Fluorescence Microscopy Parallel light comes out from the objective - - PowerPoint PPT Presentation
Fluorescence Microscopy Parallel light comes out from the objective and penetrates through the sample. Fluorescence signal is collected by the same objective. Excitation and Fluorescence is spectrally separated by set of filters.
generate monochromatic light.
by emission filter.
Broad absorption and emission spectrum of fluorophores causes bleed-through between fluorescence channels http://www.invitrogen.com/site/us/en/home/support/Research-Tools/Fluorescence- SpectraViewer.html
EMITTER MIRROR Sequential excitation with laser lines with multi-band emission and dichroic filter set.
Fluorescence signal excitation
fluorophores in the specimen.
illuminator and objective lens are positioned on the same side of the specimen.
functions both as the condenser, delivering excitatory light to the specimen, and as the
forming an image of the fluorescent object in the image plane.
the objective.
low-fluorescence glass are used to maximize light collection and provide the greatest possible resolution and contrast
human medulla rabbit muscle fibers sunflower pollen grain
control the depth of the field
cell increases the background
autofluorescence of objective glass.
Autofluorescence of Horsetail Fern (Plant Cell)
Chlorophyll and polyphenols (in plant cells) Flavins (FDH), pyridine nucleotides (NADH) Pigments, serotonin Elastin, fibrilin Aromatic amino acid side chains (Trp, Phe)
A laser point source is confocal with a scanning point in the specimen. Fluorescent wavelengths emitted from a point in the specimen are focused as a confocal point at the detector pinhole. Fluorescent light emitted at points above and below the plane of focus of the objective lens is not confocal with the pinhole and forms extended disks in the plane of the pinhole. Since only a small fraction of light from out-of- focus locations is delivered to the detector, out-of-focus information is largely excluded from the detector and final image.
back aperture of the objective and forms an intense diffraction-limited spot.
fluorescent photons from the illuminated focused spot
scanning step size.
single channel detector PMT.
to digital converter.
a timed sequence, and in multiple colors.
See tutorial in http://www.microscopyu.com/tutorials/java/virtual/confocal/index.html
(0.5 to 1.5 micrometer) optical sections through fluorescent specimens
The mouse intestine section 45 z stack reconstitution The pollen grain exterior surface
highly sensitive photon detectors that do not require spatial discrimination, but instead respond very quickly with a high level of sensitivity to a continuous flux of varying light intensity
In wide-field fluorescence optics, spatial resolution is determined by the wavelength of the emitted fluorescent light (left). In confocal mode, both the excitation and emission wavelengths are important, because the size of the scanning diffraction spot inducing fluorescence in the specimen depends directly on the excitation wavelength. Therefore, the volume both illuminated and observed is simply the product of two point spread functions (right).
The smallest distance that can be resolved using confocal optics is proportional to (1/λexc + 1/λem) , and the parameters of wavelength and numerical aperture figure twice into the calculation for spatial resolution. Spatial resolution also depends on the size of the pinhole aperture at the detector, the zoom factor, and the scan rate.
minimum resolvable distance dx,y ~ 0.4λ/NA dz ~ 1.4λn/NA2
thereby allowing higher resolution in optical sectioning.
microscope can exceed that obtainable with wide-field optics by a factor of 1.4.
diffraction disk.
effective spot diameter is slimmed down so that the disk diameter at one-half maximum amplitude is reduced by a factor of 1.4.
Dynamic Range: Analog-to Digital Converter should be adjusted that minimum and maximum signal cover the whole dynamic range of the digitizer. Signal to Noise Ratio: [I/(I+B) 1/2 ] Background is generated by photon shot noise, detector readout, and dark-current Temporal Resolution: (Number of pixels X Dwell Time) per image Usually 1 µsec per pixel and 512x512 pixels Optimum Pixel Size: ~d/2 (smaller pixels are better when signal level is too high)
Total number of photons obtained from specimen is roughly constant! We need to use our photons economically for low signal levels. Intensity Spatial Resolution Pinhole Size + - Zoom + + Scan Rate - - Temporal Resolution Objective NA + + Laser Power + + Photobleaching
Bleed-Through Correction Post Image Analysis
Narrowing emission bands reduces the bleed through but also reduces the signal Sequential image acquisition can be used if excitation wavelength are distinct
http://www.olympusmicro.com/primer/java/confocalsimulator/index.html
normally excited at 400 nm
effectively increase the intensity for 2-photon absorption.
