Cell Imaging: A Micro to Macro Adventure

by | Sep 28, 2023 | Biological Sciences | 0 comments

Bensu Sıla Çelik T., Elif Duymaz E.

Derived from the ancient Greek words micros (small) and skopein (seeing/looking), the microscope was invented in 1600 as an optical system that aims to make invisible cells and tissues visible1,2. Robert Hooke’s first observations of living structures in miniature, which he described in his Micrographia published in 1665, opened the door to a complex and fascinating world3. Lenses and microscopes designed by Antonie Van Leeuwenhoek, with their simple and sophisticated structures, contributed to the improvement of light and lens components applied in the dynamic microscope technologies of the 17th century4,5. Until today, imaging systems that provide different advantages based on phase or differential interference contrast and fluorescent labeling have been developed to eliminate resolution problems such as chromatic aberration and turbidity2,6. New research areas derived from the rapidly developing omic technologies since the Human Genome Project (HGP) have brought the need to illuminate the complex relationships in cell biology by making use of these technologies7,8. Light microscopy uses an illuminating field, fluorescent microscopy that displays images with the help of stimulating dye molecules, imaging systems such as confocal microscopy with the ability to focus on a point with a limited diffraction plane, and various microscopy methods used to examine properties such as the volumetric structures of fusion proteins, organelles and molecules in living cells9-12. 

Making a cell visible at the molecular level by microscopy often gives only the average of a population and insufficient ohmic data about cell-specific dynamics can be obtained7. Recent developments have made examining the cell in physiological conditions possible while preserving spatiotemporal resolution with super-resolution live cell imaging techniques13. On the other hand, single-cell imaging provides the opportunity to examine the differences that may occur if the growth conditions of a cell are changed in a controlled manner and to collect data14. Protein dynamics is important in studies of cell behavior. In situ, snapshots of biomolecule dynamics and data such as protein expression provide information about intracellular protein behavior. In vivo, biomolecule studies, on the other hand, use immunostaining and fixation, and fluorescent-based techniques15. This review aims to evaluate the traditional and high-resolution techniques and recent developments used for imaging living cells and their mechanics.

1. Light and Electron Microscopy

Light microscope, which forms the basis of optical microscopy based on bright field illumination, is used together with electron microscopes today due to their limited resolution16. These microscopes, which are frequently used in pathology to define tissue structure and nuclear morphology in general, help the analysis of subcellular structures such as organelles17. Fluorescence-based light microscopy has become an important option for researchers in cell studies, as it is a high-resolution method that provides imaging with specific fluorescent signal tracking in large tissue areas without the need for labeling15. The need for data that cannot be obtained by labeling has led to the use of microscopy types based on wavelengths over time. Soft X-ray tomography (SXT), which provides imaging of objects in small sizes with shorter wavelengths than an electron microscope, is a technology that provides imaging by sending concentrated photons to the sample to be examined with the help of wavelengths corresponding to the soft X-ray range (5 nm–2Å) in the electromagnetic spectrum18. 

Electron microscopy; can be studied in subgroups designed according to the example such as transmission electron microscope (TEM), scanning electron microscope (SEM), scanning transmission electron microscope (STEM), and associated light and electron microscopy (CLEM)19–21. TEM, based on the principle of passing electrons through a thin layer and magnifying the image with the help of a series of lenses, is used for detailed structural analysis studies in various disciplines from physics to biology21. The limitation of this technique is that it provides a small field of view on the x-y axis, while both beam penetration and depth of focus are limited on the z-axis22. SEM is a technology used to study surface areas and uses a high-energy electron source. It works with a mechanism that provides an image. Due to primary electrons supply energy to the sample holder that protects the sample and stimulates the secondary electrons.

On the other hand, volume scanning electron microscopy (vSEM) is a method that provides imaging with a focused electron beam scanning of the sample surface13. STEM is a system that focuses electrons on the sample through optics and captures images by scanning the optics during this process25. STEM technology enables imaging without spherical aberration in thicker sections of organisms and tissues than TEM23. The CLEM system emerged from the light and electron microscopy coalition, as studies focused on clear imaging of large samples24. The need for 3-dimensional (3D) imaging of molecules has led to the emergence of light microscopy combinations and different methods for wide study areas26-28. Examples of electron and photon source microscopy, which are frequently used today, are given in Figure 113.

