Fluorescence microscopy is an essential tool in molecular and cellular biology. It is a technique that allows one to study and visualize the cellular structures and dynamics of tissues and organelles, and macromolecular assemblies inside the cell. It was devised in the early twentieth century by various scientists like Köhler, Lehmann, Reichert and others.
The wide utilization of fluorescent proteins since their discovery have revolutionized the applications and use of the microscope in biological studies.
A fluorescence microscope uses the property of fluorescence to generate an image. It uses a high-intensity light source that excites the fluorescent molecule that may be inherently present in the sample to be studied or may be artificially labelled with a fluorescent molecule. The fluorescent molecule is called the fluorophore which is usually present in the fluorescent dye.
Therefore, one could say that any microscope that works on the same basis to study the properties of organic or inorganic substances is a fluorescent microscope.
A fluorescence microscope is a type of optical microscope that uses fluorescence (ability of a substance to emit light on excitation) and phosphorescence (ability of a substance to continue emitting light even after the removal or withdrawal of the excitation factor). It may use these properties instead of or in addition to the properties of scattering, absorption, reflection and attenuation.
The setup for the microscope may be simple as in an epifluorescence microscope or it may have a complicated design like that of a confocal microscope. A confocal microscope uses optical sectioning to provide a better resolution of the fluorescence image.
Fluorescent substances are the substances that absorb light of a particular energy and wavelength and then emit light of a longer wavelength and lesser energy.
This phenomenon of fluorescent substances can be applied to the working of the fluorescent microscope. Fluorescent dyes (also called fluorochromes or fluorophores) are molecules that have the ability to absorb excitation light at a given wavelength, and then emit light of a comparatively longer wavelength after a delayed time interval.
In practical use, the sample is stained with a fluorescent dye and then illuminated with a blue light. The blue light (short wavelength) is absorbed by the fluorophores of the fluorescent dye, and the green light (which has longer wavelength) is emitted. This change is called the Stokes shift.
The light source that is used in fluorescent microscopy is a high intensity mercury arc lamp. The lamp emits white light when then passes through a device called an ‘exciter filter’. (as shown in the figure) This device filters the emission light to reveal the location of the fluorophores. It allows only the blue component of white light (white light comprises of coloured light of all wavelengths) to pass through and prevents the passage of light of other colors.
The dichroic mirror is used to reflect the blue light and allows the green light to pass. The angle of the mirror is fixed in such a way that the blue light is reflected towards the specimen placed below. It allows the passage of green light.
Finally, when the light reaches the ‘barrier filter’, it blocks out or removes all the remnants of the residual blue light from the specimen which may not have been ideally reflected by the dichroic mirror.
Thus, enabling the observer to perceive the glowing green portions of the specimen against the jet-black background of the dark field condenser that is used. The portions of the specimen that have not been stained remain invisible to the eye and this is how fluorescence microscopy provides a sharp image for the observation of the fine and intricate components of the sample to be studied.
The essential components of the fluorescence microscope are:
Fluorescent dyes (fluorophore): Chemical compounds that have the ability to re-emit light upon excitation. Examples include; nucleic acid stain like DAPI and Hoechst, phalloidin etc.
Light source: This is provided by a bright mercury vapor arc lamp, xenon lamp or LEDs with a dichroic excitation filter, lasers etc.
Heat filter: The lamp produces infrared rays which generate considerable heat. No other major uses of the heat filter exist.)
Exciter filter: The light undergoes cooling and passes through the exciter filter which allows the passage of the shorter waves which play a role in excitation of the fluorochrome dye coated sample on the slide and does not allow the other wavelengths to pass through.
Dichroic mirror: An accurate colour filter/mirror which selectively allows the passage of light of a particular wavelength and reflects the others.
Condenser: A dark field condenser is usually used because it provides a dark background and it is easy to detect even mild fluorescence exhibited by the sample
Barrier filter: It removes all the remnants of the exiting light and is situated in the body tube of the microscope between the objectives and the eye piece.
Identify structures in fixed and live biological samples in microbiological studies.
Used in food chemistry for the assessment of the structural organization and spatial distribution of the components of food.
Used for the study of mineral like coal and graphene oxide in minerology.
Used in the textile industry for analysis of fibre dimensions.
Research methodology: The method of conducting research, by formulating problems, finding objectives, presenting result is all the crucial steps in any research. Sources of data and population consideration, ethical values, sample determination, methods executing plays a vital role before undertaking the research proposal.
