Microscope Basics and Optical Principles

Microscope

• A microscope is an instrument used to view objects too small for the naked eye.
• The term "microscope" was coined in 1628 by Johannes Faber, combining the Greek words "micro" (small) and "skopein" (to view).
• Microscopy is the science of investigating small objects using microscopes.
• The most common microscope is the optical microscope, which uses lenses to produce an enlarged image of an object.
• There are two types of optical microscopes: Simple Optical Microscopes and Compound Optical Microscopes.

Optical Principles of Microscope

• The objective lens is a high-powered magnifying glass with a short focal length, creating a virtual and enlarged image of the specimen.
• The eyepiece lens further magnifies the virtual image.
• The image is viewed with the eyes focused at infinity.
• The actual power (magnification) of the microscope is calculated by multiplying the power of the eyepiece by the power of the objective lens.

Stereo Microscope

• The Stereo or Dissecting microscope differs from a compound microscope in that it uses two separate optical paths with two objectives, each providing a different viewing angle to each eye.
• This creates a 3-D visualisation of the sample being examined.
• Stereo Microscopes are often used for examining surfaces, sorting, dissection, microsurgery, watch-making, and small circuit board manufacture or inspection.
• Larger working distances and depths of field are important for Stereo Microscopes.

Bright Field Microscopy

• Bright Field Microscopy is best suited for observing dark or strongly refracting objects.
• It has a low optical resolution, limited by diffraction to approximately 0.2 microns.
• Out-of-focus light reduces image clarity.
• Enhancements include oil immersion objectives, staining methods, and using filters on the light source.

Numerical Aperture (N.A.) and Resolution (R)

• Resolution is the ability to distinguish between two closely spaced objects.
• The resolution limit of a microscope is dependent on the wavelength of light and the numerical aperture (N.A.) of the lens system.
• The Abbe Equation: Resolution (Limit) ~ λ/NA ~ λ/2n Sinθ ~ λ/n Sinθ
• Wavelength (λ) is shorter for blue light (450nm) and longer for red light (750nm).
• A smaller wavelength or a larger N.A. results in better resolution.

Phase Contrast Microscopy

• Phase contrast microscopy enhances the visibility of transparent objects by altering the phase of light passing through the specimen.
• This creates contrast between the object and its surroundings.

Fluorescence Microscopy

• Fluorescence microscopy employs fluorescence and phosphorescence to visualize specimens.
• Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation.
• Fluorescent dyes (fluorophores) absorb excitation light at a specific wavelength and emit light at a longer wavelength.
• Applications include studying live cells, fixed cells, and localizing or measuring enzyme activity.

Confocal Microscopy

• Confocal microscopy uses a laser beam to illuminate a thin slice within tissue and reject light from other regions, creating a 3-D image.
• It is used extensively in biological and medical research.

Electron Microscopy

• Electron microscopy uses a beam of electrons to create images of specimens.
• Transmission Electron Microscopy (TEM) provides a 2-D view of thin slices with extremely high magnification and resolution.
• Scanning Electron Microscopy (SEM) provides a 3-D view of the surface of the specimen with high magnification and resolution.