9.1 Introduction

Fluorescence spectroscopy and microscopy have been used extensively in diverse areas of both scientific research and industrial applications. Particularly, fluorescence microscopy is one of the most sought-after imaging techniques in biological research field. With the advent of many novel technologies, method of fluorescence imaging has grown many folds. Our ability to visualize and resolve samples at different length scales using fluorescence imaging has improved significantly with a wide variety of microscopic techniques that are currently available. We have now access to various kinds of microscopes starting from basic epi-fluorescence microscope to highly advanced super resolution imaging techniques like photoactivated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), etc. With such superresolution microscopic techniques, we now have the ability to detect and resolve the signals at the level of individual molecules. Overall fluorescence microscopic techniques have contributed immensely to the advancement of many biological research fields.

This chapter aims to give a brief overview of a basic fluorescence microscope. First, a brief introduction to the phenomena of fluorescence is given, and major developments that have happened in this field have been highlighted. A brief account of various fluorescent molecules is discussed subsequently. The principle of fluorescence microscopy is also discussed, especially the epi-fluorescence microscopy. Finally, we give a brief overview of some advanced imaging techniques.

9.2 Phenomena of Fluorescence

Fluorescence is a manifestation of light–matter interaction. Transient electronic excitation due to the incident electromagnetic radiation leads to emission of photons of lower energy than that of the incident photon. This forms the basis of fluorescence. In particular, electrons can be excited from ground state to excited state with light of a specific wavelength. Typically, the excitation happens to a higher vibrational level of the electronic state. It quickly relaxes to the lowest vibrational level through a process known as internal conversion. Subsequently, it returns to the ground state with the emission of photon. Since the emission energy is lower than the excitation energy, the emission happens at a higher wavelength. Hence, for fluorescence emission, it is always the case that the excitation wavelength (𝛌ex) < emission wavelength (𝛌em). A more detailed description of the phenomena is explained in the following paragraph. Electronic excitation can also lead to other phenomena known as phosphorescence. Relaxation from a singlet excited state leads to fluorescence, whereas for some cases, the excited state can be a triplet state. Electrons in triplet state have the same spin orientation as that of the ground state. Since the transition from a triplet state to the ground state is forbidden, the rate of transition is slow ranging from milliseconds to seconds or even higher. A phosphorescence molecule upon excitation can glow for a long duration. This chapter mainly discusses the fluorescence phenomena.

The absorption and emission processes in fluorescent molecules are usually described with the help of Jablonski diagram named after Professor Alexander Jablonski.

A typical representation of Jablonski diagram is given in Fig. 9.1. The ground state, the first, and second excited states are shown as S0, S1, and S2 respectively. These electronic energy levels consist of several closely spaced vibrational levels. According to the Frank–Condon principle, the transitions between the electronic energy levels happen in a very short time, and therefore, the displacement of nucleus can be safely neglected. The typical transition time between the electronic energy levels is of the order of 10−15 s. Hence in Jablonski diagram, the transition between the levels is represented by vertical lines with arrows signifying the direction of transition. Upon the absorption of light of suitable wavelength, the molecule gets excited to a higher electronic energy level. Usually, the transitions happen to a higher vibrational level (Sn*) of S1 or S2. What follows this process is a rapid relaxation of the molecule to the lowest vibrational level of S1. This relaxation process (Sn*➔ Sn) is known as internal conversion. The typical time scale of this relaxation is of the order of 10−12 s. Subsequently, the molecule returns to the ground state S0 with the emission of a photon. One interesting feature is that the transition to ground state S0 typically occurs to the higher vibrational level of the ground state. Subsequently, the molecule relaxes to the thermal equilibrium state with a similar time scale of 10−12 s. A summary of different radiative and nonradiative processes associated with fluorescence and phosphorescence along with their typical timescales is given in Table 9.1.

Fig. 9.1
figure 1

Typical representation of Jablonski diagram showing the different electronic excitation states. Transition probabilities for fluorescence are very high, whereas for phosphorescence, transitions are forbidden, and hence, the rate is much slower

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Table 9.1 Radiative and nonradiative processes and their typical time scales
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The important point to note here is that both the ground state (S0) and the excited states (S1 and S2) are singlet states, that is, the spins are paired. Therefore, the transitions between these states are allowed. However, in certain cases, a spin conversion can happen in the S1 state, which may lead to the molecule being in the first triplet state designated by T in the above diagram (Fig. 9.1). In triplet state, the spin orientations are parallel in both ground and excited state, and therefore, transition between such states (singlet➔triplet or triplet➔ singlet) is forbidden. This results in a very low transition rate or a relatively larger transition time scale of the order of milliseconds to seconds. The spin configurations in singlet and triplet states are shown in Fig. 9.2.