Obtaining novel opinions and developing skills
Characterizing particular character, group or condition.
Finding interlinked connections
To test hypothesis.
In biological research following types can be included:
Identifying Research problem:
The initial step of any researcher is to identify the general are of interest. There are main two steps in formulating any research
Understanding the problem
Reshape according to analytical view.
Having guidance and restoring problems already existing and engaging oneself in discussion makes it easy for identifying the research problem.
This is basically done to get a familiarity with the problem. Literature can be conceptual, empirical, etc. There are many source of literature; it could be abstracts, journals, bibliographies, conferences, academic journals, government reports, books. This helps in formulating problems. After the review one should focus on writing a synopsis.
Formulation of hypothesis:
Hypothesis is a tentative explanation made based on the available limited evidences. Formulating hypothesis enables to find the objective as well as result interpretation. Various approaches for formulating hypotheses:
Discussion with guide, and coworkers
Assessments of records and available data
Evaluating previous studies done
After the research problem is designed then the next step is to design the research. It involves choosing various components for research Study.
Selection of sample is utmost necessary for the development of any protocol or formulation. There are mainly two types of samples, which includes Nonprobability and probability.
Nonprobability sampling: subjective methods of sampling
Probability sampling: It is simple random sampling, systematic sampling, cluster sampling
The process of gathering information enabling answer stated questions, testing hypothesis and evaluating outcomes. To maintain integrity of research accurate data collection is necessary.
Proper data collected must include:
Ability to answer research questions
Ability to repeat and validate the study
Less wastage of resources
No compromises for the fulfillment of requirement
No harm to human or subject studies
To maintain integrity there are two elements which is helpful
Before data collection
During or after data collection
Standardization of protocol
Careful documentation of protocol.
Process of applying statistical and logical techniques systematically, to describe, illustrate and evaluate data is known as data analysis.
Proper data analysis must include:
Skills to analyze data
Appropriate subgroup analysis
Acceptable discipline norms
Clearly defined objective
Reliability and validity
Appropriate category considerations
Process to evaluate the strength of evidence and providing framework for determination.
The two main steps in testing hypothesis is framing null hypothesis and alternative hypothesis
Null hypothesis: No statistical significance exists in the given set of observation. This is assumed to be true.
Alternative hypothesis: It is opposite to null hypothesis
Interpretation: After analytical and experimental study, the drawn inferences is known as interpretation. The major aspects of interpretation is
And establish explanation concepts.
Preparation of report:
There should always be the necessary documentation of each and every result. The research reports contains following elements
Description and methodology
The recommendations made
There are two types of Research reports
Technical reports: which aim to specific group of people, including scientist, researchers, guides, belonging to the area
Popular reports: which can be understood my lay man or common people, in more easy and feasible way, with less technical words.
Presentation of results: It can be done in various ways including writing research papers, presenting in conferences, writing drafts, discussing, Seminar presentation or oral presentation.
Ultraviolet (UV) spectroscopy is an essential physical instrument that utilizes light in the electromagnetic spectrum’s ultraviolet, visible, and near-infrared ranges. The Beer-Lambert law is defined as a linear relationship between absorption, absorber concentration (or absorbing species) in the solution, and path length. Therefore, for a fixed path length, UV-Vis spectroscopy may be used to determine the absorbing species’ concentration. This is a method that is very simple, flexible, quick, precise, and cost-effective. The instrument is called the UV-Vis-NIR Spectrophotometer for ultraviolet-visible (or UV-Vis) spectroscopy. This can be used for the study of liquids, gases, and solids by using radiative energy corresponding to the electromagnetic spectrum’s far and near-ultraviolet (UV), visible (Vis), and near-infrared (NIR) regions. As a result, predetermined wavelengths have been described in these regions: UV ranges in between 300 – 400 nm, whereas visible ranges between 400 – 765 nm, and Near-Infrared ranges in between 765 – 3200 nm.
