08.11.

2023
 Sample plane

Not to scale
  
𝑑= =
2𝑛 sin 𝜃 2𝑁𝐴

θ4X
θ40X θ10X
θ100X

Not to scale
 A or ACHRO (depending on brand): This signifies that the objective is an achromat.
This means that chromatic aberration was corrected for 2 colors .
PLAN: These objectives produce an image which is in focus from edge to edge.
PLANAPO: This refers to a planapochromatic objective. It produces a flat image (in
focus from edge to edge) and it is has a chromatic aberration correction for 4 colors.
160: This represents the standard tube length of 160mm.
ꝏ: Objective is infinity corrected.
0.17: This represents the thickness of the cover slip to be used in mm. Coverslips with
a deviating thickness will result is an image of lower resolution.
4, 10, 20, 40, 100: This represents the magnification of the objective. The total
magnification is calculated by multiplying the magnification of the objective with the
magnification of the ocular (eye piece), which is usually 10x. The magnification is
also indicated by the ring colors: red: 4x or 5x, yellow: 10x, green: 20x, blue: 40x, 50x
or 60x, white: 100x.

OIL: This designates an oil immersion objectives. WI: Water Immersion. Here water is used instead of oil.
0.65/ 1.25 etc:: This is the numerical aperture.
NCG or NC: These abbreviations stand for “No cover glass”. These objectives are designed to be used without a cover glass.
LWD or ULWD: These abbreviations stand for “long working distance” or “ultra-long working distance”.
P, POL or SF: These objectives are designed to be used for polarization microscopy. The objectives are strain-free (SF) and will therefore not modify the
polarization.
PL or NH: These are designation of objectives used for phase contrast microscopy. A PL (positive low) objective produces an image of a specimen which is darker
than the background, a NH (negative high) objective produces an image which is brighter than the background.
NIC or DIC: Nomarski Interference Contrast or Differential Interference Contrast objectives produce an image of a specimen which appears to be slightly 3
dimensional. If you use a filter to achieve oblique illumination, then the result will look similar.
 Apochromatic Lens/ Objective

Chromatic Aberration
 Aspherical Lens/ Objective

Lens with spherical aberration Aspherical Lens

 Resolution in Microscopy

Christiaan Huygens’ wave optics

Light travels orthogonal to the wave front. Plane Wave and Spherical wave (diverging or converging)

Huygens idea was that the elemental unit of light was not these waves, but these waves (both plane and spherical waves are
composed of infinitesimally small objects (Huygens’ wavelets), which are little points of light that are spherically distributed
but do not have any direction at all.
 Diverging spherical Wave Converging spherical Wave
Plane Wave
Point source

Bright
(nλ difference in path)

Dark
𝑛λ
( 2 difference in path)
 PSF: A wave optic description
Tube Lens
Image
Plane
Objective

Point
Source

Spherical wave
(converging)
Plane wave
Spherical wave
(diverging)

Point Spread Function (PSF): Distribution of light at the image plane of a point light source.
PSF: A wave optic description
PSF: A wave optic description

• All the points (infinite numbers) in the wavefront

behaves like a point source (wavelet) and they are
mutually coherent.
• The mutual coherence allows them to interfere with
each other and produce constructive and destructive
interference as a function of space.
 Interference of two wavelets
Tube Lens

Constructive

Destructive

Constructive
Interference of two wavelets

Constructive interference
appears bright and https://www.ibiology.org/talks/resolution-in-
destructive interference microscopy/
appears dark. The position
of image plane is not
obvious.

Effect of NA

High NA

Low NA
 Interference of wavelets

Two wavelets Three wavelets five wavelets

nine wavelets Enhanced infinite wavelets Enhanced

 Measure of Point Spread Function (PSF)

Enhanced

infinite wavelets Enhanced

XY

Enhanced
0.61λ
𝑁𝐴
XZ/YZ

2𝑛λ
(𝑁𝐴)2
1.4 NA objective, 480 nm wavelength of light

https://www.ibiology.org/talks/resolution-in-microscopy/
Maximum allowed distance between two-point sources that can be
resolved: Rayleigh Criterion (somewhat arbitrary)
2nλ/NA2

0.61λ/NA

https://www.ibiology.org/talks/
resolution-in-microscopy/

19% 26.5%
Low NA Medium NA High NA

Progress in Microscopy, by M. Francon, 1961

 Resolution in the image plane: Sampling
(Aliasing)

Sampling: Sampling: Sampling: Sampling:

sampling
Optical resolution (N): 0.61λ/NA
N = 2F
Sampling (F): 0.3λ/NA
https://svi.nl/NyquistRate
https://www.ibiology.org/tal
Image taken by the same lens but with different grain (pixel) size in the imaging device ks/resolution-in-microscopy/
 Resolution in the image plane: example

• Pixel size in a CCD is 12 × 12 μ𝑚2 .

• Suppose you are imaging with 570 nm light, using a 60X, 1.4 NA objective. Then the optical resolution limit is
(0.61λ/NA =) 0.248 μm. Now, 60X magnification would result in the diffraction limited spots to become (60 × 0.248 =
) 14.88 μm.

Solution:
1. You can use an intermediate magnification changer.
2. You can use 100X objective.
 Super resolution microscopy: STED
Stimulated emission depletion (STED) microscopy: Stefan W. Hell

Wildanger D. et. al., Optics

Express, 2009, 17 (18), 16100-
16110.

Stefan W. Hell

Qin X et. al., ACS Cent. Sci. 2019, 5 (1), 29–42

 Creation of a donut shaped beam
Neupane B et. al., Review of scientific instruments, 2013, 84, 043701: A Gaussian beam is passed through a circular π-phase
plate (πPP) or a 0-2π vortex phase plate (VPP), first. The πPP consists of two concentric regions. The size of these regions is
designed in a way that the two beam components transmitted through possess a relative phase of π and when focused,
interfere destructively at the focal plane.
As a comparison, the VPP is designed in a way that electric field of the transmitted beam around the optical axis changes
continuously from 0 to 2π. The electric field cancels out completely on the axis, yielding a donut-shaped beam.

3π/2

π/2

VPP

N. Bokor et. al., Optics Communications, 2007, 270, 145–150

 Super resolution microscopy: PALM and STROM
Photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM): Eric Betzig and
William E. Moerner