Fig. 9.2
figure 2

Ground state and excited state spins in singlet and triplet configuration

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Out of all the processes, only fluorescence and phosphorescence result in the emission of a photon; hence, these processes are known as radiative processes. All other processes are nonradiative transitions.

Another interesting point regarding the transition involving fluorescence is that the emission spectrum is very similar to the absorption spectrum only differing in the peak positions. Closely spaced vibrational energy levels and thermal motion result in a spectrally broad absorption and emission band. A typical absorption and emission spectrum of fluorescein molecule is given in Fig. 9.3.

Fig. 9.3
figure 3

The excitation and emission spectrum of Fluorescein molecule. The mean excitation peak is at 492 nm, whereas the mean emission peak lies at 520 nm. (Spectrum plotted with data collected from www.fpbase.org)

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9.3 Major Developments

Although many observations related to the phenomena of fluorescence were reported before, the significant observation of fluorescence in a molecule was done by Sir John Fredrik William Herschel in 1845 [1]. He showed that quinine solution which is otherwise colorless and transparent exhibited a blue color when irradiated with sunlight. As we know now, the ultraviolet part of the sunlight excites the quinine molecule to emit blue light. Subsequently in 1852, Sir G. G. Stokes using a very simple experimental set-up like prism and stained glass as filter described Herschel’s observation in details [2]. He showed that the ultraviolet light is only responsible for the fluorescence of quinine. This observation also led him to proclaim that fluorescence emission has longer wavelength than that of the excitation wavelength. This shift is now known as the “Stokes shift”. In the twentieth century, many important contributions to the field of fluorescence came from Nicolas and Merrit (first excitation spectrum of a dye molecule), Stern and Volmer (fluorescence quenching), Perrin (fluorescence polarization), Jablonski (Jablonski diagram), Forster (quantum theory of dipole–dipole interaction), and many others. In parallel, many compounds exhibiting fluorescence were also discovered. Mauveine (William Henry Perkin, 1856), fluorescein (Adolf Von Baeyer, 1871), and uranin (Paul Ehrlich, 1882) are some notable synthetic dye compounds. More detailed account of several research works in this regard can be found in many existing literature [3,4,5,6,7] (Table 9.2).

Table 9.2 Commonly used fluorescent probes and their excitation and emission wavelengths
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One of the most important discoveries happened in 1962, when Shimomura and his collogues gave evidence for green fluorescent proteins (GFP) [8, 9]. Subsequent investigation showed that GFP can be used as a fluorescent biomarker [10]. Subsequently, many other fluorescent proteins were developed with fluorescence emission spanning over entire visible spectrum. A concise list of some of the fluorescent proteins is presented in later sections (Table 9.3). In technological front, the first use of fluorescence dye in an optical microscope was demonstrated by the companies Carl Zeiss and Carl Reichert. After the development of protein labeling using green fluorescent proteins (GFP) and its variants, the fluorescence microscopy took a giant step toward the imaging of cellular molecules. With the advent of sophisticated microscopy techniques including PALM, STROM, etc., the fluorescence microscopy has grown leaps and bounds. We are now able to breach the optical resolution limit to the level of individual molecules using fluorescence techniques.

Table 9.3 Commonly used fluorescent proteins and their properties
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9.4 Fluorescent Molecules

Molecules that exhibit fluorescence are known as fluorescent probes, fluorochromes, fluorophores, or fluorescent dyes. Fluorophores can be naturally occurring (intrinsic) or synthetically prepared (extrinsic). Intrinsic fluorescence molecules include mainly aromatic amino acids, flavins, chlorophyll, and derivative of pyridoxal. Proteins containing aromatic amino acids such as tryptophan, tyrosine, and phenylalanine are generally fluorescent. Enzyme cofactors such as NADH are also highly fluorescent. As is the case, generally many molecules of interest are nonfluorescent. In such cases, these molecules can be studied by labeling them with extrinsic fluorescent probes. For example, ethidium bromide can be used to label the DNA. Similarly, DAPI and Hoechst 33342 also bind to the DNA and make it fluorescently labeled. Several techniques are available to fluorescently label the biomolecules [11]. A huge number of such compounds are known and are available today. Here, we give a brief account of the general characteristics of these fluorescent molecules and their classifications.

