Microscopy Dr. Bhavesh Patel Principal V.P. and R.P.T.P. Science College Vallabh Vidyanagar –

A.V. Leeuwenhoek Father of Microbiology So, is “The Geographer” Leeuwenhoek?

Metric Units of Length 1 Meter (m) = Standard unit of length 1 Decimeter (dm) = 1/10 or 0.1 or Centimeter (cm) = 1/100 or 0.01or Millimeter (mm) = 1/1000 or or Micrometer (µm) = 1/ or or Nanometer (nm) = 1/ or or Microscope Micro (Latin) = Small Skopos(Greek)=to look at

Relative Sizes TEM SEM both

Leeuwenhoek's Microscope

Light Interactions

Simple vs Compound Microscope Microscope – An optical instrument that enlarges the image of an object. Simple Microscope – Consist a single lens thus has a very limited magnification. Compound Microscope – Consist more then one lens or lens system (usually two) thus has a better magnification.

Terms Magnification – Ratio between image vs object size Apparent size of object (image) Magnification = Actual size of the object Resolving Power – Ability or power of an optical instrument to see two points clear and distinct which are otherwise very close to each other. Wavelength of light (λ) Resolving power = NA (Numerical Aperture) Numerical Aperture – It is a mathematical expression to explain amount of light that is concentrated by condenser and collected by the objective. Numerical Aperture = η sin θ η = Refractive index of the medium sin θ = half the angle of light cone entering into objective

What is Resolution?  The object of microscopy is not just to increase magnification, but to do so while retaining sufficient resolution.  Resolution is the ability to see two items as two separate things, i.e., two dots as two separate dots.  The resolution a microscope is capable of achieving is the smallest distance between two dots such that the two dots may be observed (resolved) as separate entities.  In less technical terms, lower resolution means an increased degree of fuzziness, i.e., less focusable [sic?] specimens.

Electromagnetic Spectrum

Cont…

Light Compound Microscope Compound Scope: More than One Lens (Advantage = Greater Magnification)

Light Through Compound Scope Compound Scope: More than One Lens (Advantage = Greater Magnification) Light Filter

Objective marking

Immersion Oil

Types of Microscopes

Bright- vs. Dark-Field (1/2)

Bright- vs. Dark-Field (2/2)

U.V. Microscopy  Ultra Violet rays are used in this type of microscopy.  Ultra Violet rays are invisible with low penetration and low wavelength (visible nm, UV nm)  Because of its low wave length it has low resolving power thus high resolution and magnification (double then visible microscope)  Since UV light can not penetrate through glass lenses thus quartz lenses are used for focusing UV.  Picture here can be viewed on television screen or photographic plate  UV is harmful for human being so one must take proper precaution while working with it.

U.V. Microscopy

Fluorescence Microscopy  This is also a type of UV microscopy thus here also UV light is used for illumination purpose  Here slide is stained by some fluorescent dye like Auramin-O in case of Mycobacterium  Two special filters are used in such microscope i.e. Excitation filter and Barrier filter  Excitation filter is placed near the source of illumination. Function of this filter is to allow only the UV light to pass  Barrier filter is placed between objective and eyepiece. Function of this filter is to allow visible light to pass and to restrict the UV light

Phase-Contrast Microscopy

Scanning Electron Microscope

Scanning Electron Micrograph

Transmission Electron Microscope

Shadow Casting

Freeze Fracturing TEM of a freeze fracture replica of a mouse pancreas cell. Notice, the nuclear pores on the surface of the nucleus.