Figure 1. Imaging mechanisms of ET, SXT, TEM, and vSEM microscopes13. Electron tomography is a technique that creates 2D images by digitally aligning 3D data obtained from different perspectives. Soft X-ray tomography is an imaging method that uses soft X-rays as the illumination source and collects data from different angles. Transmission electron microscopy is a method that provides images by detecting photons scattered from the sample, such as the contrast image. Volume scanning electron microscopy is a scanning electron microscope with the ability to visualize larger volumes and deeper depths of the sample. Back scattered electrons (BSE) detected by a detector provide information about the sample.

1.1. Super-Resolution Imaging

With the development of ohmic technologies, the super-resolution era began in 2000 with stimulated emission consumption microscopy (STED). The continuity of this period was ensured by the development of methods such as single-molecule localization microscopy (SMLM), stochastic optical reconstruction microscopy (STORM), and photoactivated localization microscopy (PALM)29. The super-resolution era tried to reach its goal by overcoming the limits of optical resolution and combining different microscope techniques, thus creating spatial images of micro-level structures. This approach argues that each method is a complementary feature of the other and that complementarity should be used for more efficient work30. As the basic imaging system of the period, STED is based on the principle of obtaining an image with diffraction below the diffraction threshold of the light by putting the probe-fluorophore assembly in the excited or unexcited state with the help of light. Imaging is performed with the photon flux, which is generated by the spontaneous emission generated by the stimulated emission (SE) in a few nanoseconds, between the fluorescent probe returning from its ground state to the light-excited state and then to the ground state31.

2. Scanning Probe Microscope

Scanning Probe Microscopy (SPM) is a microscopy method that can be used in cryogenic conditions to ultra-high vacuum conditions and allows to observation of the physical properties of the sample regions being scanned in real space at the atomic level. The method is derived from variations of the scanning tunneling microscope (STM) and atomic force microscopy (AFM) techniques32. SPM captures various signals by collecting the physical information on the samples. It allows for detecting and interpreting these signals at atomic and nanoscale resolutions33. Fields of view created with SPM up to 100 µm are not sufficient for metrology data; therefore, the possibility of expanding the field with the use of multiple probes is being evaluated34. On the other hand, AFM visualizes the data obtained by moving the nanoprobe needle along the substrate surface, which can provide inspection at the level of molecular bonds and can observe from micro-scale to nano-scale35,36. It is based on the principle of obtaining an image by collecting the reflection formed by the deviation of the movement of the probe on the substrate on the laser line35. In materials science, SPM is thought to play a fundamental role in the development of applications such as Kelvin probe force microscopy, scanning radiated resistance microscopy, scanning capacitance microscopy (SCM), and scanning microwave impedance microscope.37 Apart from these, scanning ion conductance microscopy (SICM), which is used in the visualization of biological interfaces, is an imaging method in which probes with ion-gated channels instead of needles are detected with a pipette and image acquisition is achieved with a temporary potential energy formation, unlike the other two methods.38,39 