9.4.1 Properties of Fluorescence Emission

The fluorescence excitation and emission spectrum displays some general characteristics across many fluorescent molecules. Although some exceptions to these characteristic features are also observed, the following features are frequently observed.

  • Each fluorescent molecule exhibits unique absorption and emission spectra, depending on the molecular structure and sometimes also on their surroundings in the sample.

  • Because of the rapid internal conversion to lowest vibrational energy level of the first excited state, the emission spectrum is independent of the excitation wavelength.

  • The excitation and emission spectrum of fluorophore can overlap. As discussed previously, the difference of the energy between excitation and emission is called the Stokes shift. This shift can be measured as the difference between the excitation and emission maxima. Depending upon the fluorophore, the Stokes shift can range from a few nanometers to several hundred of nanometers. For example, DAPI (4′,6-diamidino-2-phenylindole) exhibits an excitation peak at 345 nm, whereas the emission peak is at 460 nm. The Stokes shift for DAPI is 115 nm. But for fluorescein, the Stokes shift comes out to be just 23 nm.

  • Typically, the electron returns to higher vibrational energy levels in the ground state. This is because of the fact that the probability of an electron returning to a particular vibrational level of the ground state is similar to the probability of that electron in different vibrational levels before excitation. As a consequence, the emission spectrum is typically the mirror image of the excitation spectrum for a particular fluorescent probe. This is known as the “Mirror image rule”. It should be noted that exception to this mirror rules exists in many molecules.

9.4.2 Fluorescent Proteins

As discussed, many proteins are naturally fluorescent. Green fluorescent protein (GFP) is one of well-known names of similar proteins. Many variants of the GFP have been developed by introducing mutations into the amino acid sequences. Such mutants have different excitation/emission wavelengths as well as much better photostability. Several other types of similar protein have been discovered with excitation and emission spectrum spanning through most of the visible ranges. Yellow fluorescent proteins (YFPs), red fluorescent proteins (RFPs), etc. are some of the well-known examples. Some of the widely used fluorescent proteins along with their fluorescent properties are listed in Table 9.3. More details about the fluorescent proteins can be found out from existing literatures [12,13,14,15,16,17]. The fluorescent proteins have been widely used as biomarkers especially in live cell imaging applications [18].

9.5 Principles of Fluorescence Microscopy

Fluorescence microscopes enable the imaging of various fluorescence probes inside the samples. As discussed in the previous section depending upon the visualization requirement, a variety of fluorophores can be used to make the sample or a specific part of the sample (such as some organelles in living cells) fluorescent. Such samples can then be visualized with the help of specially designed optical microscope. The first practical realization of a fluorescence microscope was achieved by the companies Carl Zeiss and Carl Reichert at the beginning of the twentieth century. Since then, the technique has seen several advances that have led to the modern fluorescence microscopes that are being used today in many research labs. Modern fluorescence microscope improved both visualizations and quantifications of fluorescent-labeled samples. The following section discusses the principle and the components of a typical fluorescence microscope.

The basic requirement of a fluorescence microscope is an excitation source that is usually a mercury lamp/metal halide lamp/laser, an optical arrangement for selecting specific excitation and emission wavelengths, sample containing a fluorescent molecule, and detector for collecting the emitted fluorescence signal. The light from the source excites the sample containing the fluorescent probes. Subsequently, the fluorescent light emitted by the sample is collected using a detector (camera/eye). This process can be achieved essentially in two kinds of instrumental arrangements. One can use two separate objectives for excitation and emission. However, majority of biological fluorescence microscopy uses a method where the excitation of fluorescent sample and collection of emission signal are achieved using the same objective. A microscope with such an arrangement is called epi-fluorescence microscope.

