Microscopy

References

• Manual of Clinical Microbiology 10th Edition

  • CH. 2

• Veterinary Microbiology and Microbial Disease 2nd Edition

  • CH. 1 - 6

• McCurnin's Clinical Textbook for Veterinary Technicians 9th Edition

  • CH. 15

• Nikon Eclipse E200: Microscope Instruction Manual.

The Compound Microscope

• Compound Light Microscope: Microscope that uses more than one lens and a light source to magnify an image and evaluate a specimen

  • Most common type used in veterinary clinics

    • Compound: Multiple

    • Light Source: Electric bulb with tungsten filament focused into a beam by the condenser lens.

Components of a Microscope

• Base: Bottom/horizontal frame of microscope

  • Contains grooves to properly carry microscope

• Arm: Vertical frame of microscope

• Ocular Lens (Eyepieces):

  • Located at top-most place of microscope

  • Provides 10x magnification.

  • Interpupillary with Adjustment:

    • Individual eye adjust for focus at very top of microscope (used to compensate for weak eye)

• Nosepiece:

  • Revolving circular plate that holds objectives

    • When rotating between objectives, only rotate piece by circular plate (not objectives)

  • Objectives

    • Four magnification lens of varying magnifications attached directly to nosepiece

      • Multiply all by 10 to get true magnification

    • Red (4x Magnification)

      • Used in conjunction with diaphragm setting of 4

      • Requires condenser to be at very bottom/touching light source (no space)

      • Used for scanning slide

    • Yellow (10x Magnification)

      • Used in conjunction with diaphragm setting of 10

      • Requires tip of pinky space between condenser and light source

      • Used for low power field

    • Blue (40x Magnification)

      • Used in conjunction with diaphragm setting of 40

      • Requires tip of pointer finger width between condenser and light source

      • Used for high power field

      • Uses fine focus adjustments (Never course focus)

    • White (100x Magnification)

      • Only objective that requires/uses slide suspended in oil (allows for clearer view)

      • Used in conjunction with diaphragm setting of 100

      • Requires condenser to be fully at very top/away from light source

      • Requires large fine focus adjustments (Never course focus)

• Course Focus (CF):

  • Larger knob located on bottom/right side of microscope that moves mechanical stage up/down and makes big focus modifications

    • Shouldn't be manipulated after using fine focus (will lose focused image)

  • Fine Focus (FF):

    • Smaller knob located on either side of microscope (attached to coarse focus on left side)

    • Used for finer focus adjustments for sharp/detailed/defined images

• Specimen Holder:

  • Silver metal plate on mechanical stage that holds glass slide

    • Ensure slide is positioned with labeled side being to the right

  • Tip Tilt: Small lever on left side used to open/close claw

    • Don’t pry claw open

• Mechanical Stage:

  • Flat black surface where slides are placed and light is able to pass through

  • Mechanical Stage Controls: Long knob located on right side/under stage that adjusts vertical/horizontal (top = forward/backward & bottom = left/right) position of mechanical stage to move around slide view

• Condenser:

  • Lens located beneath stage that collects and focuses light onto a specimen.

    • Condenser Control Knob: Small knob on far left/back that adjusts position of condenser to change light intensity on specimen

      • Up for more light and down for less

    • Iris Diaphragm (Aperture Settings): Adjustable mechanism built into condenser that controls light amount reaching the specimen.

      • Chosen setting should match objective magnification used

• Light Source:

  • Located at base and provides emitted light/illumination

    • Ensure flush with arm to avoid “half moon” view in microscope

  • Light Source Controls: On/off switch located on far/back right side above dimmer

  • Dimmer: Very small knob located underneath light source control that adjusts light sources brightness

Functions/Characteristics of Compound Microscope

• Magnification: Measure of how much larger microscope causes image to appear

  • Allows objects not visible to naked eye to be viewed in detail

  • Expressed in form of 10x (image magnified 10-fold)

  • How to Calculate:

    • Locate power of ocular lens (written on eyepiece)

    • Determine power of objective (written on objective)

    • Multiply both powers to find total magnification

    • Formula: Eyepiece Power x Objective Power = Total Magnification

• Illumination: Proper light/regulation of light

  • Accomplished by:

    • Diaphragm (closed to open): Controls diameter of light beam before passing through specimen

      • Align diaphragm slider to correspond with objective power

      • Magnification Increase = More Light Needed

    • Condenser Position (down to up): Focuses light source directly on plane of specimen

      • High Intensity for Full Brightness (used for high power objective) = Raise Condenser

      • Low Intensity for Low Brightness (used for low power objective) = Lower Condenser

    • Light Source Intensity (low to high): Regulation of light by using Rheostat ( light control knob or dimmer knob)

      • Low Power Objective = Low Light Source Intensity

      • High Power Objective = High Light Source Intensity

      • Special Note: Immersion oil can reduce light refraction when used with 100x objective.

        • Touch Down: When objective slightly touches oil (DOES NOT TOUCH SLIDE) to focus light and allows no air between slide and objective lens for a clearer image

    • Focusing: Adjusting arrangement between optical system (lens) and object viewed for clearer image

Optical Principles

• Reverse Image: Images viewed under microscope are reversed (backward) and inverted (upside down)

  • Moving specimen right/up = image moving to left/down

  • Moving specimen left/down = image moving right/up

Care of Microscope

• General Care:

  • Keep microscope dust and dirt free using appropriate tools for cleaning

    • Use Dust Cover (plastic bag) when not in use

• Maintenance Protocols & Use Precautions:

  • Maintenance

    • Allow only professionals to handle detailed maintenance

    • Protect against contact with corrosive/abrasive materials

    • Avoid direct sunlight and moisture

    • Avoid harsh wiping

  • Precautions

    • Avoid crashing objective into slide

      • Always focus slide with lower objective magnification settings (4x and 10x) first and work your way up while adjusting mechanical stage/using course focus (Look from the side)

      • Never use course focus with higher objective magnification settings (40x and 100x)

        • Only refine image with fine focus

      • Only remove slides from stage with 4x power objective

• Cleaning Procedures:

  • Use Blower Brush to air and remove dust before wiping.

  • Use only approved Lens Paper and Cleaner to wipe/clean lens (4 lens papers)

    • Wet one lens paper with lens cleaner

      • Clean ocular lenses/condenser/light source

    • Wet second lens with lens cleaner

      • Clean all objectives

        • Clean in exact order from 4x to 100x (Do not contaminate other objectives with oil)

    • Use third and fourth lens papers to repeat process above but for drying process

• Storage Procedures:

  • Turn microscope off

  • Turn to lowest power objective and completely lower condenser/mechanical stage

  • Unplug microscope

  • Cover to protect/store

• Moving Procedures:

  • Use both hands and carry microscope by supporting features

    • One hand gripping arm and base

Focusing Techniques

• Procedure:

  1. Start with turning revolving nosepiece to lowest power objective until clicked into position

  2. Secure slide on microscope stage and center specimen under objective using only side view with eyes

  3. Move course focus knob all the way upward without touching lens with slide

  4. Look through ocular lens and adjust image with coarse focus until focused

  5. Use fine focus to refine image