Confocal microscopy
Principles of Fluorescence excitation/emission
“If light is incident on a molecule,
it may absorb the light and then emit light of a different color, a process known as fluorescence”
Encyclopedia of Biomaterials and Biomedical Engineering, 2005, 23, 1- 10
Basic setup of fluorescence microscope
• Dyed specimen is illuminated with light of appropriate wave length
Image is formed from the resulting fluorescent light
• Dichromatic mirror
- “Mirror that reflects light shorter than a certain wavelength but transmits light of longer wavelength”
- Light from main source passes through the objective to the sample
- Longer-wavelength light from fluorescing specimen passes through both the objective and dichromatic mirror
Marvin Minsky (Aug 9, 1927 – Jan 24, 2016) Artificial Intelligence (AI)
Confocal microscopy_Background
• Pioneered by Marvin Minsky in 1955
• Point-by-point image construction by focusing a point of light sequentially across a specimen and then collecting some of the returning rays
• By illuminating a single point at a time,
unwanted scattered light that obscures an image
when the entire specimen is illuminated at the same time was avoid.
“Confocal microscopy is a powerful tool for generating high-resolution images and 3-D reconstructions of a specimen.”
A laser light beam is focused onto a fluorescent specimen through the objective lens.
The mixture of reflected and emitted light is captured by the same objective the dichroic mirror The reflected light is deviated by the mirror
the emitted fluorescent light passes through a confocal aperture (pinhole) to reduce the “out of focus” light.
The focused light then passes through the emission filter and proceeds to the photomultiplier.
Confocal microscopy_Principle
“Out of focus” light is blurring the image and thus reducing the contrast/resolution
In the non-confocal image, specimen planes outside the focal plane degrade the information of interest from the focal plane, and differently stained specimen details appear in mixed color.
In the confocal image (right), specimen details blurred in non-confocal imaging become distinctly visible, and the image throughout is greatly improved in contrast.
Types of confocal microscopy
Confocal laser scanning microscopes
Spinning disk Nipkow disc confocal microscopes
Fluorophores for confocal microscopy
Confocal microscopy_Application
▶ Visualizing a wide range of alive and fixed specimens Single-label imaging & Mutiple-label imaging Z-series
Stereo imaging
Z stacks
3-D
FRET Co-localization
↓
Good
Class 1 : Ensemble techniques
: minimizing the size of the PSF during image acquisition
→ improvement resolution
Ex) Structured Illumination Microscopy (SIM)
Stimulated Emission Depletion (STED) microscopy Class 2 : Single particle localization techniques
: localizing single molecules.
Excitation
Energy transition
Photon
Excitation
Emission
Stimulated Emission Depletion Microscopy
: utilizes two lasers
1. Excitation beam : excites fluorophores
2. STED beam : a doughnut-shaped beam formed by a laser of
a longer wavelength → depletes the fluorophores
<Stimulated emission>
1. Illuminating light
: the energy gap between the excited state and the electronic ground state of the fluorophore 2. Emitted light
: same energy and phase as the
illuminating light
Silence fluorescence at the periphery, resulting in a much smaller area of
remaining fluorescence
• STED beam
: de-exciting the excited state to the ground state via stimulated emission
→ inhibit spontaneous fluorescence
• By increasing the intensity of the STED beam, the threshold intensity required to fully suppress
fluorescence moves closer to the center
→ the spot of remaining fluorescence becomes
smaller, resulting in a higher resolution for the
fluorescence image.
The stimulated emission is at longer
wavelengths than fluorescence and can be
prevented from reaching the fluorescence
detector by using appropriate optical filters.
Most of fluorophores in the focal region are excited
→ large blob
Depletion of most of the excited fluorophores in the overlapping region
→ leaving only the
fluorophores in the center of the doughnut to
fluoresce
E.coli
HeLa cell surface
Z-disk in sarcomers in fixed myofibroblasts
1. The emission spectrum of the fl uorophore must be compatible with the wavelength of the STED laser being used.
; STED laser should be in the range of the red-shifted tail of the emission spectrum of the dye
2. Fluorophores with relatively long fl uorescent lifetimes
; they allow more time to efficiently stimulate emission.