A simplified diagram showing the light paths for excitation and emission in a typical epi-fluorescence microscope is shown in Fig. 9.4. Briefly, a narrow band of wavelength matching the excitation spectrum of the particular dye is selected from the light source using an excitation filter and is incident upon the fluorescently labeled sample with the help of a dichroic mirror. The light is absorbed by the molecule and the fluorescence emission from the sample transmitted through the dichroic and selected using an emission filter. This emitted intensity is recorded by a detector. The excitation filter, dichroic mirror, and the emission are assembled together inside a filter cube. A brief account of the main components of an epi-fluorescence microscope is discussed in the following section.

Fig. 9.4
figure 4

Excitation and emission light path in an epi-fluorescence microscope. The excitation of fluorophore and the collection of emitted fluorescence signal

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Light source

A bright light source is often used. It can be either a mercury or xenon lamp. Metal halide lamps which give an improved life time are also used in many microscopes. If the lamp housing is not directly attached to the microscope, the light is introduced to the microscope with the help of an optical fiber or a liquid guide. Many modern microscopes are now using the bright Light Emitting Diodes (LEDs) as an alternate light source. In case of LEDs, combinations of several LEDs are needed in order to have the access to wider selections of excitation wavelengths. Lasers are also used as the excitation source in many arrangements particularly for advanced fluorescence imaging techniques such as confocal microscopy.

Filter Set

Using a combination of filters and dichroic mirror, the filter cube selects the appropriate excitation wavelength from the light source and projects it on to the sample. Similarly, it also allows the emitted light from the sample to the detector for observation/recording. The filter cube consists of three components, i.e., the excitation filter, the dichroic mirror, and the emission filter. The excitation filter selectively transmits a narrow band of wavelengths corresponding to the excitation wavelength of the specific fluorophore present in the sample. The dichroic mirror is essentially a long pass filter designed specifically to reflect and transmit at specific wavelength boundary. This is achieved by the multiple layers of dialectic film coated on the dichroic mirror.

The transmission properties of a filter cube corresponding to the fluorescein dye are shown in Fig. 9.5 .

Fig. 9.5
figure 5

Transmission characteristics of a filter cube corresponding to the fluorescein dye. Top image shows the absorption and emission spectrum of fluorescein. Bottom image shows transmittance of the corresponding excitation filter, dichroic mirror, and the emission filter. Shaded bands show the correspondence of the absorption and emission of the fluorophore to the filter characteristics

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Objective

The objective is one of the most important optical components in the microscope. As will be discussed subsequently, the resolution of the microscope is determined by the properties of the objectives numerical aperture (NA). Therefore, a proper selection of objective is highly essential to obtain improved image quality. Objects with high NA have better light-gathering ability, which improves the quality of the image. Both dry and immersion (water/oil) objectives are used in a fluorescence microscope. Immersion oil objectives provide better imaging capabilities as it provides a refractive index-matched medium between the objective and the microscope slides holding the specimen.

Detector

The fluorescence image of the sample can be directly visualized through eye. However, in order to record and store the image for future use, a detector attached with a computer is used. Furthermore, our eye can only visualize the range of wavelengths typically from 400 nm to 700 nm. However, a charge-coupled device (CCD) can in principle detect fluorescent signals from 400 nm up to 1000 nm. In majority of the epi-fluorescence microscopes, a charge-coupled device (CCD) or a complementary metal-oxide semiconductor(cMOS) camera is used for image recording. In advanced microscopic techniques such as confocal microscopy, photomultiplier tubes (PMTs) are used for detection, or Gallium-Arsenide photo detectors (GASP) are used.

9.6 Resolution

The most important parameter in optical microscopy is the resolution of the microscope. It refers to the ability of the instrument to distinguish two very closely spaced spatial points in the specimen. Because of diffraction, light from a point source when passed through an aperture forms spatially extended pattern known as airy pattern. It consists of a central bright disk also known as airy disk and a series of concentric dark and bright rings (Fig. 9.6). If two-point sources are very closely spaced, the airy disks of both the points will overlap which will limit our ability to distinguish the image as that of a two separate point sources.

