Microscopy Notes (Light, Electron, AFM)

Light Microscopy (Optical Microscopy)

• Purpose: Microscopy magnifies tiny cellular details and tissue characteristics using microscopes that generate images.
• Optical microscopy uses light to visualize samples and can magnify objects up to $10^3\times$. The compound microscope is the most commonly used optical microscope.
• Other major imaging modalities exist beyond optical: electron microscopy and atomic force microscopy (AFM).

Electron Microscopy and AFM

• Electron microscopy uses an electron beam instead of light, enabling magnification from two up to $5\times 10^7\times$.
• Major electron-microscopy types (based on how the electron beam interacts with the sample):
  • Transmission Electron Microscopy (TEM): generates two-dimensional images, providing information about internal structure and composition.
  • Scanning Electron Microscopy (SEM): provides information about three-dimensional topography and surface composition (e.g., bone and hair).
• Atomic Force Microscopy (AFM): uses a scanning probe that follows surface contours to capture a three-dimensional image of the sample surface; magnification can reach up to $10^6\times$.

Two-Dimensional Microscopy (2D) and Light Microscopy Basics

• The light microscope magnifies specimens and can allow viewing objects about $10^3\times$ their original size.
• Basic operating principle: a light source emits light that is focused by a condenser onto the sample.
• The light that illuminates the specimen is collected by an objective lens, which creates a magnified image that is inverted (upside down).
• The eyepiece (ocular lens) further magnifies the image before it reaches the eye.
• Additional optical elements can be introduced to ensure the image is oriented correctly for viewing.
• Compound microscopes use multiple lenses and are thus called compound.

Magnification and Resolution in the Light Microscope

• Total magnification in a compound microscope: the product of the objective lens magnification and the eyepiece (ocular) magnification:
  • $M<em>{total} = M</em>{objective} \times M_{ocular}$
  • Example: with a 40x objective and a 10x eyepiece, $M_{total} = 40\times 10 = 400\,\text{x}$.
• Eyepiece reticle (scale): projected over the image to estimate object size; tick marks represent smaller distances at higher magnification.
• Resolution: the shortest resolvable distance between two discrete objects under the microscope.
• As resolution improves, the observable distance between two points decreases.

Light Microscope Components and Key Terms

• Main components: objectives, eyepieces, specimen stage and holder, light source, field diaphragm, condenser and aperture, coarse focus knob, and fine focus knob.
• Objectives are responsible for most magnification and resolution.
• Parafocality: objectives are mounted on a nosepiece so that when switching objectives, the focal plane remains approximately the same.
• Objective specifications (often marked on the objective):
  • Magnification, numerical aperture (NA), type of immersion medium, coverslip thickness, and working distance (distance from lens tip to focal plane in the sample).
• Numerical Aperture (NA): a measure of how well an objective can gather light.
  • High-NA objectives allow light at oblique angles; low-NA objectives receive more direct light.
• Resolution depends on NA and the wavelength of light; the resolution of an objective can be related to the wavelength of light and the NA.
• Light path and components shaping illumination:
  • Light source (typically a low-voltage halogen bulb) with adjustable intensity.
  • Field diaphragm controls the illuminated area of the specimen.
  • Condenser focuses bright, uniform light on the specimen; its cone of illumination must be adjusted for the chosen objective.
  • Filters may be used along the light path.

Using a Light Microscope: Step-by-Step (Practical Procedure)

• Prepare: place the sample on the stage, center it over the objective, and secure with stage clips.
• Turn on the light source and start with the lowest powered objective.
• Focus with coarse adjustment (z-direction), then refine with the fine adjustment knob to sharp focus.
• Avoid contact: do not let the objective touch the slide or stage.
• Locate the region of interest by looking through the eyepieces and moving the slide in the x and y directions.
• Field of view decreases as magnification increases.
• Center the area of interest at low power before moving to higher power to improve finding the specimen.
• Once in focus at low power, switch to the higher-magnification objective intended for imaging.
• Lighting optimization for higher power:
  • Adjust the field diaphragm so it sits just outside the field of view.
  • Adjust the field sensor diaphragm so its settings match the objective's NA.
  • Refine focus again with the fine adjustment knob.
• You are now ready to acquire images of the specimen.

Configurations and Applications of Light Microscopy

• Light microscopy offers a wide range of configurations for different applications.
• Special-purpose setups seen in demonstrations:
  • Surgical microscope: typically suspended on a movable arm and stereoscopic; allows light to the viewer and a mounted camera; used in procedures such as kidney transplantation in mice.
  • Dissecting (stereoscopic) microscope: used for examining and manipulating larger specimens (example shown with a Drosophila larva for dissecting body wall muscles and neuromuscular junction study).
  • Inverted compound microscope: has an objective below the stage; used for microinjection techniques such as somatic cell nuclear transfer (SCNT) to generate transgenic animals and clones.

Two-Dimensional vs Three-Dimensional Imaging and Specific Techniques

• Two-dimensional microscopy visualizes structures within a single optical plane, producing flat, detailed representations.
• Bright-field (referred to as "Right field" in transcript):
  • The most common technique; transmits light through stained specimens (e.g., tissue sections, microbial smears) for even illumination.
  • Unstained specimens can be viewed but with lower contrast.
• Dark-field microscopy:
  • Isolates scattered light to produce bright images on a dark background.
  • Particularly useful for visualizing live, unstained microorganisms (e.g., microbes) and their edges/motion.
• Phase-contrast microscopy:
  • Converts phase shifts due to refractive index differences into brightness variations, enabling detailed observation of live cells, organelles, and dynamic processes.
• Fluorescence microscopy:
  • Uses fluorophores (dyes or molecules) that emit light when excited by specific wavelengths; widely used in biology and immunology.
• Notable note on terminology in transcript: “Right field” likely refers to bright-field microscopy.

Quick Reference: Key Magnifications, Imaging Modes, and Concepts

• Optical microscopy magnification: up to $10^3\times$; commonly used in the lab with the compound microscope.
• Electron microscopy magnification: from 2x up to $5\times 10^7\times$; TEM yields internal structure information (2D), SEM yields 3D surface/topography information.
• AFM magnification: up to $10^6\times$; 3D surface imaging via a scanning probe.
• Light path sequence (general): light source → field diaphragm → condenser → specimen → objective → eyepiece → eye (and optional orientation elements).
• Important relationships:
  • Total magnification: $M<em>{total} = M</em>{objective} \times M_{ocular}$.
  • Resolution improves as NA increases and with shorter wavelength light; resolution is the shortest resolvable distance between two points.
• Common practical tips:
  • Start with the lowest magnification to locate the region of interest, then switch to higher magnification for imaging.
  • Use centering at low power to improve finding the specimen at higher power.
  • Balance lighting using field diaphragm and aperture/NA alignment for best image quality.

Equations (LaTeX)

• Magnification relationship: $M<em>{total} = M</em>{objective} \times M_{ocular}$
• Example: $M_{total} = 40\times 10 = 400\;\text{x}$
• Resolution concept (qualitative): the resolution depends on the wavelength of light (\lambda) and the numerical aperture (NA); common approximations include
  • $d \approx \frac{\lambda}{2\,NA}$
  • (alternative common form) $d \approx \frac{0.61\,\lambda}{NA}$
    Note: these express how NA and wavelength influence the smallest resolvable distance.

// End of notes on microscopy as presented in the transcript