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.

Matching the Excitation with Fluorophore Absorption

Standard Filter Sets

•If a lamp is used for excitation, narrow-band (10-20 nm) excitation filter is used to generate monochromatic light. •Laser emission is reflected from dichroic mirror and sent to the sample. •Fluorescence signal from the specimen is transmitted by the dichroic and filtered by emission filter.

Filter Design

Broad absorption and emission spectrum of fluorophores causes bleed-through between fluorescence channels http://www.invitrogen.com/site/us/en/home/support/Research-Tools/FluorescenceSpectraViewer.html

Multi-Color Imaging EMITTER

MIRROR

Sequential excitation with laser lines with multi-band emission and dichroic filter set.

Wide-Field (Epi) Fluorescence Microscopy excitation

Fluorescence signal

• A bright light source is used to excite fluorophores in the specimen. • For efficient high-contrast imaging, both the illuminator and objective lens are positioned on the same side of the specimen. • In epi-illuminator, and the objective lens functions both as the condenser, delivering excitatory light to the specimen, and as the objective lens, collecting fluorescent light and forming an image of the fluorescent object in the image plane. • Fluorescence filter sets are positioned in the optical path between the epi-illuminator and the objective. • High-NA, oil immersion objectives made of low-fluorescence glass are used to maximize light collection and provide the greatest possible resolution and contrast

Epi Fluorescence Images

human medulla

rabbit muscle fibers

sunflower pollen grain

• Images are bright, but blurry and low contrast • Epi-illuminator excites the whole thickness of the specimen, unable to control the depth of the field • Bright fluorescent signal from out-of-focus objects give low-contrast • Autofluorescence (fluorescence signal from unlabeled objects) of the cell increases the background

Causes of High Fluorescent Background • Less then ideal filter sets. • Nonspecific binding of fluorophores to specimen. • Reflections and scattering in the optical pathway. • Dust and fingerprints in optics. • Fluorescence from other objects, including Raman scattering of water, autofluorescence of objective glass. • In multifluorescence applications, fluorescent bleedthrough between the channels.

Sources Autofluorescence in Cells

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)

Scanning Confocal Microscopy 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-offocus locations is delivered to the detector, out-of-focus information is largely excluded from the detector and final image.

Components of a Confocal Microscope • Epi illumination is used. • A laser beam is expanded to fill the back aperture of the objective and forms an intense diffraction-limited spot. • The pinhole aperture accepts fluorescent photons from the illuminated focused spot • Image is formed by raster scanning of the focused laser beam. • Magnification is generated by scanning step size. •Fluorescence signal is detected by single channel detector PMT. •Digital pixels are generated by analog to digital converter.

• Superior image contrast and clarity • Three dimensional view. • z stacks, yz and xz cross sections. • Five-dimensional views including information in x-, y-, and z-dimensions, in a timed sequence, and in multiple colors.

3D Scanning Confocal Microscopy • The primary advantage of laser scanning confocal microscopy is the ability to serially produce thin (0.5 to 1.5 micrometer) optical sections through fluorescent specimens • Objective can be moved in z direction with a piezoelectric motor to take z stack images.

See tutorial in http://www.microscopyu.com/tutorials/java/virtual/confocal/index.html

Image Reconstruction

The pollen grain exterior surface

The mouse intestine section 45 z stack reconstitution

Photo Multiplier Tube 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

Analog to Digital Conversion

Sampling Frequency

Resolution in Confocal Microscopy 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).

Axial and Lateral Excitation

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.

• Decreasing the pinhole size reduces the thickness of the focal plane along the z-axis, thereby allowing higher resolution in optical sectioning. • Decreasing the pinhole size also improves contrast by excluding out-of-focal-plane light. • The lateral spatial resolution in the xy plane obtainable in a confocal fluorescence microscope can exceed that obtainable with wide-field optics by a factor of 1.4. • Normally the detector pinhole is adjusted to accommodate the full diameter of the diffraction disk. • However, if the pinhole is stopped down to ¼ of the diffraction spot diameter, the effective spot diameter is slimmed down so that the disk diameter at one-half maximum amplitude is reduced by a factor of 1.4. minimum resolvable distance dx,y ~ 0.4λ/NA dz ~ 1.4λn/NA2

Image Quality and Performance 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)

Image Optimization Total number of photons obtained from specimen is roughly constant! We need to use our photons economically for low signal levels. Pinhole Size Zoom Scan Rate Objective NA Laser Power

Intensity + + + +

Spatial Resolution + Temporal Resolution + + 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

Imaging Thick Specimens

Two Photon Confocal Microscopy

• Illumination with intense 800 nm light can excite a fluorophore that is normally excited at 400 nm • Two photons must be absorbed simultaneously • Absorption efficiency is proportional to I2 of the laser.

