Lecture 2: Microscopy: Introduction to Microscopy

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Presentation transcript:

Lecture 2: Microscopy: Introduction to Microscopy Cell Biology, BIOL350 Spring 2016 Sulaiman Al-Hashmi Thursday, 4th February 2016

History Greeks & Romans Hans and Janssen Used “lenses” to magnify objects > 1000 years ago Hans and Janssen Created the “first” compound microscope (1590’s) Antonie van Leeuwenhoek and Robert Hooke Made improvements by working on the lenses Antonie van Leeuwenhoek First to describe bacteria in 1676

Human: 390 to 700 nm.

Eye power in Nature Goats have rectangular pupils Wide field of vision Dragonfly eyes are made up of 30,000 visual units, each one containing a lens and a series of light sensitive cells Assisting them with motion detection Pupil بؤبؤ العين Dragonfly يعسوب

Geckos can see colors around 350 times better than a human The eyes of a chameleon are independent from each other Look in two different directions at once Chameleon حرباء Geckos سحلية

Cat’s eyes have around 285 degrees of sight in three dimensions Cuttlefish can perceive light polarization Enhances their contrast A moth’s eyes are covered with a water-repellant Anti-reflective coating Pigeons الحمام Cuttlefish الحبار Moth فراشة Predators مفترس

Ant has two eyes, but each eye contains lots of smaller eyes Giving it a “compound eye.” Eagles have 1 million light-sensitive cells per square millimeter of the retina humans only have 200,000 A honeybee’s eye is made of thousands of small lenses.

Microscope parts

Chromatic (color)

Terminology Magnification Resolution Contrast Apparent enlargement of an object The ratio of image size to actual size 100x magnification = 100 times bigger than the actual object Resolution The ability of a microscope to show two very close points separately Clarity sharpness Contrast Difference that makes an object distinguishable. Contrast is determined by the difference in the color and brightness

Steps to use the microscope: Rotate the low power objective into place make sure the stage is all the way down. Place slide on stage make sure that the object to be viewed is centered over the hole in the stage Use the stage clips to hold the slide in place. Turn light on. Focus first with the coarse adjustment knob. Once in focus on low power, turn the nosepiece until the next higher lens is in place. At the final adjustment, use FINE adjustment knob ONLY and focus the object

Magnification Qs? A) Ocular power = 10x low power objective = 20x high power objective = 50x What is the highest magnification you could get using this microscope ? Ocular x high power = 10 x 50 = 500 B) If the diameter of the low power field is 2 mm, what is the diameter of the high power field of view in mm ? The ratio of low to high power is 20/50 So at high power you will see 2/5 of the low power field of view (2 mm) 2/5 x 2 = 4/5 = 0.8 mm

C) If 10 cells can be seen in the low power field of view, how many cells would you expect to see under high power ? At high power we would see 2/5 of the low field 2/5 x 10 cells = 4 cells would be seen under high power

Types of Optical Microscopes Reflected (bright field) Transmitted Polarized Dark Field Differential Interference Contrast Confocal Fluorescence

Light microscopy Upright microscope Inverted microscope Stereomicroscope Basic concepts of light microscope Magnification Resolution power and Numerical aperture Types of light path transmitted light reflected light

Types of light microscopes Upright Microscope Inverted Microscope Stereomicroscope

The light microscope Compound microscope Common light microscope (Labs) Contains two types of lenses Ocular Objective Most microscopes have on their base an apparatus called a condenser Condenses light rays to a strong beam A diaphragm located on the condenser Controls the amount of light coming through it.

Light pathway To magnify an object, light is projected through an opening in the stage, where it hits the object and then enters the objective. An image is created, and this image becomes an object for the ocular lens, which remagnifies the image

Upright microscopy Designed for slide sample High magnification High resolution in transmitted light sample

Inverted microscopy Lower resolution in transmitted light sample Long working distance Suitable for culture evaluation (flask, Petri dish)

Stereomicroscopes Long working distance Low magnification Observation with stereo perception

Fluorescence Microscopy Uses ultraviolet or laser as light source. When ultraviolet light hits an object, it excites the electrons of the object, and they give off light in various shades of color. Since ultraviolet light is used, the resolution of the object increases Sheep Tongue

Confocal microscopy Laser light source Scan across sample and optically section Useful on live samples (monitor real time events) Examine the organization/distribution of biomolecules within cells embryonic mouse brain Anatomy and Embryology 204:437-453, 2001 

Electron Microscopy Electron beam as source (l ~ 0.005 nm) Operate under vacuum (not all) Electromagnets instead of glass lenses Detect by fluorescent screen or photographic emulsion Two major types Transmission (TEM) and scanning (SEM) TEM: Cellular ultrastructure SEM: Surface ultrastructure Electron microscopy provides the highest resolution of subcellular structures. Many electron microscopes are operated under vacuum, so they are not suitable for examining living specimens. The principles of EM are similar to light microscopy except that – EM uses an electrons as its source instead of light and electromagnets to focus the electron beam instead of glass lenses used to focus light in a typical microscope.

TEM It has been exploited most widely in the examination of the internal structure of cells. Works like a light microscope, it transmits a beam of electrons through a thin specimen Then focusing the electrons to form an image on a screen most common form of electron microscope gives good resolution

SEM It is utilized primarily to examine the surfaces of objects This scans a fine beam of electron onto specimen and collects electrons scattered by surface This has poor resolution but gives good 3-D images

Blood The erythrocytes (red blood cells) SEM Magnification 2200:1 Blood and bacteria blood with staphylococci in an ultrathin section. The dark spots are staphylococci, erythrocytes are red and two cells of the immune system are colored in green. TEM Magnification 3000:1