11.11.2015
Imaging Techniques in
Biological Sciences:
Basics of Light and Fluorescence
Microscopy
Kimmo Tanhuanpää 121115
Light
n
In microscopy is a wave and behaves like one
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Basic Concepts
n
n
n
n
Numerical aperature (NA)
Resolution
Point spread function (PSF)
Refractive index
http://micro.magnet.fsu.edu/primer/java/nuaperture/index.html
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Resolution
Resolution = 0.61*lambda/NA
Refractive Index
Objective
n = 1.52
n = 1.52
n=1.52
n=1.52
Oil
n = 1.5
n = 1.0
Air
n = 1.52
Coverslip
Specimen
Water
n=1.33
© Microscopy UK
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Optical Aberrations
Contrast in Bright Field Images
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Fluorescence and Fluorophores
Most of the images are from Molecular Expressions Optical
Microscopy Primer: http://micro.magnet.fsu.edu/primer/index.html
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Structures of Some Fluorophores
FITC
TRITC
Pyrene fattyacid
Alexa 488 hydrazide
Fluorescent Proteins
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Detection of Fluorescence
Optical Filter Nomenclature
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Fluorescence Filter Combinations
Fluorescent Microscope
Arc Lamp
EPI-Illumination
Excitation Diaphragm
Excitation Filter
Ocular or camera
Dichroic Filter
Objective
Emission Filter
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Light source
DAPI
TRITC
GFP
Texas Red
Cy5
Objective types and markings
Objective
Type
Achromat
Spherical
Aberration
1 Color
Chromatic
Aberration
2 Colors
Field
Curvature
No
Yes
Plan Achromat
1 Color
2 Colors
Fluorite
2-3 Colors
2-3 Colors
No
Plan Fluorite
3-4 Colors
2-4 Colors
Yes
Plan Apochromat
3-4 Colors
4-5 Colors
Yes
Full list of specialized objective designations:
http://micro.magnet.fsu.edu/primer/anatomy/specifications.html
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Magnification
Objective Color Code
1/2x
No Color Assigned
1x
1.25x
1.5x
Black
Black
Black
2x
Brown (or Orange)
4x air
0.10
2.75
0.13
2.5x
Brown (or Orange)
10x air
0.25
1.10
0.30
4x
5x
10x
16x
20x
25x
32x
40x
50x
60x
63x
100x
150x
250x
Immersion Media
Oil
Glycerol
Water
Special
Red
Red
Yellow
Green
Green
Turquoise
Turquoise
Light Blue
Light Blue
Cobalt Blue
Cobalt Blue
White
White
White
Color Code
Black
Orange
White
Red
20x air
0.40
0.69
0.50
0.55
Objective Type
Plan Achromat
Magnification N.A
Plan Fluorite
Resolution
N.A
(µm)
Plan Apochromat
N.A
Resolution
(µm)
2.12
0.20
1.375
0.92
0.45
0.61
0.75
0.37
Resolution
(µm)
40x air
0.65
0.42
0.75
0.37
0.95
0.29
60x oil
1.25
0.22
1.30
0.21
1.40
0.20
100x oil
1.25
0.22
1.30
0.21
1.40
0.20
F(trans)
F(epi)
N.A. = Numerical Aperture
Correction
Magnification
Plan
Achromat
Plan Apo
Plan Fluorite
Plan Apo
Plan Apo
Plan Fluorite
Plan Apo
Plan Apo
N.A.
10x
0.25
6.25
0.39
10x
20x
20x
40x (oil)
60x
60x (oil)
100x (oil)
0.45
0.50
0.75
1.30
0.85
1.40
1.40
20.2
6.25
14.0
11.0
2.01
5.4
1.96
4.10
1.56
7.90
18.0
1.45
10.6
3.84
F(trans) = 104 • NA2/M2 ; F(epi) = 104 • (NA2/M)2
NA 1.2 water
objective 10 um in
water
PSF of a NA 1.4 oil objective 0, 1, 2, 5 and 10 um in water
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The objective type should be selected according to the
sample refractive index: water immersion for live samples,
glycerol for fixed, oil for special applications like TIRF
microscopy and for fixed samples when glycerol optics is not
available.
Correction collar (if available) should be adjusted
individually for each sample.
Camera
Color cameras are sensitive only on
the visible spectrum area. B/W
cameras are limited by the CCD
quantum efficiency and optics
transmission
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Anatomy of a Microscope
Sampling
●
Nyqvist sampling theorem states that image must be
sampled at least 2 times the resolution
For optimized sampling rates, see
http://support.svi.nl/wiki/NyquistCalculator
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Deconvolution
On a typical wide field fluorescence image 90%
of fluorescence can be out of focus.
Inverse filter deconvolution of a fluorescent 4 µm
sphere Image volume 22 x 23 x 12.5 µm. A stack of
25 images seen from the side.
What does deconvolution do on a real sample?
Mitotracker green in live astrocytes.
3D blind deconvolution with Media
Cybernetics Autodeblur
PSF
calculated
from the
original data
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Old tenets are still applicable
“When you employ the microscope,
shake off all prejudice, nor harbour
any favorite opinions; for, it you do,
‘tis not unlikely fancy will betray you
into error, and make you see what
you wish to see”
“Remember that truth alone is
the matter that you are in
search after; and if you have
been mistaken, let not vanity
seduce you to persist in your
mistake.”
Henry Baker, chapter 15, “Cautions in viewing
objects” at The Microscope Made Easy, 1742
Further resources
http://micro.magnet.fsu.edu/primer/
http://virtual.itg.uiuc.edu/training/LM_tutorial/
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