The Laser Scanning Microscope

The term laser scanning microscope is used for a number of very different instruments. Scanning can be accomplished by galvano-driven mirrors in the beam path, by piezo-driven mirrors, by a Nipkow disc, or by a piezo-driven sample stage. This section refers to microscopes with fast beam scanning by galvano-driven mirrors. Greatly simplified, the optical principle of these microscopes, is shown in Fig. 5.71. 

Laser-scanning microscopes can be classified by the way they excite and detect fluorescence in the sample. One-photon microscopes use a NUV or visible CW laser to excite the sample. Two-photon, or Multiphoton , microscopes use a femtosecond laser of high repetition rate. The fluorescence light can be detected by feeding it back through the scanner and through a confocal pinhole. The principle is termed confocal or descanned detection. A second detection method is to divert the fluorescence directly behind the microscope objective. The principle is termed direct or nondescaimed detection. 

With a Ti Sapphire laser or another high-repetition rate femtosecond laser, the sample can be excited by simultaneous multiphoton absorption [132, 164, 278, 282, 343, 471, 472]. For biological specimens, three-photon or higher-order excitation is rarely used. Nevertheless, such microscopes are normally called Multiphoton microscopes. 

Two-photon excitation was predicted by Maria Goppert-Mayer in 1931 [189] and introduced into laser microscopy by W. Denk, J.H. Strickler, and W.W.W. Webb in 1990 [132]. The wavelength of two-photon excitation is twice the absorption wavelength of the molecules to be excited. Because two photons of the excitation 

Nondescanned (or direct ) detection solves a problem endemic to fluorescence scattering in deep sample layers. Fluorescence photons have a shorter wavelength than the excitation photons and experience stronger scattering. Photons from deep sample layers therefore emerge from a relatively large area of the sample surface. To make matters worse, the surface is out of the focus of the objective tens. Therefore the fluorescence cannot be focused into a pinhole. 

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Images can also be obtained by scanning the sample by a piezo-driven scan stage. The principle is the same as in the laser scanning microscope, but the optical beam scanner is replaced with a scan stage that moves the sample. The sample scanning technique is shown in Fig. 5.96. 

In the Scan Sync In mode, the scanner controls the TCSPC module. The scan controller is programmed to scan the image with a defined number of pixels per line and lines per frame. The scanner delivers a frame pulse when the scanning starts, a line pulse at the beginning of each line, and a pixel pulse at the transition to the next pixel within a line. The recording then runs in the same way as described for the laser scanning microscope with fast beam scanning. 

A variant of the confocal microscope is the laser scanning microscope (LSM, Fig. 8.9). Here, the effective focus acts as a three-dimensional probe, which is scanned in subsequent height steps two-dimensionally through a transpar-... 

Wolleschensky R, Feurer T, Sauerbrey R and Simon U 1998 Characterization and optimization of a laser-scanning microscope in the femtosecond regime Appl. Phys. B 67 87-94... 

Figure 4. DDC (A), serotonin (B), and tyrosine hydroxylase (C) immunore-activity in the posterior region of a wild-type Drosophila ventral ganglion. Tyrosine hydroxylase (TH) encodes the rate-limiting step in dopamine biosynthesis and is a marker for dopamine cells. B and C are the same CNS assayed for both serotonin and TH. M, medial dopamine neurons VL, ventrolateral serotonin neurons DL, dorsolateral dopamine neurons. Short unmarked arrows in C show vacuolated cells that do not contain DDC immunoreactivity. The immunoreactivity in these cells may represent a nonspecific cross-reactivity of the rat TH antibody. The length bar in A is 50 pM. The images are confocal projections generated on a Molecular Dynamics-2000 confocal laser scanning microscope.

Historically, this has been the most constrained parameter, particularly for confocal laser scanning microscopes that require spatially coherent sources and so have been typically limited to a few discrete excitation wavelengths, traditionally obtained from gas lasers. Convenient tunable continuous wave (c.w.) excitation for wide-held microscopy was widely available from filtered lamp sources but, for time domain FLIM, the only ultrafast light sources covering the visible spectrum were c.w. mode-locked dye lasers before the advent of ultrafast Ti Sapphire lasers. 

