Advanced Optical Flow Cytometry

Methods and Disease Diagnoses

Valery V. Tuchin, Valery V. Tuchin

• Publisher: Wiley-VCH
• Year: 2011
• ISBN:
  9783527634293
• Edition: 1
• Topic: Ciencias biológicas
• Subtopic: Biofísica

A detailed look at the latest research in non-invasive in vivo cytometry and its applications, with particular emphasis on novel biophotonic methods, disease diagnosis, and monitoring of disease treatment at single cell level in stationary and flow conditions.
This book thus covers the spectrum ranging from fundamental interactions between light, cells, vascular tissue, and cell labeling particles, to strategies and opportunities for preclinical and clinical research. General topics include light scattering by cells, fast video microscopy, polarization, laser-scanning, fluorescence, Raman, multi-photon, photothermal, and photoacoustic methods for cellular diagnostics and monitoring of disease treatment in living organisms. Also presented are discussions of advanced methods and techniques of classical flow cytometry.

1

Perspectives in Cytometry

Anja Mittag and Attila Tárnok

1.1 Background

Cytometry is the general term for quantitative single cell analyses. Without cytometric analyses, work in modern life sciences would be unthinkable. Since its introduction, cytometry has been influencing and promoting development in biology and medicine. A high number of molecular parameters are analyzable within heterogeneous cell systems by cytometry. If the normality of a heterogeneous cell system is known, changes can be identified. Hence, biological alterations induced by malignancies, infections, and so on, are diagnosable. Such phenotypic changes allow for understanding disease-related (or induced) alterations of molecule expression patterns and hence, the functionality of the whole biological system. This interest to unravel molecular properties of single cells of healthy and diseased organisms (and to compare them) led to the development of the first cytophotometric instruments in the middle of the last century [1].

Analyses in those days were usually based on different light absorption capabilities of cell constituents of cells fixed on microscopic slides, with or without staining (e.g., Feulgen). Since these analyses were very time consumptive (5–10 min per nucleus or cytoplasm region), measurements of high cell numbers were simply not possible [2]. This technology was followed by instruments for blood cell counting with a higher throughput where cell concentrations were enumerated by counting electrical voltage pulse during cell transit [3].

Application of fluorescence dyes opened the way for obtaining more information per cell. In 1961, the first use of fluorescence for quantitation was reported [4]. Since then, development of new instruments was focused toward fluorescence analysis. In 1969, the first impulse cytophotometer (ICP-11) (Phywe GmbH, Göttingen [5]) was commercially launched where the fluorescence (resulting from mercury arc lamp excitation) of several thousand cells per second was measured by photomultiplier tubes (PMTs). Later, lasers were employed as stable light sources for excitation of fluorescence dyes. The first flow cytometer equipped with two lasers was available in 1976 [6]. Several fluorescence dyes could now be measured simultaneously. The basic principle of this technology is still applied in modern flow cytometers: cells are separated by sheath fluid, (hydrodynamically) focused, and excited by (laser) light in flow. The scattered and emitted or absorbed light is measured.

The demand for comprehensive analyses and with it the simultaneous detection of several parameters on many (thousands to millions of) individual cells in one sample led to further developments in the field of cytometric analyses. More lasers as well as detectors were included to be able to perform three- or four-color measurements (plus information of scattered light) routinely.

This was sufficient for many applications, or at least, it had to be. Detailed cellular subtyping, coexpression of specific markers, cytokine analyses of certain cell types, cell–cell interaction, and so on, are, in the majority of cases, not possible by using only four fluorescence parameters. The list of applications is long where multiparametric analyses are essential.

