Optical Microscopy
罗敏敏
NIBS, Tsinghua Univ, & CIBR
Seeing is believing
Outline
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Introduction
The development of microscope
The beam path in microscope
Compounds of a microscope
Contrast enhancing techniques
Confocal
Multi-photon microscope
Fiber-optic fluorescence imaging
Supra-resolution microscope
What do we want?
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Clear (resolution)
Sensitive (better detector or camera)
Fast
Non-invasive
Why do you need a microscope?
Our eyes can focus a subject
at a distance between approx.
20cm to infinity.
our eyes need a view
angle to recognize the
details of a subject.
We can not directly see
any details of the specimen.
A “magnifying glass” is an
older tool for seeing small
subjects.
Single lens only provides
limited magnification.
We need the compound lens: microscope!
How does microscope magnify?
Microscopes magnify in two steps: two
different kinds of ray paths as below.
Finite tube length microscope
Infinity corrected microscope
A: object
2:objective
3:tube lens
4:intermediate image
5:eyepiece
We can get bigger view angle from microscope.
Total magnification= objective magnification × eyepiece magnification
Total magnifications Up to 2000×, but magnification
is not enough, resolution determines what we see.
What does “resolution” mean?
The limit up to which two small
objects are still see separately is
used as a measure of the resolving
power of a microscope.
Because of diffraction, one sharp
point may be imaged as a slight
blurred spot with diffraction rings,
which is called “Airy disks”.
There is a distance which can right
separate two “Airy disks”: Rayleigh
distance.
Rayleigh distance =1.22λ/（2×N.A.）
Light wavelength
Visible light 400-700 nm
0.18 – 0.3 micron (N.A. = 1.4)
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X-ray 0.01-10 nm
0.4-43 A
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About NA
The numerical aperture (NA)
of objectives is an important
parameter, and it increases
with magnification, up to
about 40×.
Higher NA, higher resolution.
40×/0.75
40×/1.3oil
How to get ideal resolution?
Are the objective and the specimen clean?
Do the cover slips have the correct thickness?
Is the objective suitable for the specimen?
The development of microscope
Anton van Leeuwenhoek (1632-1723)
1 Joseph Von Fraunhofer (1787~1826): creation of chromatic error
correction.
2 Ernst Abbe (1840~1905): theoretical basis of microscopic image
formation.
3 Otto Schott (1851~1935): a glass chemist.
4 August Kohler(1866~1948): invention of the kohler illumination system.
The beam path in microscope
Eyepiece
Sample
Light source
The beam path in transmitted light
Objective 40× vs 4×
Camera
Eyepiece
Light source
Sample
The beam path in reflected light
components of a modern microscope
Light source
Mercury lamp
Mercury line
Contrast enhancing techniques
BF
Dark field
BF
PH
Wide-field Fluorescence
microscope
Beam path of fluorescence microscope
1) Excited light source: mercury
lamp, LED
2) Fluorescence cube: excited filter,
dichroic mirror, emission filter
How to get fluorescence through exciting
Excitation spectra
Emission spectra
Laser line
Micro-optical sectioning tomography
Li et al., Science 2010
Virus injection
Thick section preparation
Immunostaining
Lin et al. Luo Lab 2018 Nature Methods
Clearing
Imaging & reconstruction
Light-sheet fluorescence Microscopy
激光片层扫描显微技术
Light-sheet fluorescence Microscopy
Confocal
Zeiss LSM880 Inverted, CIBR
Leica SP8 upright, CIBR
Drawbacks of tradition microscopy
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Light from mercury or xeon
light sources excites the
entire field. Emitted
fluorescent light can be
observed directly or
acquired with a CCD camera.
Pictures tend to be blurry
because of horizontal and
vertical interference.
Out of focus blurring
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Vertical Z-axis blur. For samples with certain
thickness(>2 micros), fluorescence is emitted by
samples above or below the focus plane and thus
produces blurring.
Horizontal XY-axis blur. Diffraction from lights
emitted by areas adjacent to the region of
interests (ROI) on the same focal plane.
Advantages of confocal microscopy
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Dramatically reduces out
of focus blur at both XYaxis and Z-axis.
Confocal microscopy at the
same provides the ability
to perform optical
sectioning. Can perform 3D image reconstruction for
thick samples (like pollen
or fly brain)
Multi-color fluorescent
confocal imaging
Gao Lab at NIBS
Difference between confocal and
traditional microscopy
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For traditional microcopy,
light is applied to the entire
field. Thus there is no delay
in image acquisition.
For confocal, light application
and detection are applied by
point-by-point scanning. Thus
it is impossible to look into
the eye-pieces for images
and there is certain delay
for image acquisition.
Pinhole
detector
pinhole
laser
scanner
specimen
Beam path of confocal
Why confocal needs a pinhole
Laser Scanning Microscopy
confocal laser scanning microscopy
multiphoton laser scanning microscopy
notes
cell
with
probe
focal
plane
X
l
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n
e
s
c
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t
Y
X
X
t
s
c
a
n
s
scanning
mirror
X
Y
s
c
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n
s
X
X
Y
s
c
a
n
Y
t
laser
b
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j
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k
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2
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Spin-disc confocal
Olympus IX71 Live-cell station
Rao Lab
Multi-Z confocal microscopy
-37.5 μm
-12.5 μm
-12.5 μm
+37.5 μm
Extended depth
A. Badon et al., Video-rate large-scale imaging with Multi-Z confocal microscopy. Optica 6, 389-395 (2019).
Zeiss LSM880-Airy scan
K. Korobchevskaya, H. Colin-York, B. Lagerholm, M. Fritzsche, Exploring the potential of Airyscan microscopy for live cell
imaging. 4, 41-41 (2017).
