LIGHT MICROSCOPY basic LESSON 3 Types of Light

LIGHT MICROSCOPY basic LESSON 3

Types of Light Microscopy 1. 2. 3. 4. 5. 6. Bright-field microscopy Phase Contrast microscopy Differential Interference Contrast (or Nomarski) Dark-field microscopy Fluorescence microscopy Confocal Scanning microscopy

Light path of a bright-field microscope

Components of a bright-field microscope

Objectives

Objectives 10 - magnification factor (10 X) 0. 25 - Numerical Aperture (N. A. ) 160 - tube length (mm) 0. 17 - thickness of the cover slip (mm)

How small object can be seen with current technologies OPTICAL PRINCIPLES OF MICROSCOPY

Maximizing resolution!! important goal in microscopy

Resolution (=Resolving power) Ability to see fine details The distance between two particles at which they can be seen as separate objects The maximal resolving power of the LM ≈ 0. 2μm Objects smaller or thinner than 0. 2 μm ? Electron Microscopy

Limit of Resolution The minimum resolvable distance between two points Abbé equation R = 0. 61λ/ N. A. λ = wavelength of light (0. 4~ 0. 7 μm) N. A. = numerical aperture (lens property; 0. 25 -1. 32) Resolution smaller. improves as r becomes ◦ wavelength of light (the shorter, the better) ◦ numerical aperture of the objective lens (the higher, the better) 0. 2μm (=200 nm) for Light Microscopy 2 nm for Electron Microscopy

Numerical Aperture (N. A. ) • a measure of its ability to gather light and resolve fine specimen detail at a fixed object distance N. A. = nsinμ μ = one-half the angular aperture (A) n = refractive index of the medium between lens and object (from 1. 00 for air to 1. 52 for specialized immersion oils. ) • angular aperture (A) : how much of the illumination that leaves the specimen actually passes through the lens.

N. A. = nsinμ

N. A. = nsinμ μ = one-half the angular aperture(A) n = refractive index of the medium between lens and object (from 1. 00 for air to 1. 52 for specialized immersion oils. ) Refractive index : a measure if the change in the velocity of light as it passes from one medium to another

Refraction

Oil Immersion

N. A. = nsinμ n = refractive index of the medium between lens and object Objective n = 1. 52 n = 1. 51 n=1. 52 Oil n = 1. 5 n = 1. 0 Air n = 1. 52 Coverslip Specimen Water n=1. 33 • allows very little refraction of the light rays as they go through the slide, specimen, coverslip, oil and through the glass objective lens of your microscope.

Oil Immersion • highest power objective lenses (i. e. 100 X lens) • refractive index very close to that of glass.

Thus the resolution of the microscope is R = 0. 61λ/ N. A. = 0. 61λ/ nsinμ a function of • the light (the shorter, the better), • the refractive index of the medium through which the light passes (the bigger, the better), • the acceptance angle of the lens (the bigger, the better).

Learning Resources (Textbooks) Junqueira’s Basic Histology ; pp 4 - 6 2. Becker’s World of the Cell ; pp 1 – 5 3. Looking at the Structure of Cells in the Microscope 1. http: //www. ncbi. nlm. nih. gov/books/NBK 26880/ “The Light Microscope Can Resolve Details 0. 2 µm Apart”