Methanol immersion reduces spherical aberration of water dipping lenses at long wavelengths used in multi-photon laser scanning microscopy

Dipping objectives were tested for multi-photon laser scanning microscopy, since their large working distances are advantageous for thick specimens and the absence of a coverslip facilitates examination of living material. Images of fluorescent bead specimens, particularly at wavelengths greater than 850 nm showed defects consistent with spherical aberration. Substituting methanol for water as the immersion medium surrounding the beads corrected these defects and produced an increase in fluorescence signal intensity. The same immersion method was applied to two representative biological samples of fixed tissue: mouse brain labeled with FITC for tubulin and mouse gut in which the Peyer’s patches were labeled with Texas Red bilosomes. Tissue morphology was well preserved by methanol immersion of both tissues; the two-photon-excited fluorescence signal was six times higher than in water and the depth of penetration of useful imaging was doubled. No modification of the microscope was needed except the provision of a ring to retain a sufficient depth of methanol for imaging

Advanced Fluorescence Microscopy Techniques-FRAP, FLIP, FLAP, FRET and FLIM

Fluorescence microscopy provides an efficient and unique approach to study fixed and living cells because of its versatility, specificity, and high sensitivity. Fluorescence microscopes can both detect the fluorescence emitted from labeled molecules in biological samples as images or photometric data from which intensities and emission spectra can be deduced. By exploiting the characteristics of fluorescence, various techniques have been developed that enable the visualization and analysis of complex dynamic events in cells, organelles, and sub-organelle components within the biological specimen. The techniques described here are fluorescence recovery after photobleaching (FRAP), the related fluorescence loss in photobleaching (FLIP), fluorescence localization after photobleaching (FLAP), Forster or fluorescence resonance energy transfer (FRET) and the different ways how to measure FRET, such as acceptor bleaching, sensitized emission, polarization anisotropy, and fluorescence lifetime imaging microscopy (FLIM). First, a brief introduction into the mechanisms underlying fluorescence as a physical phenomenon and fluorescence, confocal, and multiphoton microscopy is given. Subsequently, these advanced microscopy techniques are introduced in more detail, with a description of how these techniques are performed, what needs to be considered, and what practical advantages they can bring to cell biological research

Multi-confocal Fluorescence Correlation Spectroscopy : experimental demonstration and potential applications for living cell measurements

We report, for the first time, a multi-confocal Fluorescence Correlation Spectroscopy (mFCS) technique which allows parallel measurements at different locations, by combining a Spatial Light Modulator (SLM), with an Electron Multiplying-CCD camera (EM-CCD). The SLM is used to produce a series of laser spots, while the pixels of the EM-CCD play the roles of virtual pinholes. The phase map addressed to the SLM is calculated by using the spherical wave approximation and makes it possible to produce several diffraction limited laser spots, either aligned or spread over the field of view. To attain fast enough imaging rates, the camera has been used in different acquisition modes, the fastest of which leads to a time resolution of 100 $\mu$s. We qualified the experimental set-up by using solutions of sulforhodamine G in glycerol and demonstrated that the observation volumes are similar to that of a standard confocal set-up. To demonstrate that our mFCS method is suitable for intracellular studies, experiments have been conducted on two stable cell lines: mouse embryonic fibroblasts expressing eGFP-actin and H1299 cells expressing the heat shock factor fusion protein HSF1-eGFP. In the first case we could recover, by analyzing the auto-correlation curves, the diffusion constant of G-actin within the cytoplasm, although we were also sensitive to the complex network of interactions with F-actin. Concerning HSF1, we could clearly observe the modifications of the number of molecules and of the HSF1 dynamics during heat shock

Multimodal wide-field two-photon excitation imaging: characterization of the technique for in vivo applications

