**Meet the editor**

Dr. Stefan G. Stanciu received a PhD degree in Electronics and Telecommunications from the University Politehnica of Bucharest (UPB) in 2011. Currently, he is conducting his research activities as Scientific Researcher at the Center for Microscopy-Microanalysis and Information Processing of UPB. His main research interests revolve around scanning laser and scanning

probe microscopies and connected image processing and computer vision topics. Stefan's current research agenda focuses not only on optical characterization of cells, tissues and biomaterials using combined biophotonics approaches but also on the design and development of novel image classification frameworks for automated disease diagnostics.

## Contents

### **Preface XI**


Chapter 7 **Skin Wound Healing Revealed by Multimodal Optical Microscopies 151** Gitanjal Deka, Shi‐Wei Chu and Fu‐Jen Kao


Chapter 9 **Microscopic Investigations on Woody Biomass as Treated with Ionic Liquids 203** Toru Kanbayashi and Hisashi Miyafuji


## Preface

Chapter 8 **Automated Identification and Measurement of Haematopoietic Stem Cells in 3D Intravital Microscopy Data 181**

Chapter 9 **Microscopic Investigations on Woody Biomass as Treated with**

Alberto Casu, Elisa Sogne, Alessandro Genovese, Cristiano Di Benedetto, Sergio Lentijo Mozo, Efisio Zuddas, Francesca Pagliari

Chapter 11 **In Situ Transmission Electron Microscopy Studies in Gas/Liquid**

Chapter 12 **Advanced Scanning Tunneling Microscopy for Nanoscale Analysis of Semiconductor Devices 299** Leonid Bolotov and Toshihiko Kanayama

Chapter 13 **Electron Orbital Contribution in Distance‐Dependent STM**

Chapter 14 **Wavefunction Analysis of STM Image: Surface Reconstruction**

Chapter 15 **Application of Scanning Acoustic Microscopy to Pathological**

Chapter 16 **Applying High-Frequency Ultrasound to Examine Structures and Physical Properties of Cells and Tissues 405**

Hirokazu Sakamoto, Eiichi Mori, Hideyuki Arimoto, Keiichiro Namai, Hiroyuki Tahara, Toshio Naito, Taka-aki Hiramatsu, Hideki Yamochi

**of Organic Charge Transfer Salts 355**

Reema Adel Khorshed and Cristina Lo Celso

Toru Kanbayashi and Hisashi Miyafuji

Chapter 10 **The New Youth of the In Situ Transmission Electron**

**Ionic Liquids 203**

**VI** Contents

**Section 2 Non-Optical Microscopy 221**

**Microscopy 223**

and Andrea Falqui

**Environment 267** Fan Wu and Nan Yao

**Experiments 329** Alexander N. Chaika

and Kenji Mizoguchi

**Diagnosis 381** Katsutoshi Miura

Frank Winterroth

The word "microscopy" has its origins in the Greek language where the term "mikro" means "small" or "little", while "skopein" means "to look at,", so literally "microscope" means *an instrument for looking at small things*. So why the interest in such instruments? That's because *humans* have a deeply *curious* nature, and an infinity of questions that cross their minds cannot be answered without looking at small objects and scenes that are impos‐ sible to be seen with the naked eye. Although the underlying mechanisms of curiosity are not yet well understood, no one can argue that curiosity is equivalent to the desire to learn and to gain knowledge. This desire made Zacharias Jansen and his father, Hans, two Dutch spectacle makers, to be thrilled to see in the 1590s that by placing several lenses in a tube, an enlarged view of an object near its end could be observed, much larger than it was possible with any simple magnifying glass. Not much later, in the seventeenth century, the English‐ man Robert Hooke was constructing the first microscope and using it to see and describe biological organisms. His book *Micrographia* , published in 1665, was the first illustrated vol‐ ume on microscopy containing detailed accounts of 60 "observations" of objects examined microscopically. *Micrographia's* illustrations of a world were not accessible to the human eyes before created sensation, taking its readers by storm. For example, Samuel Pepys, a Fel‐ low of the Royal Society, was writing in his diary: "Before I went to bed, I sat up till 2'o clock in my chamber, reading of Mr. Hookes Microscopical Observations, the most ingeni‐ ous book that I ever read in my life." It is in *Micrographia* where the term "cell" was first introduced, a term that is currently used to describe the basic structural and functional bio‐ logical unit of living organisms, the smallest unit of life that can replicate independently. Looking at thin cork slices, Hooke remarked structures resembling to pores, which to him looked similar to the small room in which a monk inhabited, the cellula, thus deriving the name: ". . . I could exceedingly plainly perceive it to be all perforated and porous, much like a Honey-comb, but that the pores of it were not regular. . . . these pores, or cells, . . . were indeed the first microscopical pores I ever saw, and perhaps, that were ever seen, for I had not met with any Writer or Person, that had made any mention of them before this. . .." These structures that puzzled Hooke were the dead cells of cork; although Hooke himself was going to observe as well living cells later, it was someone else who was to witness for the first time a live cell under a microscope, Antonie van Leeuwenhoek. The Royal Society in London was releasing a first letter from this self-educated Dutch scientific explorer 8 years later after *Micrographia* had been published. This letter, entitled "A specimen of some Observations made by a Microscope", contrived by M. Leewenhoeck in Holland, lately com‐ municated by Dr. Regnerus de Graaf", presented microscopic observations on mold, bees, and lice. Further letters followed in which he provided his findings on different subjects us‐ ing microscopes developed by him, the details of most of which he refused to reveal, prefer‐ ring instead to provide his interpretations of the imaged scenes. Many of his letters dealt

