Microscopy: Magnification, Contrast, and Resolution

• Microscopy principles include magnification, contrast, and resolution.

Magnification

• Refers to the number of times an object's image is enlarged.

Contrast

• It's the difference in light intensity between the background and the specimen, enhancing visibility with techniques like wet mounts of colorless cells in a dark background.

Resolution

• Also known as resolving power, refers to the capacity to distinguish two points as separate entities at a specified distance.
• For example, resolution of 0.2µm means objects at least 0.2µm apart can be seen distinctly; objects closer than 0.2µm will appear merged.
• Shorter wavelengths improve resolution.
• Van Leeuwenhoek's lenses achieved 1 micrometer resolution in the 1700s.

Light Manipulation

• Diffraction is the bending of light only.
• Refraction is the scattering and separation of light particles.
• Both diffraction and refraction describe bending of light.

Oil Immersion

• Cedar wood oil is used with oil immersion objectives (OIO) to prevent light scattering as it passes through the slide, specimen, and OIO.

Refractive Index

• It measures a substance's light-bending capacity.
• Staining alters the refractive index of a specimen, creating contrast, and enhancing image clarity.
• When light passes through a specimen and medium, light rays change direction (refract) which increases image contrast between the medium and the object being viewed.
• Light rays spread out from the specimen and enter the objective lens, leading to image magnification.
• Immersion oil is applied between the glass slide and OIO lens, improving resolution by preventing light refraction.
• Without immersion oil, images appear fuzzy due to light refraction as it enters the air from the slide.
• Using oil has the same effect as increasing objective lens diameter.
• Unstained cells have low contrast and are difficult to see. Refractive indices of different materials:
• Glass: 1.517
• Water: 1.333
• Cedar wood oil: 1.516
• Air: 1.0003
• NSS: 1.334

Total Magnification

• Achieved by multiplying the objective lens magnification by the eyepiece magnification.

• Example magnification values using an Olympus compound light microscope:

  • Scanner (4X) with 10X eyepiece: 40X total magnification
  • LPO (10X) with 10X eyepiece: 100X total magnification
  • HPO (40X) with 10X eyepiece: 400X total magnification
  • OIO (100X) with 10X eyepiece: 1000X total magnification.

• An eyepiece with 10X magnification means the object is magnified 10 times

• 1000X magnification means an object has been enlarged 1000 times.

Microscope Use

• The light source can be a mirror or electric type.
• Start with the lowest objective lens (scanner) before using higher magnification lenses.
• Refine the image using lower power objectives before moving to OIO to avoid blurry images.
• Check the power source, light source, and regulate light intensity using the iris diaphragm.
• Carry the microscope with two hands, one on the arm and one on the base.
• View ocular lenses with both eyes open.
• Clean the microscope (ocular, objective lenses, and stage) before and after use with lens cleaner or alcohol.
• Upon returning the microscope, rotate the nosepiece to the lowest objective (scanner) in focus.

Glass Slides

• Scanner and LPO can be used with coarse adjustment
• HPO and OIO require fine adjustment only due to their proximity to the stage.
• Fine adjustment is used to refine the details of cells on a microscope.

Digital Microscope

• Princess summer uses a digital microscope in the Philippines.
• It is connected to a computer to enhance images.
• It is a light microscope, electronic type.
• It features additional lenses, making it up to 6,000x magnification.