Multi-Modality Microscopy

The following sections are included:

• Preface

• Contents

The following sections are included:

• Light Microscopy

• Image Acquisition

• Data Correction

• Video Enhanced Contrast

• Image Processing

• Deconvolution

• Three-Dimensional Rendering

• References

The following sections are included:

• Introduction

• The Problem of Spheres

• 'Tube' Length and Magnification

• What to Do?

• Intermediate Summary

• Detecting Spherical Aberration

• How Do You Correct Spherical Aberration?

• Upon Reflection

• Conclusion

• References

The following sections are included:

• Introduction

• Aberration Introduced by Refractive Index Mismatch

• Compensation by Alteration of Immersion Medium Refractive Index

• Validity of the Debye Approximation in the Presence of Spherical Aberration

• Conclusion

• Acknowledgements

• References

As permitted by the size limitations of this publication we will survey and describe a selection of image restoration algorithms. The main emphasis lies on the iterative group as they are based on rigorous mathematical foundations and provide for restorations with limited data. Common topics, like finding the optimal regularization parameter, the influence of data discretisation and the significance of an accurate point spread function (PSF) are dealt with accordingly.

To control image quality for iterative deconvolution methods, an image quality measure is described. It is based on information theory. The method allows for a more accurate and objective description of the image quality in digital microscopy than previously possible, since it takes into account the signal distribution, the transfer characteristic of the system and an estimate of the noise. Computer simulations of fluorescent beads serve as an example.

We describe a simple modification to a confocal microscope in order to introduce additional quantitative image contrast modes. In particular quantitative information about specimen birefringence, dichroism and orientation may be extracted without compromise of image acquisition speed. The basis of the method is to illuminate the specimen with circularly polarised light and to detect the image signal through a rotating analyser. The act of rotating the analyser is to introduce a time dependent modulation onto the image signal from which information about the state of polarisation may be extracted using lock-in detection techniques. A novel electro-optic implementation of a rotating analyser is described.

The following sections are included:

• Introduction

• Materials and Methods

• Results and Discussion

• Summary

• Acknowledgements

• References

An innovative microscope system, named Morphinscope, that integrates the function of a confocal microscope, a photon tunneling microscope, a laser based phase-shifting interferometry microscope, and an ellipsometer was developed to facilitate the measurement of inhomogeneous surface profile. Design, configurations, and experimental data of this newly developed instrument were all detailed.

A novel Zeeman laser scanning confocal microscope (ZLSCM) is proposed. It has the same configuration as the conventional laser scanning confocal microscope (LSCM) in which a Zeeman laser in conjunction with a Glan–Thompson analyzer are used. In our system, the analyzer and the band pass filter which play the roles of a polarization gate and a coherence gate enhance the collection efficiency of the weak scattering photons and suppress the multiple scattering photons simultaneously. Comparing with the conventional LSCM, the improvement on the depth resolution of ZLSCM in a scattering medium is discussed and demonstrated experimentally.

The following sections are included:

• Introduction

• Materials and Methods

• Results

• Discussion

• Summary

• Acknowledgements

• References

Three-dimensional (3-D) optical memory with photorefractive materials is discussed for ultra-high density/capacity memory exceeding the classical limit of a conventional optical recording system. Bit data are recorded as highly localized refractive index variation in 3-D volume using a focused laser beam. We show recording and reading results using various recording materials and optical configurations. A multi-structured optical recording medium using a photoisomerization polymer and a transparent film has been developed for reflection confocal reading. Two-photon recording is also demonstrated.

Single 2,5-dioctyloxy p–phenylene vinylene (DOO–PPV) molecules embedded in a polystyrene matrix are located and observed by scanning confocal microscopy. The time-dependent fluorescence of DOO–PPV single molecule shows the multiple-step bleaching. The fluorescence intensity from single molecules during excitation with rotating polarized light is also observed. The fluorescence variation follows the excitation polarization with a DC background observable in some molecules. These experiments indicates that the single polymers should be described as an assembly of semi-independent chromophores on a rigid backbone rather than as a single quantum system. Variations in the Huang-Rhys factor at a single polymer level are also observed.

The following sections are included:

• Introduction

• Materials and Methods

• Results and Discussion

• Conclusion

• Acknowledgements

• References

The following sections are included:

• Introduction

• Experimental Results

• Conclusion

• Acknowledgements

• References

The following sections are included:

• Introduction

• Natural Spheroids or Engineered Cell Aggregates

• Micro-capsules

• Porous Scaffold-based 3-D Tissue Constructs

• Micro-Fluidic Channels

• Three-Dimensional Tissue Slices

• Conclusion and Future Work

• References

The following sections are included:

• Appendices

• Index