Principle: A light beam travels through an object and is determined by the light’s wavelength hitting the detector. The calculated wavelength provides valuable data on the chemical structure and the number of molecules (present in the intensity of the measured signal). Thus, it is possible to obtain both quantitative and qualitative information. Information can be obtained from a wavelength range of 160 to 3500 nm as radiation transmittance, absorbance, or reflectance. Incident power absorption promotes electrons to excited states or anti-bonding orbitals. Photon energy must equal the energy required by electrons to be promoted to the next higher energy state in order for this transition to occur. This method forms the fundamental operating theory of spectroscopy of absorption. Three types of ground-state orbitals can theoretically be involved:
1. The molecular orbital σ (bonding)
2. π (bonding) orbital molecular
3. Atomic Orbital n (non-bonding)
The anti-bonding orbitals, besides, are:
The orbital σ* (sigma star)
The orbital π* (pi star)
A transition from the s bonding orbital to the σ anti-bonding orbital involving an electron excitation is called the transition from σ to σ*. The excitation of a lone pair electron (non-bonding electron pair) to an anti-bonding π orbital is likewise expressed by π to π*. Electronic transitions that occur due to UV and visible light absorption are:
from σ to σ*;
from n to σ*;
from n to π*;
from π to π*.
Fig: Electron transitions in UV-Visible spectroscopy.
Higher energies are included in the transitions s to σ* and n to σ* and thus typically occur in far UV regions or weakly in 180 to 240 nm. Thus, in the UV zone, saturated groups do not demonstrate good absorption. Unsaturated core molecules undergo transitions n to π* and π to π*; these transitions require lower energies and thus occur at longer wavelengths than transitions to anti-bonding orbitals σ*.
Through the following types of absorption instruments, the UV-Vis spectrum can be recorded:
Single spectrometer beam
Spectrometer with double beams
All three types of spectrometers have a common light source (mostly tungsten lamps), a smallholder, and a detector. However, besides, a filter can be used to choose one wavelength at a time. This filter is also called a monochromator. A monochromator between the source of light and the specimen is part of the single beam spectrometer. For both wavelengths, the specimen is independently analyzed. The double beam spectrometer uses a single light source, a monochromator, a splitter, and a set of mirrors to direct the beam towards the reference and the sample under investigation. In contrast, a simultaneous spectrometer uses an array of diodes at all wavelengths to simultaneously detect absorbance. The quickest and most potent of the three is this.
Fig: Single and double beam UV-Visible spectrometer
Fig: Simultaneous UV-Visible spectrometer
Instrumentation: The light source (UV and visible), monochromator (wavelength selector), sample level, and detector are the essential components of a spectrometer. As a light source, a tungsten filament, continuous throughout the UV field, is usually used. Usually, the detector is a photodiode or CCD. To filter light of a specific wavelength, photodiodes go with monochromators to be fed to the detector. The visible lamp must be switched off when tracking the UV spectrum’s absorption, and vice versa. Figure 6 contains a UV-Vis-NIR Spectrometer diagram.
Components of Instrumental
A. Source of UV :
1. In its operating wavelength range, the power of the radiating source does not differ.
2. The continuous UV spectrum is created at low pressures by electrically exciting deuterium or hydrogen.
3. The UV light generation process involves creating an excited molecular species split into two atomic species and one UV photon.
4. The emission wavelengths are in the 160 to 375 nm range of both deuterium and hydrogen lamps.
5. The cuvettes’ content needs to be chosen so that the light incident is not absorbed since this would result in errors in the absorption spectrum obtained. Thus, typically, quartz is used.
B. Light Source (Visible)
1. As a visible light source, a tungsten filament lamp is used.
2. In the 350 to 2500 nm wavelength range, this lamp can produce light.
3. The energy, i.e., released, is directly proportional to the fourth power of the operating voltage in a tungsten filament lamp.
4. Thus, a highly stable voltage must be added to the lamp to achieve stable emissions.
5. By using electronic voltage regulators or constant-voltage transformers, voltage stability is assured.
6. Tungsten/halogen lamps contain small amounts of iodine, including the tungsten filament, contained within a quartz ‘envelope.’
7. The iodine reacts with sublimation-formed gaseous tungsten and creates a WI2 volatile compound.
8. They decompose when WI2 molecules touch the filament and redeposit tungsten back on the filament.
9. The tungsten/halogen lamps typically have a lifespan twice the traditional tungsten filament lamp.
10. Due to their high performance, tungsten/halogen lamps are used in modern spectrophotometers, and their output extends to the UV region as well.
C. Without Cuvettes
1. The monochromator source is used; light is separated into two sections of equal intensity by a half-mirror splitter before reaching the sample.
