is a fundamental aspect of scientific analysis.

Spectrophotometry

• Spectrophotometry measures the concentration of solutes in solutions.
• It measures the amount of light absorbed by a solution in a cuvette within a spectrophotometer.
• A spectrophotometer is a machine measuring the amount of light or electromagnetic radiation transmitted or absorbed by the solution.

The Absorption Process

• Some molecules in solution absorb light at specific wavelengths.
• The energy absorbed depends on the amount of the substance present.
• The wavelength of transmitted light determines the observed exit color.
• Transmitted energy is expressed as percent transmittance (%T).
  • %T = (I/I₀) x 100.
  • I is the intensity of transmitted light.
  • I₀ is the intensity of incident light.
• If all light is absorbed, %T = 0. If no light is absorbed, %T = 100.

Beer-Lambert Law

• Beer-Lambert law quantifies compounds based on their ability to absorb light.
• Absorbance is directly proportional to concentration (Beer's Law).
• Absorbance is directly proportional to the path length of the light through the sample (Lambert's Law).
• The Beer-Lambert law equation is A = abc.
  • A is absorbance
  • a is molar absorptivity constant
  • b is the cell path length (cm)
  • c is the concentration of the analyte.

Spectrophotometer Components

• Wavelength selector (monochromator): Selects a specific wavelength of light. Methods include prisms, gratings, and color filters.
• Sample cell (cuvette): Holds the sample. Usually round or square. Made of material that doesn't absorb light in the desired range.
  • Glass is used for visible light.
  • Quartz is used for ultraviolet light.
• Light detector: Measures the amount of light passing through the sample. Often uses a photomultiplier tube (PMT).

Types of Spectrophotometers

• Single-beam instruments: Measure the sample and then blank alternatively.
• Double-beam instruments: Continuously compare sample and blank simultaneously. Correct automatically for changes in electronic signal or light intensity of the light source.

Turbidimetry and Nephelometry

• Turbidimetry measures the turbidity or cloudiness of a solution by measuring the intensity of transmitted light. 
• Nephelometry measures the intensity of scattered light.
• Both methods utilize a near-ultraviolet light source (290-410 nm).  
• In turbidimetry, the detector is in line with the incident light, measuring the transmitted light.
• In nephelometry, the detector is at an angle (30° or 90°) to measure scattered light.

Fluorometry

• Fluorometry measures light emitted by molecules that absorb and re-emit light. Fluorescent compounds have specific excitation and emission wavelengths, often in the ultraviolet region.
• The intensity of fluorescence is directly proportional to the concentration of the fluorophore and the intensity of the excitation source.
• Typically uses high-intensity UV light sources such as mercury vapor, halogen, or xenon arc lamps.
• Used in immunoassays and flow cytometry.

Chemiluminescence

• Chemiluminescence measures light emitted from a chemical reaction.
• Certain compounds, like luminol, react with an oxidizing agent (like H₂O₂) in the presence of a catalyst to produce excited-state products.
• The release of light occurs during relaxation to the ground state.
• Used in immunoassays, such as western blotting.
•  Luminometers measure the flashes of light produced during chemiluminescence.

Atomic Absorption Spectrophotometry (AAS)

• AAS measures the absorption of light by ground-state atoms in a flame.
• A defined wavelength of light is used to quantify elements like iron, lead, calcium, magnesium, zinc, and copper in body fluids.
• AAS is a reference method and is accurate and sensitive.
•  Limited in clinical labs due to high instrument cost.
• Flameless AAS utilizes a graphite furnace for atomization, offering higher sensitivity than flame methods.

Electrochemical Measurements

• Electrochemical measurements are used to determine the relationship between electrical and chemical energy. 
• These measurements often involve redox reactions, where electron transfer occurs.
  • Oxidation: loss of electrons.
  • Reduction: gain of electrons.
  • Redox: combination of oxidation and reduction reactions.
  • Oxidizing agent: accepts electrons.
  • Reducing agent: donates electrons.

Reference Electrodes

• A reference electrode maintains a constant potential for comparison during electrochemical measurements.
• The standard hydrogen electrode (SHE) represents the standard for electrochemical potentials.
• SHE uses a platinum electrode immersed in an H⁺ ion solution.

Indicator Electrodes

• Indicator electrodes respond to the analyte in the solution.
• Common examples include glass membrane electrodes and ion-selective electrodes (ISEs).

Microscopy

• Microscopy magnifies images of small objects, making them visible to the naked eye.

Introduction to Microscopy

• Microscopes are essential in various clinical labs for analyzing stained blood smears, tissue sections, cell counts, urine sediments, cellular reactions, microorganisms, etc.
• Simple microscopes can resolve below 1 micrometer (µm).
• Compound microscopes can resolve down to about 0.2 µm.

Types of Microscopes

• Microscopes are generally classified as light microscopes, electron microscopes. -Light microscopes include bright-field, dark-field, phase-contrast, polarizing, and differential interference contrast (DIC) microscopes and fluorescence microscopes, and confocal laser scanning microscopes. -Electron microscopes include Transmission electron microscope (TEM) and Scanning electron microscope (SEM).

Compound Microscope Parts

• Ocular lenses (eyepieces): Magnify the image.
• Objective lenses: Lenses positioned close to the specimen, providing varying magnifications (scanning, low power, high power, oil immersion).
• Condenser: Focuses or directs light onto the specimen.
• Diaphragm: Adjusts the amount of light striking the specimen.
• Coarse and fine focus adjustments: Used to sharpen the image.

Illumination Sources

• Light microscopes use visible light, ultraviolet light, or lasers.
• Confocal laser scanning microscopes use focused laser beams of narrow wavelengths.
• Electron microscopes use an electron beam to illuminate specimens.

Magnification in Light Microscope

• Total magnification = magnification of objective lens x magnification of eyepiece lens.
• Example magnification tables provided.

Phase Contrast Microscopy

• Used to view unstained samples or transparent cells, such as urine sediments.
• Background appears gray while the specimens stand out brightly.

Dark-Field Microscopy

• Light is blocked in the center of the condenser.
• Specimens appear bright against a dark background.
• Used to view living cells or active microorganisms, such as sperm and parasites.

Bright-Field Microscopy

• Used to view stained specimens.

Differential Interference Contrast (DIC) Microscopy

• Produces three-dimensional images of unstained specimens.
• Used to identify substances, like crystals in urine.

Fluorescence Microscopy

• Uses ultraviolet (UV) light to illuminate specimens stained with fluorescent dyes.
• Dyes absorb short-wavelength light and emit longer wavelengths.
• Used to identify microbes and assess antibodies/diseases, like syphilis or lupus.

Confocal Laser Scanning Microscopes (CLSM)

• High-resolution microscopes using a laser beam.
• Can examine fluorescently stained specimens.
• Create three-dimensional images.

Electron Microscope

• Utilizes electrons to visualize specimens in high magnification and resolution.
• Includes transmission electron microscope (TEM) and scanning electron microscope (SEM).

Transmission Electron Microscope (TEM)

• High-resolution technique with electron beam passing through a thin specimen layer.

Scanning Electron Microscope (SEM)

• Produces images of a sample's surface by scanning with a focused electron beam.

Test your knowledge on the principles of spectrophotometry and microscopy. This quiz covers key concepts such as the Beer-Lambert law, microscope functions, and different types of lenses. Ideal for students in biology or chemistry courses.