Advantages of confocal
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Lower background, higher sensitivity,
and better images (most of time)
Optical sectioning, 3D imaging
Problems of confocal
3D Deconvolution
1. Nearest Neighbours Deconvolution
2. Constrained Iterative Deconvolution (CI) (PSF Key)
The idea behind 3D Deconvolution
3D deconvolution configuration
Multi-photon microscope
Olympus FVMPE-RS multi-photon
Two-photon excitation
MPM has been applied to a
variety of imaging tasks and
has now become the
technique of choice for
fluorescence microscopy in
thick tissue and in live animal.
principle Isaac Abella 1962; implementation Winfried Denk and James Strickler in Webb lab at Cornell in 1990
Schematic of a MPM
Schematic of a MPM
In vivo imaging
OSN fiber
Soma
Basal dendries
Tan and Zhan, Luo Lab 2010
2-phton in vivo imaging
40 µm
µm
40
In vivo imaging in primate brain
Li et al., Tan Shiming Lab 2017 Neuron
Optic-fiber fluorescence imaging
Mark Schnitzer, 2004
Li and Yu, 2009
GRIN (gradient index) lens
Mark Schnitzer, 2009
High-speed, miniaturized fluorescence
microscopy in freely moving mice (1.1g)
Mark Schnitzer, 2008
The use of Ca2+ imaging: one example
Ziv et al., 2013; Nature Neuroscience
fast high-resolution, miniaturized two-photon
microscope (FHIRM-TPM)
~3 g; 0.64 um x-y and 3.35 um z, 40 Hz at 256 × 256 pixels, FOV of
Fiber photometry
to amplifier
& DAQ
PMT
GFP BP
ND
488 nm laser
DM
lens &
commutator
optical fiber
Li et al., in preparati
Nanomicroscopy
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STED
PALM/STORM
SI (structured illumination)
STED (Stimulated Emission Depletion)
microscopy 受激发射损耗显微镜
Hell, S. W. and J. Wichmann (1994). "Breaking
the diffraction resolution limit by stimulated
emission." Opt. Lett. 19(11): 780-782.
STED
Gerald Donnert, Jan Keller, Rebecca Medda, M. Alexandra Andrei, Silvio O. Rizzoli, Reinhard Lührmann, Reinhard Jahn, Christian Eggeling,
Stefan W. Hell (2006). "Macromolecular-scale resolution in biological fluorescence microscopy". PNAS 103 (33): 11440–11445.
Features
High resolution (best 5.8 nm, typically
30-50 nm)
 Living cells
 Fast
Problems:
 Very complicated equipment
 Dye quench/bleach
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PALM/STORM
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In conventional biological imaging, diffraction places a limit on the minimal
xy distance at which two marked objects can be discerned
Photoactivated localization microscopy (PALM) and stochastic optical
reconstruction microscopy (STORM) optically resolve selected subsets of
protect fluorescent probes within cells at mean separations of <25
nanometers.
It involves serial photoactivation and subsequent photobleaching of
numerous sparse subsets of photoactivated fluorescent molecules (protein
for PALM and cy5 for STORM). Individual molecules are localized at near
molecular resolution by determining their centers of fluorescent emission
via a statistical fit of their point-spread-function.
The position information from all subsets is then assembled into a superresolution image, in which individual fluorescent molecules are isolated at
high molecular densities. In this paper, some of the limitations for PALM
imaging under current experimental conditions are discussed.
PALM: photo-activated localization
microscopy 光敏定位显微镜
Science 2007
STORM (stochastic optical reconstruction
microscopy) 随机光学重构显微镜
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Cy3-cy5 dye pair, cy5.5, cy7
Switch off by red light, on by green light
Optical resolution 20 nm (theoretical 4 nm, stage drift/focus drift
3D is possible.
Hours for a large picture
B. Huang, W. Wang, M. Bates, X. Zhuang, "Three-dimensional Super-resolution Imaging by Stochastic Optical
Reconstruction Microscopy", Science 319, 810-813 (2008)
M. Bates, B. Huang, G. T. Dempsey, X. Zhuang, "Multicolor Super-Resolution Imaging with Photo-Switchable
Fluorescent Probes", Science 317, 1749-1753 (2007)
M. J. Rust, M. Bates, X. Zhuang, "Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy
(STORM)", Nature Methods 3, 793-795 (2006)
STORM
Structured illumination microscopy
(结构光显微镜）
SIM Pros and Cons
Pros:
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X-Y-Z resolution of 110 × 110 × 360 nm for 488nm excitation in the 3D
mode; lateral resolution of 100 nm in the TIRF mode (82 nm if NA=1.7). For
nonlinear probes, theoretically unlimited.
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No special sample preparation or fluorophore requirements
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As fast as 3 sections/sec/color in 3D mode, or 6 frames/sec in TIRF-SIM
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2-color 3D live cell imaging capability
Cons:
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Post-processing of images is required for reconstruction.
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Samples for 3D SIM should be thinner than 12 µm.
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The SIM reconstruction algorithm suffers from motion artifacts, if the
sample moves while the illumination pattern changes. However, motion less
than 100 nm/s is well tolerated.
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Photobleaching and phototoxicity can severely restrict imaging time.
The future:
1: Super-resolution
2: Faster. Zeiss LSM Live line-scanning confocal
3: Better probes.
4: Miniaturization.