3. Fluorescent Microscopy

Fluorescent imaging systems are based on the mechanism of the absorption of photon energy by indicators (fluorophores or fluorochromes) and the emission of this absorbed energy as another photon for a few nanoseconds. Since light with a short wavelength has higher energy, the photon emitted from the indicator generally has a longer wavelength than the photon incident on the indicator and carries less energy10.  The groundbreaking confocal microscopy technique, which achieves maximum resolution of a defined area by eliminating out-of-focus light, is made possible through the combination of fluorescent microscopy with a laser source and scanning mirrors10. Replacing the laser source in this method with ultrafast lasers leads to two or multiple photon excitation. Two-photon excitation is an event that occurs with the simultaneous absorption of two photons of a fluorophore, which can absorb a single photon in the ground state. Two-photon fluorescence microscopy, which provides focus according to the intensity of the photon flow, increases the contrast of the focused point by blurring other surfaces marked with fluorescence thanks to this feature. The major limitation of two-photon excitation is its sensitivity to tissue movement in live imaging40-41. Combining the fluorescent microscope with laser scanning hardware, these devices, also called laser scanning microscopes, have led to the development of fluorescent resonance energy transfer (FRET), fluorescence lifetime imaging microscopy (FLIM), and post-photobleaching fluorescence recovery (FRAP) techniques42. FRET; is a method that provides imaging by radiation-free energy transfer to a molecule in the ground state near the fluorophore in the excited state, allowing it to observe cellular processes field43; it also has a more sensitive nanometer sensitivity than other microscopy44. FLIM; provides spatial and temporal quantitative information about protein-protein interactions. In general, FLIM is used to clean and better interpret contamination in FRET data43-45. On the other hand, FRAP is a method used to measure the recovered fluorescent probe to examine protein motility and intracellular structures46.

In light plate fluorescence microscopy (LSFM), unlike confocal microscopy, real images are obtained from larger tissues by using a thin light layer instead of a laser source47. Finally, single-molecule localization microscopy (SMLM) generates images by computationally constructing a time course and identifying their trajectories from diffraction data of specific fluorescent molecules. SMLM generally provides imaging of biological structures at a molecular scale48.

4. Live Cell Imaging

Live cell imaging is an imaging method that provides a real-time examination of biological processes occurring at the cell level and allows them to be visualized. Although single-celled organisms can be studied more easily than complex organisms, there may be problems in visualizing some biological processes. In live cell imaging systems, the environment in which the sample will be examined is important49. The biggest challenge of live cell imaging is that it requires small-distance imaging. To overcome this limitation, super-resolution microscopy methods and molecular detection methods have been developed over time50. Optimization of live cell imaging systems consists of disciplinary steps. The importance of a methodological approach in obtaining more efficiency from these systems is also emphasized (Figure 2)51. In line with the industrial needs for large-scale imaging of cells, the approach of creating 3D models and conducting experiments on these models was primarily used; however, the development of optical microscopy platforms has been a cornerstone in the creation of this complex system. Devices developed by different biotechnological companies such as Cell Voyager 7000S, Opera Phenix, and IN Cell Analyzer 6000, which have 3D imaging capabilities, have carried the use of high-efficiency imaging systems beyond pharmaceutical research52.

Figure 2. Steps to Successful Data Acquisition in Biological Processes51. Cell imaging steps include successive hardware setup and experimental design stages to obtain healthy data as a result of studies.

4.1. High-Efficiency Imaging

High Throughput Scanning (HTS); is defined as an automated workflow consisting of sample processing, microscopy-based image acquisition, image processing, and statistical data analysis. Automation of the system allows the systematic examination of millions of samples at the beginning of the experiment53. HTS is primarily designed for use in drug research. This is because imaging can focus precisely on a single mechanism and many chemically synthesized molecules can be detected in a short time based on fluorescence and chemiluminescence. A typical HTS can screen as many as 10,000 compounds per day54,55. HTS, which also allows functional screening in living cells, is a very costly method; In addition, there are technical difficulties to be overcome, such as limitations in usage areas, the need for more target purification of the multiple sample plates used, and the repetitive use of the created libraries56.

4.2. Multiscale Fluorescent Imaging of Living Specimens

Modern fluorescent imaging methods allow researchers to image molecular contrast and specificity over spatio-temporal scales over other techniques57. Systems such as rotating disk confocal, epifluorescence, and total internal reflection fluorescence microscopy (TIRF), which can provide fluorescence-based live cell imaging, basically have hardware based on several variables. Hardware excitation and emission light path, aperture, objective lens, and camera features of each microscopy system in the fluorescent microscopy class are of particular importance58. Fluorescent imaging systems generally isolate molecules that separate fluorescence well by cycling chloroform between the bright and dark states. The emission of this decomposed group is localized; furthermore, fluorescence from a single molecule provides a localization with nanometer precision and well below diffraction, resulting in a super-resolution image57.