• Pulsed infrared lasers (Ti-Sapphire) compress photons in time domain and effectively increase the intensity for 2-photon absorption. • Excitation occurs only in focus, no pinhole is required to exclude out of focus fluorescence. • The use of near IR permits examination of thick specimens, up to 0.5 mm.

Imaging Thick Specimens

• Absence of out-of-focus absorption allows more of the excitation light photons to reach the desired specimen level. • The red and infrared light employed in two-photon excitation undergoes less scattering than shorter wavelengths. • The effects of light scattering are less detrimental to two-photon microscopy than to confocal microscopy.

Imaging Thick Specimens

One Photon vs. Two Photon Confocal Microscopy •Image Resolution: No difference •Thick Specimens: Two Photon can study 10 times thicker specimens than one-photon. •Thin Specimens: Two Photon slightly increases the photobleaching of dyes. •Absorption Spectrum: Two-photon absorption spectrum can be different from singlephoton. •Focal Spot Size: Two-photon can be used effectively for localized photochemistry applications. UV-switchable probes (caged-GFP or caged-ATP). •Lasers: Two photon requires mode-locked ultrafast lasers. Ti-Sapphire covers 720-900 nm. NdYLF is at 1047 nm.

Limitations of Two Photon Microscopy: •Cost, alignment •Local heating of water. •Phototoxicity of IR excitation and near-UV emission. •Development of new ultrafast lasers that cover all of the wavelengths for common probes.

Spinning Disk Confocal Microscope • Raster scanning method slows down image acquisition • To generate multiple focused spots on the specimen plane, collimated laser beam is passed through a disk that contains multiple pinholes. • Disk is rotated rapidly to generate thousands of confocal spots in less than a second. •Yokogawa introduced microlenses in pinholes to boost light gathering efficiency. •Every point in the specimen receives same amount of illumination. •Live cell imaging with confocal microscopy!

Nipkow Disk

• Initial design. • A Nipkow disk contains thousands of small pinholes arranged in rows of outwardly spiraling tracks. • Pinholes of the Nipkow disk have very low light transmission. • In the Petráň spinning disk 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 • Single-sided spinning disk utilized the same set of pinholes for both illumination and detection. • The spinning disk was located in 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.

Each disk contains 20,000 pinholes (with a 250micrometer spacing) arranged in a series of nested spirals. The upper disk contains microlenses that direct and focus light onto perfectly aligned 50micrometer pinholes in the lower disk. The light throughput of this system approaches 50% as opposed to the 4-6%.

• Laser light is shaped by a 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. • Individual microlens elements 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). • The pinhole spiral patterns are designed so that a single image is created with each 30-degree rotation of the disk. • The specimen is fully rasterscanned by partially overlapping images of the pinholes.

Advantages of the Spinning Disk • A real confocal image is generated • Image can be recorded by multi-channel detector (CCD camera) • CCDs can yield higher SNR compared to photomultiplier tubes. • Time resolution is greatly improved. >100 images can be taken per second instead of 90 percent of the illumination light does not pass through the disk, resulting in high background levels.

Acousto Optic Tunable Filter AOTF functions as an electronically tunable excitation filter to simultaneously modulate the intensity and wavelength of multiple laser lines from one or more sources. 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 oscillating wavefront, photoelastic effect. The alternating ultrasonic acoustic wave induces a periodic redistribution of the refractive index through the crystal that acts as a transmission diffraction grating

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.

λcenter = V • B/f The spectral resolution of a tunable filter is 2-6 nm

V is the acoustic wave velocity, Δn is the birefringence of the acousto-optic crystal, and f is the acoustic wave frequency.

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 orthogonal to that of the incident beam. Diffracted beam is separated by an output polarizer (Analyzer Beamsplitter).

•The narrowband diffracted light and incident broadband light is physically separated, and because they exit the crystal through different pathways, polarizers are not required for operation. •Because the two orthogonally polarized firstorder beams do not separate until they leave the crystal, and then diverge at a fixed angle, the diffraction angle does not change with wavelength. •The first-order diffracted component is allowed to illuminate the specimen (typically only one diffracted output is used), while the zeroth-order beam is blocked. •By utilizing crystals having larger birefringence 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.

Advantages of AOTFs 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.