Physical Biochemistry Light Microscopy

Snell’s Law
n1 sinΦ1 = n2 sin Φ2
n = refractive index of medium (nair = 1, nwater = 1.33, noil = 1.5, nglass = 1.5)

Thin Lens Equation

f = focal length [m]

1/f = optical power [dioptre – inverse meters]
s1 = distance from lens to object
s2 = distance from lens to image

SPECIAL CASES

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Physical Biochemistry Light Microscopy

THE COMPOUND MICROSCOPE

Angular Magnification (M)

Near Point - shortest distance at which eye can accommodate (250mm)
M = angleb
Using a suitable combination of lenses, there is no limit to the magnification that can be attained

Factors limiting the smallest details that can be seen in a microscope:

 Optical Resolution – Rayleigh resolution criterion

Immersion Lenses:

Example of how immersion affects theta:

Blue light λ = 400nm
Object in oil ‘n’ = 1.5
Maximum possible angle = sinΘ ~1

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Physical Biochemistry Light Microscopy

 Detector Sampling – Nyquist criterion

o It is necessary to sample a sine wave at at-lest twice its frequency in order to be able to
reproduce it faithfully: fsampling > 2 fsine
o Detector sampling can limit the overall reproduction of detail
o Detector element spacing should be less than Dx/2 of the optical system (as projected
onto the detector plane) so as not to lose any information due to sampling
Less than two samples per cycle results in the detection of a false signal (aliasing)

‘Useful’ Magnification:
It is not the magnification but the optical resolution of a microscope that is important
Magnification can be adjusted so that the resolution of the optical system and the sampling work
well together.

Design constraints on magnification:

 Resolution – should match that of the detector
 Field of view to be imaged – larger field of view = lower magnification
 Size of image – detectors have finite sizes which limits the amount of magnification

In visual light microscopy the maximum useful magnification is ~2000X

(Electron microscopy is 1000000X)

Lens Aberrations:
All real lenses have defects (aberrations) which limit the optical resolution of the system

Monochromatic Aberration (one wavelength)

 Spherical: Different refraction at the edges of a lens
 Coma: Similar to spherical – different refraction strength at different points in the lens
 Astigmatism field curvature distortion

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Physical Biochemistry Light Microscopy

Chromatic Aberration (multiple wavelengths)

Also includes all monochromatic aberrations
Refractive index varies with wavelength (blue light is refracted more than red)
Refractive index is a function of wavelength
This causes ‘smudging’ in the image plane
Can be a significant problem depending on the solvent / embedding medium
Chromatic aberrations can be compensated for using multiple lenses

Modern optical equipment used in light microscopy can have an objective lens containing 10
lenses in order to compensate for these aberrations the more lenses there are – the greater the
level of compensation. Modern objective lenses can approximate the theoretical behaviour quite
well and the resolution is close to the theoretical limit.

Samples will have perturbations of refractive indices within them leading to new aberrations. The
only way to correct for this is by carrying out in situ calibration. Beads can be used that fluoresce
in blue green and red light and are imaged in all three colours. The imaged spheres will not
overlap due to the process described above. A computer works out how to make them overlap
and applies this function to the data thereby correcting the residual aberration.

In electron microscopy the lenses are poor and all aberrations are present and significant.
Recently spherical aberrations and some others have been corrected but this is still a dominant
issue.

Specimen Contrast:
Fluorescent staining is the most common way to get contrast between subject and
solution as they are usually difficult to distinguish from one another. Microscopy works on the
basis that contrast is created from the specimen absorbing different amounts of light. Cells do not
have much difference in absorbance than the aqueous solution so conventional microscopy does
not provide much contrast. Other techniques include:
 Phase contrast & differential interference contrast (DIC)
o Makes use of the fact that there may be a large difference in refractive index between
the cell / solution and even within the cell (organelles etc.)
o Used to visualise refractive index differences
 Polarisation microscopy
o Linearly polarised light will interact differently with ordered specimens depending on the
alignment of the plane of polarisation
o Useful for visualising muscle filaments

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Physical Biochemistry Light Microscopy

 Dark field
o Useful for specific tasks
o Condenser lens collects light from a source to illuminate the specimen
o A stop is placed before the lens – thereby preventing most of the light from reaching the
lens other than that at a very oblique angle (at the edges of the lens)
o This results in only the sharp edges being seen or areas of the sample where there is a
fast change of refractive index
o Scattering from sample is imaged scattering is at a maximum
at the interface of refractive indices
o e.g. Cell edges (the cell body itself is not visualised)
o Can be useful to highlight sharp details within a specimen

Summary:
Numerical Aperture (NA) = n sin (Θ)

Lateral Resolution =

Highest Lateral Resolution ~ 0.1-0.2 μm

This resolution is useful for intracellular study but not for molecular study unless fluorophores are
used as labels.

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