fluorescence.
reach the desired specimen level.
scattering than shorter wavelengths.
to confocal microscopy.
photon.
probes.
down image acquisition
spots on the specimen plane, collimated laser beam is passed through a disk that contains multiple pinholes.
thousands of confocal spots in less than a second.
in pinholes to boost light gathering efficiency.
receives same amount of illumination.
microscopy!
very low light transmission.
microscope, illumination of the specimen and detection of the resulting images occur in tandem using pinholes on opposite sides of the disk, to prevent high intensity scattered and reflected light
the same set of pinholes for both illumination and detection.
the intermediate image plane, but placed at a slight angle so that excitation light could be reflected away from the optical axis and trapped by a beam stop.
specialized lens to adjust the intensity distribution of the Gaussian beam towards the center and is then projected onto the microlens disk with a collimating lens.
gather a substantial amount of the incoming light and focus it through the dichromatic beamsplitter onto an area covering approximately 1,000 pinholes on the lower Nipkow disk (an area spanning 7 x 10 millimeters).
designed so that a single image is created with each 30-degree rotation of the disk.
scanned by partially overlapping images of the pinholes. Each disk contains 20,000 pinholes (with a 250- micrometer spacing) arranged in a series of nested
direct and focus light onto perfectly aligned 50- micrometer pinholes in the lower disk. The light throughput of this system approaches 50% as
crosstalk). This artifact increases the background signal for thicker specimens, and reduces the axial resolution of the system.
dim fluorescent specimens.
background levels.
Birefringent crystal whose optical properties vary upon interaction with an acoustic wave The transducer generates a high-frequency vibrational (acoustic) wave that propagates into the crystal. The crystal lattice structure is alternately compressed and relaxed in response to the
The alternating ultrasonic acoustic wave induces a periodic redistribution of the refractive index through the crystal that acts as a transmission diffraction grating AOTF functions as an electronically tunable excitation filter to simultaneously modulate the intensity and wavelength of multiple laser lines from one or more sources.
Diffraction occurs over an extended volume of the crystal rather than at a planar surface, and only a limited band of spectral frequencies are affected. Changing the frequency of the transducer signal applied to the crystal alters the period of the refractive index variation, and therefore, the wavelength of light that is diffracted. The relative intensity of the diffracted beam is determined by the amplitude (power) of the signal applied to the crystal. Two orthogonally polarized beams do not separate until they leave the crystal, and then diverge at a fixed angle, the diffraction angle does not change with wavelength. V is the acoustic wave velocity, Δn is the birefringence of the acousto-optic crystal, and f is the acoustic wave frequency. λcenter = V • B/f The spectral resolution of a tunable filter is 2-6 nm
The interacting acoustic and optical waves are collinear. The acoustic wave is launched along a principal axis of the crystal. The incident beam passes through a polarizer and follows the same propagation path along the crystal axis, interacting collinearly with the acoustic waves. A narrow band of spectral wavelengths is diffracted into a polarization direction
Diffracted beam is separated by an output polarizer (Analyzer Beamsplitter).
broadband light is physically separated, and because they exit the crystal through different pathways, polarizers are not required for
the crystal, and then diverge at a fixed angle, the diffraction angle does not change with wavelength.
allowed to illuminate the specimen (typically
zeroth-order beam is blocked.
values, this deflection angle (the angle separating the diffracted and undiffracted beams) is increased, achieving adequate separation between the diffracted and undiffracted beams without using polarizers.
Control of the intensity and/or illumination wavelength on a pixel-by-pixel basis while maintaining a high scan rate The illumination intensity can not only be increased in selected regions for controlled photobleaching experiments, but can be attenuated in desired areas in order to minimize unnecessary photobleaching.