1.2 Basics of Cytometry

The beauty of labeling specific markers or cellular functions with fluorescence dyes lies in its multicolor approach and therewith the feasibility of simultaneous analysis of many parameters. If cells are stained with different “colors,” each single color can be distinguished from each other and multiple information can be obtained for single cells. Admittedly, differentiation of more than three colors by the eye is almost impossible, but light detectors in cytometers (PMTs or camera), in combination with appropriate bandpass filters, are able to detect wavelength ranges (i.e., specific “colors”) of interest. Discrimination of fluorescence dyes is hereby possible by defining certain wavelength ranges, suitable for specific fluorescence dyes. However, the usually very broad emission spectra of fluorescence dyes make it sometimes hard to differentiate between dyes in one detection filter owing to spectral overlap. This problem is known for fluorescence dyes such as fluorescein isothiocyanate (FITC) and phycoerythrin (PE) but can be mathematically solved by compensation, that is, “purification” of specific fluorescence from unwanted signals. Another novel possibility to overcome the problem of spectral overlap is multispectral analyses (known from microscopy) although it is rarely used in flow cytometry (FCM) [7].

In general, there are two different types of cytometric analyses named by the analytical technique: FCM and slide-based cytometry (SBC). As previously mentioned, cytometry has its roots in the analysis of cells on a slide. Owing to the higher throughput, development moved to the FCM, although now, with higher computing and storage capacity of workstations, cytometry by microscopy has been revitalized. Nevertheless, both methods are quite similar and identical in many details.

1.2.1 Flow Cytometry

It is apparent from the name cytometry that cells are analyzed in flow. Generally, cells in suspension are sucked or pressed into the cytometer by overpressure or mechanical pumps. Covered with sheath fluid, cells are separated (like pearls on a string) and move actively to the place of analysis. Lasers (or also light emitting diodes nowadays) excite the cell (i.e., the fluorescence dye on it) and the emitted fluorescence is detected by PMTs. On the basis of specific characteristics (mainly fluorescence of a certain label but also light scattering properties), separation of wanted cell types and its concentration and purification can be accomplished. However, fluorescence-activated cell sorting (FACS) is necessary for that, which can be time consumptive. In “normal” FCM, the sample is usually lost after analysis. Up to 50 000 cells can be measured per second, although the normal throughput is usually around 1000−10 000 cells per second.

Fluorescence information of the cells can be displayed as histograms and dotplots. Clever experimental setup (marker selection, fluorescence combination) and smart gating strategies allow for extraction of multiple information out of a three- or four-color staining. Nevertheless, detailed subtyping or functional information (e.g., activation) of specific cellular subtypes can hardly be obtained by such low-color analyses [8].

Although the main principle in FCM has not dramatically changed since its beginning, there are of course some new developments besides increasing number of detectors and lasers. There is not only hydrodynamic focusing of cells (with the need for utilization of sheath fluid) but also focusing derived from acoustic radiation pressure forces [9] or the utilization of photodetectors for sensing the position of particles in the sample stream without sheath fluid [10]. Without sheath fluid (but with a unique flow cell design), even usage of FCM in space is conceivable [11]. Another development is the implementation of imaging in FCM. There are flow cytometers available that are able to capture images of analyzed cells in flow for morphological analysis [12].

Cellular analyses in FCM, however, are restricted to cell suspensions. Solid tissue or adherent cells cannot be analyzed, that is, not without prior trypsinization or disintegration of tissue. For these types of specimens, SBC was developed.

1.2.2 Slide-Based Cytometry

There are two major types of SBC systems: camera-based detection in combination with lamp illumination (e.g., [13]) and laser excitation and fluorescence detection via PMTs (e.g., [14]). However, mixed systems, for example, lasers and camera, are available, too. No matter which modality is used for excitation and detection, the core of these instruments is a fluorescence microscope. But that does not mean that every fluorescence microscope is capable of cytometric analysis. Cytometric analysis means quantitative analysis of the whole cell, that is, it requires optics with a relatively low numerical aperture. Analysis of single slices of a cell, as, for example, in confocal microscopy, is not cytometric. Moreover, analyses using microscopes with lamps or diodes as excitation source and no corrections (optical or software solutions) for light excitation intensity, that is, stability of the excitation light, are also not cytometric. Owing to unstable excitation intensity, one cannot be certain that the resulting fluorescence intensity of cells in different fields of view (or different samples, irrespective of the same acquisition setups) will provide the same fluorescence intensity. Qualitative statements about existing fluorescences are possible but no quantitative conclusion can be made about cell activation or other marker expression of cells. Prerequisites for cytometric analyses with microscopes are stable excitation power, even illumination of the sample, and a steady and sensitive detection of the emitted fluorescence.

