Optical Microscopy. Lecture 1. Concepts we will discuss in this lecture: . Natures of light Mechanism of Optical Imaging system The Use of Lenses and the Problem of Lenses Spatial Resolution. Some Properties of Light. Both lasers and white light sources used in microscopy. Laser.
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Both lasers and white light sources used in microscopy
White light contains all, or most, of the colors of the visible spectrum.
Lasers are Monochromatic
(very narrow frequency distribution)
Both white light, lasers used in microscopy techniques
Plane where electric field vector lies, E=Eºcos(ωt)
Perpendicular to direction of propagation
Parallel to floor
H,V (s,p) 90 degrees
out of phase
elliptical polarization: less than 90 out of phase
This nature used extensively
In microscopy: pol microscopy, DIC, SHG
Constructive, destructive interference
0, 180 degrees Limiting cases for complete constructive, Destructive interference, respectively
Underlies image formation in almost all forms of microscopy:
phase, DIC, polarization,
Some advanced forms of confocal
Good for modeling
Not real form
Wave, particle duality physically important
Some phenomenon described by both
Focal length is negative
To an eye on the right-hand side, these diverging rays will
Appear to be coming from the point F’: the second focal point.
water (20° C)
where q1 is the angle of incidence, q2 is the angle of refraction
Only need 2 rays
Real image: if rays intersect and unite in image plane
and can be projected onto some surface in image plane
Two-lens Imaging system
Virtual Image: if rays diverge, but backwards extensions
converge and intersect behind specimen
Eye is part of optical
system of microscope
Infinity optics allows insertion of
Filters, analyzers without changing tube length, or final image
Basic Formulae in air
This is called dispersion
All but quartz
These values are tabulated (e.g. CVI Laser, Melles Griot)
Spherical Aberration could also be caused by the use of the cover glass-slip.
A correction collar might be found on the objective to set the thickness of the glass-slip.
If no correction collar can be found, the objective is corrected for a 0.17 mm glass-slip.
Astigmatism and coma are caused by imperfection in the lens manufacturing.
light from an object must either be different in intensity or color (= wavelength) from the background light:
Both used in light and fluorescence microscopy
diffraction occurs at the objective lens aperture
Numerical Aperture (N.A.)
From diffraction theory
N.A. = n sinq
NA= radius/focal length
~250 nm in visible
Resolution only determined by NA and wavelength
Visible region used for
Light microscopy small
Part of EM spectrum
Resolution limit :λ/2
Visible good for
Ideal for micron sized
EM, X-ray cannot
do live imaging
Spacing of Grating and Diffraction Pattern
Inverse relationship (transform) of
object spacing (or size) and diffraction pattern
a sinθ = nλ
n = 0, 1, 2, 3 …
d = f λ / a
Fringe spacing in the image:
d2 = f’ λ / d1 = f’ λ a / f λ = M a
Requires at least one of the first order diffraction spot in order to form the image.
Abbe showed need for central and diffracted spot
Appear same size
Impossible to remove
Separated exactly by
Absence of light between
Rings is due to
Light from each point of the object is spread out in the microscope because light diffracts at the edges of the lens
Central spot is 0th order diffraction or Airy disk
Contains 84% of power
Con inter at P’
Destr at P’’
Smaller for full
Maxima larger, max, min further apart:
Covers more cone cells
or camera pixels:
The resolution of a microscope is the shortest distance two points can be separated and still be observed as 2 points.
MORE IMPORTANT THAN MAGNIFICATION !!
Limits on NA and Resolution?
Air: NA= 0.95 fora =70 degrees
Immersion increase n:
NA= 1.4a =67 degrees (oil) n~1.5
1.2 (water) n=1.33
Higher index materials for greater resolution?
Some exist: methyl iodide, smelly, toxic
Also need higher index coverslips, slides
Useful Magnification (total) = 500 to 1000 • NA (Objective)
More mag does not help, and
decreases image quality through artifacts, diffraction
Limit comes from rod separation in the eye
Axial resolving power
Defined only by NA2
Small Depth of Field at high NA
Focusing critical at high NA
f = image distance / effective diameter of the lens