Fig. 9.6
figure 6

Airy pattern formed by point sources when passed through an aperture. Left image shows the 2D image of a point source, and the right image shows the typical intensity profile of the airy pattern

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Generally accepted criteria for resolution is the Raleigh criterion which says that the two points are said to be resolved when the maximum of the airy disk of the one point lies at the first minimum of the airy pattern of the second point [19]. Accordingly, the lateral resolution of a microscope is given by the following expression.

$$ {d}_{\mathrm{lateral}}=\frac{0.61\kern0.5em \lambda }{NA} $$

where d is minimum spatial length scale that can be resolved, 𝛌 is the emission wavelength, and NA refers to the numerical aperture of the corresponding objective. As it is evident, the NA of the objective is a crucial determinant of the resolution of the microscope. It is defined as the

$$ \mathrm{NA}=n\ \mathrm{Sin}\ \alpha $$

where n is the refractive index of the immersion medium, and α is the one half of the opening angle of the given objective as shown in Fig. 9.7. Higher NA essentially means a better light collection ability for the objective lens (Fig. 9.8).

Fig. 9.7
figure 7

Rayleigh criteria for resolution

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Fig. 9.8
figure 8

The numerical aperture of objective is Sin of the half of the opening angle of the objective

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The resolution criterion discussed above is valid for imaging a lateral plane in the sample. When it comes to the resolution along the propagation of light (axial resolution), the expression is modified to

$$ {d}_{\mathrm{axial}}=\frac{2\kern0.5em \lambda }{NA^2} $$

The lateral resolution is proportional to NA of the objective, whereas the axial resolution is proportional to (NA)2. Hence, axial resolution is inferior to the lateral resolution for a particular objective and wavelength.

9.6.1 Nyquist Criterion

While recording an image in a microscope, one important criterion to consider is the sampling. An optical microscope is an analogous system which produces a continuous wave signal (i.e. the emitted fluorescence intensity from the samples through the optical components of the microscope). This signal is recorded as a digital output using a camera (CCD, CMOS, PMT, etc.). Therefore, for the digital image to be meaningful, the signal must be sampled appropriately so as to record the smallest resolvable features present in the sample. According to Shannon’s sampling theorem, an analog signal can be reconstructed appropriately if it is sampled with Nyquist criterion that is: Sampling interval must be at least twice the maximum frequency measured [20, 21]. For the case of optical microscopy, Nyquist criterion can be stated as the smallest resolvable feature in the sample (Rayleigh criterion) should be imaged by at least 2.3 pixels in the imaging detector. For example, the Nyquist criterion demands the following minimum condition for the pixel size of the detector for a particular objective magnification.

$$ \mathrm{Mag}\ \left(\mathrm{Objective}\right)\times \frac{0.61\kern0.5em \lambda }{NA}=2.3\times \mathrm{pixel}\ \mathrm{size}\ \mathrm{of}\ \mathrm{the}\ \mathrm{camera} $$

In wide-field fluorescence imaging, the detector (Camera) has fixed pixel size. Hence, we should be careful in using objective magnification so as to satisfy the Nyquist criterion. In confocal microscopy where we use the PMTs, the pixel size can be adjusted, thereby giving us the advantage of always satisfying the Nyquist criterion.

9.7 Advanced Microscopic Techniques

While a basic epi-fluorescence microscope is immensely useful in visualization and quantification of fluorescent samples, it is very much limited when it comes to the resolving features that are very small, for example, two actin filaments of a cell spaced very close to each other. From the resolution criterion discussed above, it is apparent that the theoretical resolution that can be achieved in a basic fluorescence microscope is not adequate for several imaging purpose. A simple estimation shows that for 𝛌 = 500 nm and typical NA = 1, the resolvable length scale turns out to be around 315 nm (refractive index is taken to be 1 that of air). In practice, because of optical aberrations, achieving this resolution also becomes challenging. The problem is more severe when the depth resolution along the z-direction (perpendicular to the sample plane) is considered. In a typical epi-fluorescence microscopic technique (also known as the wide-field microscopy), the fluorescence signal is collected not only from the point of interest (focal plane) but also from the out of focal plane resulting in glare. This limits the ability to control the depth of field. This limitation can be overcome by using spatial filtering techniques, which is achieved using pinholes specifically placed to get rid of the out of focus signals, thereby eliminating the glare. This essentially forms the basis of confocal microscopy. The principles of confocal microscopy are beyond the scope of this current chapter, and the readers are advised to refer to many excellent texts available in that context [22]. Similarly, other advanced fluorescent techniques have been developed which can enhance both the visualization and quantification in fluorescence signals manifolds. Foster resonance energy transfer (FRET), fluorescence lifetime imaging (FLIM), total internal reflection microscopy (TIRF), spinning disk microscopy, and structured illumination microscopy (SIM) are examples of such advanced techniques.