Fig. 9 Surface modification of cells with ssDNA-PEG-lipid. (a) Real-time monitoring of PEG-lipid incorporation into a supported lipid membrane by SPR. (r) A suspension of small unilamellar vesicles (SUV) of egg yolk lecithin (70 pg/mL) was applied to a CH3-SAM surface. A PEG-lipid solution (100 pg/mL) was then applied, (ii) Three types of PEG-lipids were compared PEG-DMPE (C14), PEG-DPPE (C16), and PEG-DSPE (C18) with acyl chains of 14, 16, and 18 carbons, respectively, (b) Confocal laser scanning microscopic image of an CCRF-CEM cell displays immobilized FITC-oligo(dA)2o hybridized to membrane-incorporated oligo(dT)20-PEG-lipid. (c) SPR sensorigrams of interaction between oligo(dA)2o-urokinase and the oligo (dT)2o-PEG-lipid incorporated into the cell surface, (i) BSA solution was applied to block nonspecific sites on the oligo(dT)20-incorporated substrate, (ii) Oligo(dA)20-urokinase (solid line) or oligo(dT)20-urokinase (dotted line) was applied...

Fig. 10 Confocal laser scanning microscope images of islets with urokinase (UK) immobilized on the membrane. The green fluorescence indicates positive immunostaining for UK. (a) Islets were modified with oligo(dT)2o-PEG-lipid (C16) or (b) oligo(dT)2o-PEG-lipid (C18) then, oligo (dA)2o-UK was added to the media, (c) Unmodified islets with (left) and without (right) oligo (dT)20-PEG-lipids added to the solution. Insets. Bright field images. Scale bars 100 pm...

Excitation spectra of D API and Hoechst 3 3 342 are too short for most of the lasers and mirrors that are supplied with commercially available laser scanning microscopes, although these dyes can be imaged in conventional fluorescence microscopes with Xenon or Mercury arc discharge lamp or when using HeNe laser/UV system or multiple photon microscopy... 

BT 20 cells incubated in serum free medium for 10 hours with the vector/ DNA complexes (DQAplexes, C-DQAplexes). For control, cells were exposed to naked DNA and empty vesicles. The cells were then stained with Mitotracker Red CMXRos (Molecular Probes) for five minutes to enable the visualization of mitochondria followed by confocal fluorescence microscopic analysis on a Zeiss Meta 510 Laser Scanning Microscope. 

Fig. 23 Confocal laser scanning microscopic image of rhodamine-labeled SiP coated with PMMA brush The diameter of silica particle core is 230 nm, and the Mn of the graft polymer is 256000...

Light-matter interactions can be described via an induced polarization, i.e., the induced dipole moment per unit volume. Ultrafast laser pulses, which are used in laser scanning microscopes, have high enough intensity to induce a nonlinear polarization in various materials. For intense optical electric field E, the polarization vector P can be expanded in the power series (Boyd 1992)... 

The experiments have been performed on a setup that used the ps-OPO-based CARS system described above and a femtosecond Tiisapphire laser in conjunction with a commercial laser scanning microscope (Carl Zeiss, model LSM-510). The peripheral nerve samples were gained from C57/B6 wild-type mice. After removing the skin from the lower extremities from freshly sacrificed mice, the saphenous nerve is exposed as it runs very conveniently for excision along the saphenous vein, without too much additional fatty tissue and a favorable tissue thickness of less than 20 m. A 500- m long piece is excised and freed from additional fatty tissue as well as the collagenous nerve sheath. The myelinated nerve tissue is fixed for 3-5 hr in 4% PEA or 10% formalin and mounted on 100-pm thick coverslips that are treated with 3-aminopropyltriethoxysilane or a chromium potassium sulfate solution. After... 

Moriguchi, K., Utsumi, M., Maeda, H., Kameyama, Y., and Ohno, N. 1999. Confocal laser scanning microscopic observation and cytochrome oxidase activity in the hamster submandibular gland using microwave irradiated fixation. Proc. Scanning 27 161-162. 

Marttin, E., et al. 1997. Confocal laser scanning microscopic visualization of the transport of dextrans after nasal administration to rats Effects of absorption enhancers. Pharm Res 14 631. 

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