3. Small molecule organic dye : ATTO,DY dye
Fluorescent proteins (FPs) : GFP , YFP
1. large and can potentially perturb localization and function of the tagged protein.
2. Not nearly as photostable as
organic dyes, making them more
susceptible to photobleaching
1. Scanning rate : 4~12s/point ~ 25s/point
2. High resolution can be achieved routinely ( < 50 nm) 3. Signal can be collected at far-field
4. Incorporates most widely used fluorescence technique 5. Can image live biological sample
6. Optical system is simple to understand
7. Can scan the sample in z direction for 3 D image
Yeast Mitochondria Mammalian Golgi
3 D Image
• The diffraction limits the resolution of the microscope.
• The light that passes through the aperture is not rounded by diffraction.
• Objects below 200-300 nm cannot be observed.
Diffraction limit
Ensemble measurements
Targeted approach
- SIM (structured illumination microscopy) -STED(stimulated emission depletion)
Single-molecule measurements
Random approach
- PALM (photo activated localization microscopy) - STORM (stochastic optical reconstruction microscopy)
• Super-resolution techniques allow images to be taken with a higher resolution than the diffraction limit.
• Objects below 200-300 nm can be observed.
B
Figure. Microtubules overlapped in the conventional fluorescence image (left panel) are resolved in the super-resolution image (right panel).
(A) A conventional, diffraction-limited image cannot separate the merging PSFs (point spread functions) of two adjacent fluorescent emitters.
(B) The fluorophores are successively activated (solid lines) and deactivated (dotted lines) and the centroid positions of the detected PSFs are determined with high accuracy.
Figure.Principle of localization-based super-resolution methods.
Figure.Principle of super-resolution imaging based on single-molecule localization.
(a) The target structure shows a hypothetical desely labeled filamentous network of intracellular structures.
(b)-(e) Diffrent fluorophore emitted the light and be localized.
(f) Final image can be obtained by repetition of (b)- (e).
• STORM uses pairs of cyanine (Cy) dyes typically coupled to secondary antibodies
• The photo-switchable dye
- Dyes act asactivator and reporter pairsto cycle multiple times betweenthe dark and light states.
1) Activator : relatively short wavelength dye for photoactivation 2) Reporter : relatively long wavelength dye for image capturing
Activator : Cy2 Reporter : Alexa647
Application examples
The actin cytoskeleton in fixed cellswith resolutions of 10 nm in the lateral direction and 20 nm in the axial direction
Xu et al. 2012.
In living cells, STORM has been used to image at resolutions of 30 nm in the lateral direction and 50 nm in the axial direction,
at a temporal resolution of 1–2 s.
Jones et al.
2011.
2 μm
100 nm
100 nm
50 nm
Fig.STORM images of transferrin in live cells.
Future work
Contrast ratio
Fluorescen t
probes
Imaging system
Switching time
Acquisition speed
Localization accuracy
목차
1. 의료영상기기
2. MRI Scan의 원리
3. MRI 사용사례
4. MRI의 한계점
5. Q&A
1. 의료영상기기
의료영상기기
원리
방사선 방사성 동위원소 자기장 초음파
진단법
CT X선 PET SPECT MRI fMRI 초음파
영상
형태
3D 2D 3D
방사능
매우
높음 높음 낮음 없음
용도
해부학적 진단 생리학적 진단 해부학
적 진단 생리학 적 진단
(뇌)
적 진단 해부학
2. MRI Scan의 원리
M agnetic R esonance
2. MRI Scan의 원리
I maging
2. MRI Scan의 원리
3. MRI 사용사례
3. MRI 사용사례
3. MRI 사용사례
3. MRI 사용사례
3. MRI 사용사례
4. MRI의 한계점
1. 촬영 받는 사람의 불안감
2. 강력한 기계 작동소음, 시간, 움직임 3. 검사실의 낮은 온도
4. 강한 자기장