We report fast, non-scanning, wide-field two-photon fluorescence excitation with spectral and lifetime detection for in vivo biomedical applications. We determined the optical characteristics of the technique, developed a Gaussian flat-field correction method to reduce artifacts resulting from non-uniform excitation such that contrast is enhanced, and showed that it can be used for ex vivo and in vivo cellular-level imaging. Two applications were demonstrated: (i) ex vivo measurements of beta-amyloid plaques in retinas of transgenic mice, and (ii) in vivo imaging of sulfonated gallium(III) corroles injected into tumors. We demonstrate that wide-field two photon fluorescence excitation with flat-field correction provides more penetration depth as well as better contrast and axial resolution than the corresponding one-photon wide field excitation for the same dye. Importantly, when this technique is used together with spectral and fluorescence lifetime detection modules, it offers improved discrimination between fluorescence from molecules of interest and autofluorescence, with higher sensitivity and specificity for in vivo applications

Confocal and multiphoton imaging of intracellular Ca²⁺

This chapter compares the imaging capabilities of a range of systems including multiphoton microscopy in regard to measurements of intracellular Ca<sup>2+</sup> within living cells. In particular, the excitation spectra of popular fluorescent Ca<sup>2+</sup> indicators are shown during 1P and 2P excitation. The strengths and limitations of the current indicators are discussed along with error analysis which highlights the value of matching the Ca<sup>2+</sup> affinity of the dye to a particular aspect of Ca<sup>2+</sup> signaling. Finally, the combined emission spectra of Ca<sup>2+</sup> and voltage sensitive dyes are compared to allow the choice of the optimum combination to allow simultaneous intracellular Ca<sup>2+</sup> and membrane voltage measurement

Two-photon imaging through a multimode fiber

In this work we demonstrate 3D imaging using two-photon excitation through a 20 cm long multimode optical fiber (MMF) of 350 micrometers diameter. The imaging principle is similar to single photon fluorescence through a MMF, except that a focused femtosecond pulse is delivered and scanned over the sample. In our approach, focusing and scanning through the fiber is accomplished by digital phase conjugation using mode selection by time gating with an ultra-fast reference pulse. The excited two-photon emission is collected through the same fiber. We demonstrate depth sectioning by scanning the focused pulse in a 3D volume over a sample consisting of fluorescent beads suspended in a polymer. The achieved resolution is 1 micrometer laterally and 15 micrometers axially. Scanning is performed over an 80x80 micrometers field of view. To our knowledge, this is the first demonstration of high-resolution three-dimensional imaging using two-photon fluorescence through a multimode fiber

Development of high-speed imaging techniques for C. elegans nervous system studies

We report high-speed imaging techniques for C. elegans nervous systems studies. We introduce C. elegans, the main model organism in this dissertation, and neuroscientific and biomedical studies using C. elegans involving calcium imaging, nerve regeneration, and drug screening. We review technologies including confocal microscopy and microfluidic devices used in the neuroscientific and biomedical studies We discuss development of a high-speed laser scanning confocal microscope capable of flexible control of imaging conditions, fast imaging speed, and large field-of-view. We provides the design principles used in the development of the confocal microscope including the optical, electrical, and software implementation, and the details of the confocal microscope we built based on the design principles. We present the performance characterization of the confocal microscope, then a few sample images obtained with the confocal microscope. We present development of time-lapse volumetric confocal imaging of whole animal C. elegans Ca²⁺ dynamics. We provide the design of the time-lapse volumetric confocal imaging system including a microfluidic device to accommodate the whole animal within the field-of-view of the imaging system. We examine the feasibility of the volumetric confocal imaging of a whole animal, and demonstrate imaging of the whole animal C. elegans neurons’ response to NaCl within a 630 × 150 × 25 μm³ volume at 2 Hz rate. We report a high-throughput automated imaging platform for C. elegans nerve regeneration study. We describe the design of the automated imaging platform and the automation flow, and characterizes the performance of the platform. The imaging platform can obtain high-resolution 3D confocal images of 20 animals in 10 minutes. We show sample images of C. elegans anterior lateral microtubule nerve regeneration examples acquired via the automated imaging platform. We demonstrate a planar laser activated neuronal scanning platform (PLANS), a high-throughput animal examination system for drug screening. We explain the construction of PLANS involving the optics, the microfluidic device, and the electronics. The PLANS system can scan an animal in less than 5 ms with a spatial sampling resolution of 3 μm FWHM. We show sample scanning results of a Huntington’s disease model of C. elegans. We summarize the studies discussed in this dissertation, and suggest relevant future research to follow up on the studies.Electrical and Computer Engineerin