Simple to Complex – Life’s Levels of Organization Our journey begins here.
Properties of light
Amplitude : minimum displacement of wave from an equilibrium position
When a light wave decrease or increases in amplitude, it is observed by our eye in the form of a difference in the intensity of light i.e darkness or brightness.
Light ray passing through medium– there is decrease in amplitude
The decrease – greater/lesser depending on refracting index of media
Light ray can pass through a cell but various parts of cell have different refractive indices (RI).
Cytoplasm – causes little or no decrease in amplitude- transparent
Nucleus has higher RI- decrease in amplitude – darker in microscope
2) Frequency: It refers to the number of times a wave crest passes a particular point in one second.
Frequency remains constant for a light wave.
Light waves or identical frequency of coherent rays can combine or interfere with each other.
3) Wavelength: it is the distance between two wave crests
Our eyes are sensitive to different wavelengths of light in the visible range (380-740 nm) & register them in the form of colour
The light spectrum
Wavelength ---- Frequency
Blue light 488 nm short wavelength high frequency high energy (2 times the red) Red light 650 nm long wavelength low frequency low energy Photon as a wave packet of energy
Properties of Light
Reflection:- light bounces off an object
We see colors of objects based on the wavelengths of light reflected by the surfaces of the object
Absorption:- black object absorbs light than reflect light will gain heat more rapidly than white objects which reflect than absorb light
Bending of light as it passes through one medium to another of different density
Such as from air in to glass microscope lens
Degree of bending depends on relative refractive indices of the media
Principles of Microscopy
Refractive Index :- bending of light from one medium to another of different densities
Given by RI= speed of light in a given medium
speed of light in vacuum
Light is also bent as it passes through a glass microscope lens
So shape of lens determines how exactly light is bent
A convex-convex lens (one that is curved outward on both on both sides) will bent parallel light rays so that light theoretically is focused at a single (focal) point.
In light microscopy, visible light is bent by a series of ground glass microscope lens to achieve magnification
Magnification- enlargement of object
Two lenses magnify images – ocular lens & objective lens
Image of specimen much larger than object itself
Total magnification is the product of magnification of individual lens
Resolution: degree to which detail in specimen is retained in magnified image
Capacity of microscope or any other instrument to distinguish between images of two pointed objects lying very close together
Unaided eye – 0.1 mm apart
Microscope - 0.2 µm apart
Limit of resolution
Limit of resolution:
Minimum distance at which two objects appears as two distinct object
Resolution Actual What We Might See Even if we magnify an image of two objects, we can not distinguish them unless we have adequate resolution .
Limit of resolution = 0.61 λ
NA is light gathering capacity of objective
NA (Numerical aperture) = n sin α
constant Wavelength of illumination Numerical aperture Refractive index of air or liquid between specimen & lens Sine of semi-angle of the aperture
Limit of resolution inversely proportional to resolving power (N.A)
So higher N.A lower limit of resolution
Sin α value cannot exceed 1 & R.A of instrument is constant
40X objective RA= 1.6, 100X objective RA= 1.6
So limit of resolution = 0.61 X wavelength ( λ )
1 X 1.4
So limit of resolution directly dependent on wavelength
In case of red light with a wavelength of 6000 ---- limit of resolution = 0.25 µm
But if violet light which has wavelength of 4000 is used, then limit of resolution = 0.17 µm
Thus NA is constant & only way to decrease the wavelength of the light
For this purpose UV light can be used
But UV cannot pass through glass lenses thus necessitating the use of quartz
Resolution vs. Magnification
light as source of
limited resolution (loses resolving power at magnifications above 2000X)
COARSE ADJUSTMENT KNOB
Reading an objective
How Does a Microscope Work? the eye the image the specimen Each lens magnifies the image, increasing its overall size A lens is a bi-convex disk that bends light The farther the light rays are bent, the larger the image appears The bent rays produces an image
How Does a Microscope Work? the eye the image the specimen The image is always seen upside down and backwards from its actual position
Know and be able to use the vocabulary of the microscope
ability of lens to gather light
Know and be able to use the vocabulary of the microscope contd…
Depth of field
between lens & stage
bottom to top of slide
all objectives in reasonable focus at the same time
Care of microscope
Carrying a microscope: one hand grabs the arm and the other hand supports the bottom of the base.
DO NOT TOUCH THE LENS! The oil from your hands can etch the glass.
CLEAN THE LENS WITH LENS PAPER ONLY! Other paper has fiber that can scratch the lens.
Putting away the microscope: rotate to the 4X objective and roll the nosepiece away from the stage so that the space between the stage and nosepiece is at a maximum.