9.8 Application of Fluorescence Microscopy in Biological and Biophysical Research

The fluorescence microscopy has been used extensively to probe both biological and material samples. In this section, we give a brief account of major applications of fluorescence imaging techniques mainly in biological and biophysical research. In particular, fluorescence microscopy is used extensively to study the intracellular distribution, dynamics, and molecular mechanisms of several macromolecules in cells and tissues.

9.8.1 Immunofluorescence and Live Cell Imaging

The techniques of immunofluorescence first developed by Albert Coons in the 1940s permit the visualizations of many cellular components of various cell and tissues. Specifically, it uses the basic immune chemistry of antigen–antibody reaction to tag the fluorescence probe to virtually any cellular proteins [23, 24]. Briefly, the specimen (Cells/tissue) is fixed using a standard protocol and then subsequently labeled with an appropriate antibody tagged with fluorescent probe. In direct immunofluorescence, only one antibody is used (primary). It recognizes the target antigen in the specific region and binds it making. In secondary/indirect staining, two antibodies are used. The unlabeled primary antibody binds to the target molecule, and the secondary antibody tagged with a fluorophore binds to the primary antibody. Cell and tissue morphology along with the organization of specific proteins can be studied using such immunofluorescent techniques.

The major disadvantage of immunofluorescent method is that the cells/tissues need to be fixed. Therefore, the dynamical characteristics of the living system cannot be inferred directly from immunostained images. In order to get that information, live cell imaging is performed.

Fluorescence microscopy is one of the primary imaging tools to probe living cells. Depending upon the target proteins/organs, one or more fluorescent proteins are expressed in the cells. In other method, the fluorescent probe can also be added as tracer to tag a protein/organelle of interest. In either of the method, we have access to multitude of fluorescence probes to selectively image a target molecule of a living cell. One of the primary aspects that is studied using fluorescence microscopy is the dynamic intercellular organization in response to mechanical, chemical stimuli. One such example is the organization the acto-myosin network in rat embryonic fibroblast cells as a function of substrate rigidity [25].

Some fluorescent dyes can permeate a living cell, thereby giving us the ability to tag an intracellular target of interest. Whereas the elective permittivity of dyes like fluorescein has been used to study sensory neurons of Caenorhabditis elegans (Perkin 1986). Polar dyes cannot permeate a cell membrane; hence, they can be effectively used to mark the cellular integrity. Altogether, fluorescent proteins can be used as both biomarkers and biosensors, thereby revealing useful details regarding many molecular and cellular processes [26].

9.8.2 Reconstituted Lipid Membranes

Fluorescence technique has been extensively used in systems of reconstituted lipid membranes especially in the form of giant unilamellar vesicles (GUVs) and supported lipid bilayers (SLBs) [27,28,29,30]. Majority of the studies in such systems are focused on investigating the phase separation of lipid bilayer. Because of biased partitioning of fluorescently labeled lipids in different phases, it is easy to visualize the domains. Such studies have led to a better understanding of the lipid raft hypothesis. The fluorescence microscopy data have been used to construct the phase diagrams of lipid mixtures. Another aspect which is often studied is the lateral structure of lipid bilayer. Many investigations have been reported in regards to the temperature-dependent lateral structures in giant vesicles of different phospholipids, binary and ternary mixtures of lipids and sterol [30, 31]. Fluorescence microscopy has also been used to study the lipid–protein interactions. Particularly, a variety of peptides and toxins that are harmful to living cells have been investigated in vesicular membrane [32,33,34,35,36].

9.9 Conclusion and Future Perspective

Since its first realization, the fluorescence microscopy has helped tremendously in obtaining fundamental insights into the structure and functions of biological systems. In particular, the imaging ability at high resolutions has helped us understand the steady-state structure and organization of intracellular components. In addition, the live cell imaging has revealed the dynamical aspects such as mobility, turnover, and various biochemical and biomechanical responses. The development of single-molecule techniques has given us ultrafine structures beyond the optical resolution limit. Such methods are still being refined and may eventually lead us to a simultaneous imaging of ultrafast processes at ultrafine structural details.