6. Microscopy

COMPOUND LIGHT MICROSCOPE

  • Key components include:

    • Diopter adjustment

    • Interpupillary distance adjustment

    • Nose piece

    • Objectives

    • Stage

    • Aperture iris diaphragm

    • Condenser

    • Condenser focus knob

    • Ocular (eyepiece)

    • Tube body

    • Arm

    • Light source

    • Field diaphragm

    • Mechanical stage

    • Coarse adjustment

    • Fine adjustment

    • Stage adjustment

    • Condenser centering adjustment

    • Rheostat

    • Base

INTRODUCTION TO THE COMPOUND LIGHT MICROSCOPE

  • The compound light microscope, commonly known as the brightfield microscope, is used for examining cellular structures that cannot be seen with the naked eye.

    • Key factors:

    • Resolution achieved by the microscope is critical.

    • Resolution is the ability to see structures as separate and distinct.

    • Understanding the microscope is essential for sufficient magnification and good resolution.

BASIC MICROSCOPY TERMINOLOGY

  • Resolution:

    • Indicates how small and how close individual objects can be and still be recognized as distinct individual objects.

    • Involves the separation of two distinct points/objects. Higher resolution indicates more detail visible.

  • Magnification:

    • Total magnification = Objective magnification × Ocular magnification

    • Example: With a 10x ocular lens and a 10x objective lens, the total magnification is 100x.

    • Expressed in terms of diameters; for example, 10x means the diameter of an object is magnified to 10 times its original size.

    • The ocular lens magnifies the image created by the objective lens, and using a higher power eyepiece can magnify any possible errors in your objective magnification.

  • Numerical Aperture (NA):

    • NA of a lens system measures its light-gathering ability.

    • It is an index of the resolving power of a lens and its ability to render the finest detail distinctly visible.

    • Higher NA allows more light rays to enter the objective lens, improving resolution.

    • NA depends on the radius of the lens and its focal length.

  • Resolving Power:

    • Ability of a lens to separate two distinct points to provide resolution.

    • Limit of usable magnification; further magnification without resolution is called empty magnification.

    • Depends on the angle of light rays that can enter the objective lens, refractive index, and wavelength of light used.

DEFINITIONS AND FUNCTIONS

  • Definition:

    • Capacity of the objective lens to render the outline of an object distinct.

    • Definition depends on both the object and illumination; resolving power is a function of the lens.

PARTS OF THE MICROSCOPE
1. BASE

  • Transformer:

    • Usually located in the base and steps down the voltage for the illuminator.

  • Rheostat:

    • Dimmer switch for regulating light intensity; controls the current entering the illuminator.

  • Illuminator (lamp):

    • Provides major illumination for the specimen.

    • Positioned at the back of the base; requires proper alignment and may utilize tungsten or tungsten-halogen bulbs.

2. CONDENSER

  • Located under the stage directly over the light; collects light and focuses it on the object being examined.

    • Should ideally have the same NA as the objective lens used.

    • Its position can be adjusted to optimize light focus and maximize resolving power.

  • Centering Screws:

    • Utilized for centering the condenser over the light; essential for achieving Kohler illumination.

  • Filter Holder:

    • Attaches to the bottom of the condenser for holding color-selective filters.

    • Filters used can include blue (for improved image quality), green (for black and white photomicrography), and neutral density filters.

  • Aperture Iris Diaphragm:

    • Composed of overlapping metal leaves; adjusts light beam diameter and can reduce spherical aberration.

    • Should not be used to control brightness—this is done via the rheostat.

3. FIELD DIAPHRAGM

  • Controls the circle of light in the field of view and aids in microscope alignment.

4. CONDENSER ADJUSTING KNOB

  • Raises and lowers the condenser to focus the light on the specimen.

5. FOCUSING KNOBS

  • Coarse and fine adjustment knobs for focusing on the specimen.

    • Coarse adjustment: largest knob, used when low power objective is in place.

    • Fine adjustment: used once initial focusing is achieved for sharper focus.

6. MECHANICAL STAGE

  • Platform supports the slide; can move in two directions using co-axial adjustment knobs.

7. REVOLVING NOSEPIECE

  • Holds and allows switching between objective lenses.

8. MICROSCOPE HEAD

  • Contains prisms to split light into two beams for binocular viewing.

9. BODY / OPTICAL TUBE

  • Holds lenses in alignment; the standard mechanical tube length affects resolution and magnification.

10. OBJECTIVE LENS SYSTEM

  • Responsible for primary magnification.

    • Quality affects resolution and contrast, marked by specifics like tube length, magnification, NA, and cover glass thickness.

    • Common objectives:

    • Low-power (10X)

    • High-power (40X)

    • Oil immersion (100X)

11. OCULAR LENS (EYEPIECES)

  • Eyepieces typically have a magnification of 10X and should be adjusted for interpupillary distance.

COVERSLIP GLASS THICKNESS AND QUALITY

  • Coverslips protect lenses and create even thickness for viewing.

  • Thickness affects focused view; tolerance usually engraved on the lens (e.g., oil immersion lens: 0.17 ± 0.01 mm).

SLIDE THICKNESS AND QUALITY

  • Slides should be colorless and between 0.9 to 1.1 mm thick.

  • Important for ensuring light rays are focused on the specimen.

OTHER TYPES OF MICROSCOPES

  • Brightfield microscopes require stained objects for detail; other types can view without staining.

    • Phase Contrast:

    • Enables viewing of unstained, live preparations; aligns objectives and condenser precisely.

    • Polarizing:

    • Utilizes polarizers to view birefringent objects against a black background.

    • Darkfield:

    • Reflects indirect light off objects for improved visibility; often used for spirochetes.

    • Fluorescence:

    • Detects specific wavelengths emitted from fluorescent objects.

    • Electron:

    • Provides high magnification through electron beams; includes transmission (TEM) and scanning (SEM) variations.

    • Inverted:

    • Light source above the specimen; useful for observing reactions in test tubes.

MICROSCOPY TROUBLESHOOTING

  • Common issues include:

    • Insufficient light due to closed diaphragms or improper positioning of condenser.

    • Incomplete illumination under low power due to objective misalignment.

    • Glare from overexposed lighting.

    • Poor definition due to dirty optics.

    • Double images due to interpupillary distance misalignment.

    • General discomfort from improper adjustments.

Note: Regular maintenance involves careful handling, cleaning protocols, and proper storage of the microscope. Always prioritize the integrity of optical components to maintain clarity in observations.