4.3. Raman Microscopy

Super-resolution techniques have created a different perspective from another microscopy when it comes to Raman microscopy or spectroscopy (RS). The structured illumination method in RS provides flexibility in optical properties such as imposition in sample selection, making it a preferred imaging system for improving spatial resolution. In addition, it has been observed that the spectral separation of layers can be visualized in complex and multicomponent samples.55 This method is based on the collection of a Raman photon from a fiber end and its detection by the spectrograph.56 Discovered in 1928, Raman scattering is a concept that refers to a frequency shift of light from a molecular and cellular sample. The Raman photon scattered from the beam incident on the samples represents the energy of the specific molecular vibrations, revealing the sample’s detailed chemical composition. The biggest difficulty of RS is scattering the mentioned Raman photon at very weak energy. RS, which can detect cancer cells without the need for an external contrast agent, can also be used to monitor the differentiation of stem cells in stem cell studies57,58.

When it comes to Raman microscopy or spectroscopy (RS), super-resolution techniques have offered a different perspective compared to other microscopies. The structured illumination method used in RS provides flexibility in optical characteristics such as sample selection, making that is a preferred imaging system for improving spatial resolution. Additionally, it has been observed that layer spectrum differentiation can be visualized in complex and multi-component samples59. This method is based on the collection of Raman photons from one end of a fiber and their detection by a spectrograph60. Raman scattering, discovered in 1928, is a concept that refers to a frequency shift of light from a molecular and cellular sample. The Raman photon scattered from the samples represents the energy of their specific molecular vibrations, revealing the detailed chemical composition of the sample. The greatest challenge of RS is the very weak energy of the Raman photon. RS can detect cancer cells without the need for external contrast agents and can also be used to image stem cell differentiation in stem cell studies61-62.

5. Biophotonics

Biophotonics is a discipline whose purpose is the study, understanding, and manipulation of biological matter, which includes the research and applications of the science of light and the orientation of its applications to technology63. High-resolution optical microscopy is a very important element in the discipline of biophotonics. Optical imaging enables the visualization of biological samples on a very large spatial scale64.

6. Cell Imaging and Bioinformatics

The data that emerged with the development of cell imaging techniques has increased the necessity of improving image quality and automation of data processing, except for imaging in biological sciences. Hypotheses built on increasingly complex biological systems can be proven with image data, which is defined as big data. Computational biology is built on two software designs, modularity, and abstraction. Modularity involves sequencing and subdividing data. Abstraction, on the other hand, simplifies the algorithmic data in each module and encourages its reuse65. Today, software, machine learning, or algorithm-based tools such as CytoPacq, DeepCell Kiosk, CellProfiler 3.0, and DeepBacs have been developed to quantitatively evaluate microscopy images66-69.

The leaps from the micro to the macro to gain knowledge about life’s physiological and pathological conditions by examining the biological systems, processes and interactions in the living world led to the invention of microscopes and making the unknown visible in the 17th century. Today, sophisticated microscopy methods have been developed to be used in various fields, from materials engineering to biological sciences. These methods have taken their place in the history of humanity as the heroes behind the scenes in the realization of technological breakthroughs with the human being themselves, the environment, and cyclical world processes. The information obtained from the unique worlds revealed through a lens is tried to be interpreted by scientists and with the help of bioinformatics tools.

Advancements in the study of biological systems, processes, and interactions, from micro to macro scales, to gain knowledge about the physiological and pathological conditions of life, were made possible by the invention of microscopes in the 17th century, which allowed the invisible to become visible. Today, a wide range of microscopy techniques, equipped with cutting-edge technology, have been developed for use in diverse fields, from materials engineering to the biological sciences. These methods have played a crucial role in the advancement of human knowledge about ourselves, our environment, and the cyclic processes of the world, as well as in technological breakthroughs. The unique worlds revealed through a lens are being interpreted by scientists and bioinformatics tools to better understand the underlying phenomena.

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