This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Advantages:Advantages: - Intuitive image interpretation- Applicable to samples in natural
environment- In general non-destructive- Easy to use
Optical MicroscopyOptical Microscopy
Disadvantages:Disadvantages:- Abbe diffraction limit- Large sample area
exposed to illumination light
1.2 if 2
0.61 min
NA
NA
Total Internal Reflection FluorescenceMicroscopy (TIRFM)
- Intensity Measurement: Controlling the depth resolution via changing incident angle or wavelength
Filter out scattering light
“ What bother the TIRF ?” It can provide a better depth resolution than confocal microscopyconfocal microscopy, especially when it comes to selecting fluorescent molecules close to the biosensing surface.
Four Plasmonic Effects:Four Plasmonic Effects:- Surface plasmons - Particle plasmons- Inter-particle coupling - Gap mode
→ → To increase sensitivity (less than 1 pg/mmTo increase sensitivity (less than 1 pg/mm22) ) → → To increase local EM field (about 10To increase local EM field (about 105 5 times)times)
- - SPR Phase MicroscopeSPR Phase Microscope for Living Cell Membrane Images for Living Cell Membrane Images with No Fluorescent Labelswith No Fluorescent Labels
- - Plasmon-enhanced TIR Fluorescence MicroscopePlasmon-enhanced TIR Fluorescence Microscope for for
Dynamic Living Cell Membrane ImagesDynamic Living Cell Membrane Images
- GFP-tagged TM on the melanoma cell membrane near the chip surface is excited by the evanescent wave for TIR or surface plasmon wave for SPR;
- The enhancement of fluorescence is observed apparently btw the two images. The experimental results show that the fluorescence intensity can be enhanced about 3.0 fold.
- Because of the variant distance btw the cell membrane protein TM and the collagen-coated surface, different surface plasmon effects can be applied to interpret the phenomenon of fluorescence emission or quenching.
TIRFM Plasmon-enhanced TIRFM
Melanoma-GFP-tagged TM cell
L.-Y. He et al., Proc. SPIE 6088 (2006).
K.-F. Giebel et al., Biophysical Journal 76 (1999) 509.
A Goldfish Glial CellInterference Reflection Microscope
SPR Intensity Microscope
Ag Al
Lateral Resolution Limited by Lateral Resolution Limited by Propagation Length of Surface Plasmon WavePropagation Length of Surface Plasmon Wave
Applications:• Single molecule to cell detection• Nanolithography• Super-resolution data storage• Near-field optical interaction on nanoparticles, nanoclusters, and localized surface plasmon.• Nanophotonics• Surface photochemistry
Evanescent Wave and Nano-scanning Tip Techniques to Break “Diffraction Limit”• Lateral spatial resolution: ~20nm• Longitudinal spatial resolution: ~50nm
• Confined by a metal aperture• Within short distance beyond the screen.
Liquid operation of NSOM opens the way to directly visualise and quantify the size and composition of membrane domains, like lipid rafts, in solution. Fluorescence image of a dendritic cell in buffer solution collected in confocal mode (A) and NSOM mode (B).
Single molecule detection on cells by NSOM. This figure shows a 40 nm optical resolution near-field ‘zoom-in’ on the indicated area (3.2 mm2) in the bright-field image of a fibroblast expressing LFA-1-GFP. GFP excitation is accomplished using 488 nm light (Ar-Kr laser line) linearly polarized along 90°.
Apertureless NSOMApertureless NSOM
Adaptive Photonics Lab, NCKUAdaptive Photonics Lab, NCKUR. Fikri et al., Optics Letters 28 (2003) 2147.
In apertureless near-field scanning optical microscopy (ANSOM), the probe vibration is often used to increase the detected signal that can be detected at by a lock-in amplifier. The realistic model of ANSOM should take into account the scan of the probe as well as the probe vibration and the material properties.
Excitation pulses are followed by stimulated emission depletion pulses for fluorescence inhibition. After passing dichroic mirrors and emission filters, fluorescence is detected through a confocal pinhole by a counting photodiode.PNAS 97 (2000) 8207.
The role of the STED beam is to induce the transition L2 L3 by stimulated emission and to deplete the excited fluorescence.
Comparison btw Confocal & STEDComparison btw Confocal & STED
Reducing the fluorescence focal spot size to far below the diffraction limit: (a) spot of a confocal microscope (left) compared with that in a STED microscope (right) utilizing a y-oriented intensity valley for STED (upper right insert, not to scale) squeezing the spot in the x direction to 16 nm width. (b) The average focal spot size (squares) decreases with the STED intensity following a square-root law. Insert (right) discloses the histogram of the measured spot sizes rendering the 26 nm average FWHM.
Negative Refraction Makes a Perfect Negative Refraction Makes a Perfect LensLens
A negative refractive index medium bends light to a negative angle with the surface normal. Light formerly diverging from a point source is set in reverse and converges back to a point.
- Break diffraction limit & improve single particle detection without near-field tip- Lateral spatial resolution: ~100nm
Enhanced Lateral Resolution by Control of the Size Enhanced Lateral Resolution by Control of the Size & Distribution of Metal Nanoparticles& Distribution of Metal Nanoparticles