with the description of specific forms of microorganisms, which he referred to as "animal‐ cules." These included protozoa and other unicellular organisms, like bacteria. Some of Leeuwenhoek's initial findings were met with both skepticism and open ridicule, but this was until Hooke was to return to his microscopes, which he had given up because of eye strain, and verify Leeuwenhoek's observations and confirm his findings. Leeuwenhoek was also the first to find and describe in his letters the sperm cells of animals and humans and to see that the fertilization process requires the sperm cell to enter the egg cell, which put an end to previous theories of spontaneous generation that revolved around the idea that cer‐ tain forms of life such as fleas could arise from inanimate matter. Antonie van Leeuwenhoek was elected to the Royal Society in February 1680, and although he considered this to be a high honor, he did not attend the induction ceremony in London and never attended the meetings of the Royal Society. By the time of his death in 1723, Leeuwenhoek had written more than 550 letters to different scientific institutions, of which around 200 letters had been published by the Royal Society. Ever since those times, microscopes represent tools of the utmost importance for a wide range of disciplines. Without them, it would have been im‐ possible to stand where we stand today in terms of understanding the structure and func‐ tions of organelles and cells, tissue composition and metabolism, or the causes behind various pathologies and their progression. Our knowledge on basic and advanced materials is also intimately intertwined to the realm of microscopy, and progress in key fields of mi‐ cro- and nanotechnologies critically depends on high-resolution imaging systems. While Hooke and Leeuwenhoek were placing efforts on looking at small things with microscopes that relied on optical magnification, at this time a wide variety of imaging systems are avail‐ able, relying on various contrast mechanisms. Light and optical magnification remain fun‐ damental for the microscopy realm, but "looking" at small things is now possible also by using nanostructured probes that are scanned across a sample's surface to assess its topogra‐ phy or sense various other properties, by using beams of accelerated electrons to interact with a sample of interest and provide information on its structure, by exploiting sound in‐ teraction with matter, and by many other approaches. This volume includes 16 chapters that address highly significant scientific subjects from diverse areas of microscopy and analysis. Nine of these chapters deal with optical microscopy topics, while the remaining seven refer to nonoptical microscopy subjects. The authors present in this volume their work or review recent trends, concepts, and applications, in a manner that is accessible to quite a broad readership audience from both within and outside their specialist area. I am confident that this volume will be of great value not only to those actively involved in the addressed fields but also to those with passive but constant interest in these scientific areas and to those who will have their first encounter with microscopy and analysis when reading the contained chapters. In the end, I would like to express my deepest thanks to each of the authors for his or her fine contributions to this project.

#### **Stefan G. Stanciu, PhD**

Center for Microscopy-Microanalysis and Information Processing University Politehnica of Bucharest Romania

**Section 1**

**Optical Microscopy**

with the description of specific forms of microorganisms, which he referred to as "animal‐ cules." These included protozoa and other unicellular organisms, like bacteria. Some of Leeuwenhoek's initial findings were met with both skepticism and open ridicule, but this was until Hooke was to return to his microscopes, which he had given up because of eye strain, and verify Leeuwenhoek's observations and confirm his findings. Leeuwenhoek was also the first to find and describe in his letters the sperm cells of animals and humans and to see that the fertilization process requires the sperm cell to enter the egg cell, which put an end to previous theories of spontaneous generation that revolved around the idea that cer‐ tain forms of life such as fleas could arise from inanimate matter. Antonie van Leeuwenhoek was elected to the Royal Society in February 1680, and although he considered this to be a high honor, he did not attend the induction ceremony in London and never attended the meetings of the Royal Society. By the time of his death in 1723, Leeuwenhoek had written more than 550 letters to different scientific institutions, of which around 200 letters had been published by the Royal Society. Ever since those times, microscopes represent tools of the utmost importance for a wide range of disciplines. Without them, it would have been im‐ possible to stand where we stand today in terms of understanding the structure and func‐ tions of organelles and cells, tissue composition and metabolism, or the causes behind various pathologies and their progression. Our knowledge on basic and advanced materials is also intimately intertwined to the realm of microscopy, and progress in key fields of mi‐ cro- and nanotechnologies critically depends on high-resolution imaging systems. While Hooke and Leeuwenhoek were placing efforts on looking at small things with microscopes that relied on optical magnification, at this time a wide variety of imaging systems are avail‐ able, relying on various contrast mechanisms. Light and optical magnification remain fun‐ damental for the microscopy realm, but "looking" at small things is now possible also by using nanostructured probes that are scanned across a sample's surface to assess its topogra‐ phy or sense various other properties, by using beams of accelerated electrons to interact with a sample of interest and provide information on its structure, by exploiting sound in‐ teraction with matter, and by many other approaches. This volume includes 16 chapters that address highly significant scientific subjects from diverse areas of microscopy and analysis. Nine of these chapters deal with optical microscopy topics, while the remaining seven refer to nonoptical microscopy subjects. The authors present in this volume their work or review recent trends, concepts, and applications, in a manner that is accessible to quite a broad readership audience from both within and outside their specialist area. I am confident that this volume will be of great value not only to those actively involved in the addressed fields but also to those with passive but constant interest in these scientific areas and to those who will have their first encounter with microscopy and analysis when reading the contained chapters. In the end, I would like to express my deepest thanks to each of the authors for his

**Stefan G. Stanciu, PhD**

Romania

University Politehnica of Bucharest

Center for Microscopy-Microanalysis and Information Processing

or her fine contributions to this project.

VIII Preface
