LIGHT MICROSCOPY basic LESSON 3

Types of Light Microscopy 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 condenser - lens system that aligns and focuses the light from the lamp onto the specimen

Objectives

Objectives 10 - magnification factor (10X) 0.25 - Numerical Aperture (N.A.)  160 - tube length (mm) 0.17- thickness of the cover slip (mm)

Optical Principles of Microscopy 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 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 improves as r becomes smaller. 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 Abbé equation

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 The rays now pass straight through without deviation

N.A. = nsinμ n = refractive index of the medium between lens and object Objective n = 1.52 n = 1.5 n = 1.51 Oil Air n = 1.0 n=1.52 n = 1.52 Coverslip n=1.33 n=1.52 Specimen Water One or two drops of oil are placed on top of the coverslip and the 100X objective lens is brought into position so that it touches the oil and creates a "bridge" of oil between the slide and objective lens. 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. 100X lens) refractive index very close to that of glass. The oil can get messy and must be wiped up when you are finished.

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 Becker’s World of the Cell ; pp 1 – 5 Looking at the Structure of Cells in the Microscope http://www.ncbi.nlm.nih.gov/books/NBK26880/ “The Light Microscope Can Resolve Details 0.2 µm Apart”