Confocal alignment

Bouchard, M.B., et al. Swept confocally-aligned planar excitation (SCAPE) microscopy for high-speed volumetric imaging of behaving organisms. Nat. Photon. 9(2), 113-119(2015)... 

Homo-FRET is a useful tool to study the interactions in living cells that can be detected by the decrease in anisotropy [106, 107]. Since commonly the donor and acceptor dipoles are not perfectly aligned in space, the energy transfer results in depolarization of acceptor emission. Imaging in polarized light can be provided both in confocal and time-resolved microscopies. However, a decrease of steady-state anisotropy can be observed not only due to homo-FRET, but also due to rotation of the fluorescence emitter. The only possibility of discriminating them in an unknown system is to use the variation of excitation wavelength and apply the... 

In terms of beam delivery, the DLW method is based on optical microscopy, confocal microscopy [4,6,13] and laser tweezers [14] (for reviews on laser tweezers see [ 15,16]). These techniques allow for a high spatial 3D resolution of a tightly focused laser beam with optical exposure of micrometric-sized volumes via linear and nonlinear absorption. In addition, mechanical and thermal forces can be exerted upon objects as small as 10 nm molecular dipolar alignment can be controlled by polarization of light in volumes of with submicrometric cross-sections. This circumstance widens the field of applications for laser nano- and microfabrication in liquid and solid materials [17-22]. 

Zucker RM and Lerner J Wavelength and Alignment tests for Confocal Imaging systems Microscopy Research and Technique. 68 5 307-319 (2005). 

Figure 14.5 (a-b) SEM images of stripe patterns widi 4 p and 0.6 p width, respectively, realized by aligning 200-nm-wide triangular nanoprisms by e-beam lithography (c-d) Confocal microscopy images of the NC / PMMA blend film deposited onto the same arrays. 

Fig. 8 Left-. C2C12 cells were seeded on aligned and randomly oriented fibers and on a glass control. Confocal images taken at 30 min, 60 min, 2 h, 5 h, and 24 h after seeding are shown, with increasing time from top to bottom. Actin is stained green and nuclei are stained blue. Scale bar. 25 pm. Right Quantification of cell aspect ratio is shown for all time points. All data sets are significantly different from each other except those indicated by NS [52]...

IRM can be performed by either epi-illumination or confocal laser imaging. In this chapter, IRM imaging is performed by standard epi-illumination due to the need for simultaneous TIRF imaging which requires a total internal reflection of the laser beam. A proper alignment of the epi-illumination is critical for IRM imaging. 

The confocal epifluorescent detection scheme we use is common among many who use this detection mode and features a cube-and-rail assembly system, specifically the microbench system from LINOS Photonics (Milford, MA) on an optical breadboard to maintain proper alignment of components (Figure 45.13). The excitation source is a multiline argon ion gas laser (model Reliant 150 m. Laser Physics, West Jordan, UT) that features user-selectable wavelengths (457,488, and 514 nm)... 

Optical Alignment The most serious problem, especially for studying the electrochemical interface of two condensed phases, is detection sensitivity. Many problems are caused by the optical alignment related to the spectro-electrochemical cell and sample. Since the confocal microprobe Raman system has much higher sensitivity than other Raman systems, it is used most frequently for spectroelectrochemical studies. This will be taken as an example for the detailed discussion that follows. 

Without making careful and correct optical alignment of the electrochemical cell, it is not possible to take advantage of the confocal microscope and the surface Raman signal may even not be detectable. For example, the solution layer of the electrochemical system could dramatically affect the collection efficiency of the microscope and hence the detection sensitivity. An investigation of the effect of the thickness... 

To fully integrate the confocal concept with portable, stand-alone micro-/nanodevices, inexpensive yet functionally equivalent miniature microscopes are required. Diode lasers, microlenses, pinhole structures, and PMT detectors must be reliably fabricated then aligned... 

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