Even though cytometric analyses were slide based at the beginning and the modern concept of SBC was presented in the 1980s, the first type of such instruments, the laser scanning cytometer (LSC), became commercially available a decade later [15]. The reason for this was probably the time needed for image analyses in the past. However, higher processing power and storage capacities of modern computers promoted development in this field.

Unlike FCM, samples in SBC analyses are fixed on a slide or plate. Although mainly developed for tissue analysis, LSC was used for many different applications, for example, cell cycle studies [16–18], apoptosis [19, 20], immunophenotyping [21–24], tumor analysis in solid tissue [25], fine-needle aspirate biopsies [26], circulating tumor cell analysis [27], stem cell analysis [28, 29], or study of the effects of drugs [30, 31].

The principle of LSC analysis is comparable to FCM. Fluorescence dyes on (or in) cells are excited by laser light and the emitted fluorescence is split into certain wavelength bands by optical filters and detected by PMTs. The deviation from FCM is that cells remain on the slide and can be further analyzed or even cultured.

LSC allows for studying growth and the variety of expression of specific markers during development of cells in their “natural” environment [31]. Possible effects of cell preparation, for example, stress or activation caused by detachment of cells from the surface (like for FCM analyses), can be avoided. Moreover, interactions between single cells can hardly be observed on detached cells. This applies for cell cultures as well as tissue sections. Another advantage is that cells can be traced and analyzed during culturing [29].

1.3 Cytomics

Since the complexity of biological systems is very high, a multiplicity of different information from cells, their interaction, and triggered reactions (e.g., by external stimuli or diseases) is necessary to understand such systems. For this purpose, different concepts were and still are under development to get a better insight into biological processes in organisms. Cytomics is one of these concepts. Its aim is to characterize single cells in cell systems and to unravel the interactions of cells within these systems [32]. Another concept is systems biology. The aim of systems biology is similar to that of cytomics, but it focuses more on understanding intracellular behavior, that is, the interaction of single cellular constituents such as genes, proteins, metabolites, and organelles and in silico modeling [33]. Interconnection of different analyses is very important for this purpose, that is, to obtain all needed information and combine them appropriately. In contrast to other concepts like genomics (characterization of genome [34]), proteomics (analysis of proteome [35]), lipidomics (cellular lipid constituents [36]), or other -omics, where only certain components of cells are in the focus of interest, cytomics and systems biology focus on interaction of cells and cellular constituents.

Always, biological conditions are the result of the interaction of all components of a complex system. Therefore, such a system must be analyzed as a unit to unravel its secrets. For example, different developmental stages of an organism have the same genome but are different (also phenotypically) in their protein composition [37]. Cytomics and systems biology start there and go even further. Not only single components are under investigation but also the relations and interactions between different components. Therewith, changes in cell systems can be understood – from work flows within the cell (systems biology) to interactions of the whole system (cytomics). If these actions are known, alterations (even before clinical manifestation) can be classified and can lead to predictive and preventive individualized medicine [38, 39]. Cells are the elementary building units of an organism and hence, their analysis is the easiest way to identify diseases or reasons for diseases. Alterations to healthy conditions can be found by differential screening, that is, examining a multiplicity of cell types for phenotype, activation, or cytokine production (multiparameter analyses), and extracting important and relevant cell types and marker combinations for a further diagnostic panel. However, it is clear that the mass of information obtained from multiparametric cytometry must be analyzed appropriately to find causal connections. Bioinformatics tools, that is, algorithms for cluster analyses, can be applied [40–42].

1.4 Cytometry—State of the Art

Routine are still fluorescence analyses with a relat...