Instrumentation

• MICROSCOPE: an instrument for viewing objects that are too small to be seen by the naked/unaided eye.
• MICROSCOPY: the science of investigating small objects using such an instrument.
• 1590 – 1 S T microscope was probably constructed by Zacharias Jansen.
• 1665 – Robert Hook’s microscope: separated objective, eyepiece, & light source of light.
• 1676 – Antonie van Leuuwenhoek: simple microscope.
• 1847 - carl Zeiss initiated the mass production of microscope.
• 1933 – 1 st electron microscope was constructed.
• MAGNIFICATION: degree of enlargement, number of times the length, diameter, of an object is multiplied.
• RESOLUTION/RESOLVING POWER: ability to reveal closely adjacent structural details as separate & distinct, a measure of the clarity & level of detail in the image.
• PARFOCALITY: lenses can be interchanged with minimal adjustment of focus, similar focal points in the same plane, most microscopes are designed to be parfocal.
• BRIGHT-FIELD MICROSCOPE: objects appear dark against a light background, routinely used in clinical laboratory.
• DARK-FIELD MICROSCOPE: objects appear bright against a dark background, replaces the condenser with a condenser that contains opaque disk, used for Treponema pallidum.
• PHASE CONTRAST MICROSCOPE: for low refractive indexes, allows visualization of transparent/unstained specimen.
• Smears involve a small drop of suspension containing cells being placed near an end of a slide and is spread across the slide by the edge of another slide.
• Dispersion devices in Spectrophotometry cause different wavelengths of light to be dispersed in different angles.
• Absorbance (A) is the amount of light absorbed as incident light passes through a sample.
• Path length of the sample (I) is the distance the light travels through the sample.
• Analytical devices are a set of instruments used to analyze and measure the characteristics of a substance or sample.
• Concentration of the substance in the solution (c) is a component in Spectrophotometry.
• Covered slides contain cell suspensions or processed histology tissue covered by a coverslip.
• Impression preparations involve a new clean slide being slightly pressed on the surface of the examined tissue and attached cells are observed.
• Concentric circles in an optical field include the central, peripheric, and peripheral circle.
• Beer-Lambert Law states that absorbance is directly proportional to the concentration of the solution.
• Molar reabsorptivity (𝑝) is the proportionality constant or a compound that is the measure of the absorption of radiant energy.
• The optical field is divided clockwise into 4 quadrants.
• INVERTED MICROSCOPE: has a changed order of optical part & source of light, optics is under slide & light source is above, used for monitoring the cell cultures.
• Components of Spectrophotometry include a light source which emits a broad spectrum of light, a monochromator which isolates individual wavelengths of light, and a dispersion device which separates the incoming light into its various component wavelengths.
• Spectrophotometry is a process which measures the amount of light that can pass through a solution to determine the concentration of the light absorbing substance in the solution.
• FLUORESCENCE MICROSCOPE: allows visualization of naturally fluorescent microorganisms or those stained by a fluorescent dye including labeled antigens & antibodies.
• Fluorometry determines the concentration via excitation of the substance via electromagnetic radiation.
• Interference filters: selectively transmit/reflect a certain range of wavelengths with dielectric films.
• Wavelengths bend as they pass a sharp corner.
• Material: glass, quartz, plastic.
• In single beam mode, there is only one light path that passes through both the reference & sample cells, alternating between measuring the reference (blank) & the sample.
• Classes: single beam, double beam.
• Diffraction gratings are most commonly used as optical components with closely parallel lines/grooves/rulings on its surface (15,000 or 30,000 per inch).
• CONFOCAL MICROSCOPE: creates high-contrast, high-resolution images, particularly in thick specimens, laser light source is typically used to illuminate the specimen.
• Absorption filters: absorb short wavelengths and transmit long wavelengths.
• Double beam in time: one photodetector with chopper/slitter.
• In double beam mode, there are two separate paths for sample & reference, allowing for simultaneous measurement of blank & sample.