2. One component (or sample beam) passes through the cuvette with the material solution studied in a transparent solvent.
3. The second beam, or reference beam, passes through a comparable cuvette with only a solvent.
4. Containers of the reference and sample solution have to be transparent towards the moving beam.
D. The Detectors
1. The detector measures the light intensity emitted by the cuvette and sends it to a meter to record and show the values.
2. The strength of light beams is measured and compared by electronic detectors.
3. Two detectors have multiple UV-Vis spectrophotometers-a phototube and a photomultiplier tube, and reference and sample beams are simultaneously monitored.
4. The photomultiplier tube is the detector used widely in UV-Vis instruments.
5. It requires a photoemissive cathode (when photons strike it, electrons are released from the cathode), several dynodes (when one electron strikes it, a dynode emits several electrons) anode.
6. The photon incident hits the cathode after it reaches the tube.
7. Furthermore, the cathode releases different electrons, accelerated to the first dynode (whose potential is 90V more positive than cathode).
8. The first dynode is struck by the electrons, resulting in multiple electrons’ emission with each incident electron.
9. To create more electrons accelerated towards dynode three, and so on, these electrons are then accelerated towards the second dynode.
10. At the anode, all the electrons are finally collected. By this time, 106 to 107 electrons had been formed by each initial photon.
11. Amplified and calculated are the resulting current. Photomultipliers have rapid reaction times and are extremely sensitive to UV and visible radiation.
12. Photomultipliers, however, are only used for low-power radiation because high-power light will destroy them.
One example of a multichannel photon detector is the linear photodiode array. These detectors will simultaneously measure both elements of a beam of scattered radiation. A linear photodiode array consists of many tiny photodiodes of silicon produced on a single chip of silicon. The number of photodiodes on a chip can vary from 64 to 4096 sensor components, but 1024 photodiodes are the most common. There is a storage capacitor and a switch for each diode. It would help if you sequentially scanned the individual diode- capacitor circuits.
Fig: Photodiode array
Charge-Coupled Devices (CCDs) are like detectors for the diode series, but they consist of an array of photo capacitors instead of diodes.
The reference beam’s strength should have little to no absorption and is called I0, while the sample beam is called I. Within a short time, the spectrophotometer immediately analyses all wavelength components. In order to assess concentration as well as molecular structure or structural changes, this approach is acceptable. It is also used before and after contact with a substrate or molecule to analyze changes in vibrational and conformational energy levels.
1. The instrument’s precision is the most significant benefit for chemists and astronomers who use UV-VIS spectrometers.
2. Also, small UV-VIS spectrometers may provide highly accurate readings, which are essential when preparing chemical solutions or recording the celestial body’s movement.
3. It is quick to use UV-VIS spectrometers. Telescopes are connected to most UV-VIS spectrometers used in astronomy.
4. In chemistry, most of those users are similar in size to electron microscopes and require the same necessary skills to be used.
5. Since they are easy to handle, there is little risk of inappropriate use of a UV-VIS spectrometer.
1. The primary downside to using a UV-VIS spectrometer is the time it takes to plan for one to be used. Setup is vital for UV-VIS spectrometers.
2. The region must be cleared of any visible light, electronic noise, or other external pollutants that could interfere with the spectrometer’s reading.
3. UV-VIS spectrometers are easy to use and provide precise results if the room has been appropriately prepared ahead of time.
4. However, even a little bit of outside light or vibration from a small electronic device may interfere with the results you hope to achieve when using a UV-VIS spectrometer if the room has not been appropriately prepared.
X-ray spectroscopy is a tool that detects and analyses photons with wavelengths in the X-ray section of the electromagnetic spectrum or particles of light. It is used to help scientists understand an object’s chemical and elemental properties. Many distinct X-ray spectroscopy techniques are used in science and technology, including archaeology, astronomy, and engineering. These approaches are used separately to construct a complete image of the substance or entity being studied.
1. In 1901, a German physicist, Wilhelm Conrad Roentgen, was awarded the first Nobel Prize in physics for the discovery of X-rays in 1895.
2. According to the SLAC National Accelerator Laboratory, his new invention was rapidly put to use by other scientists and doctors.
3. Between 1906 and 1908, Charles Barkla, a British physicist, conducted research that contributed to his discovery that X-rays could be typical of individual substances. He also received a Nobel Prize in physics for his work, but not until 1917.
4. In fact, the use of X-ray spectroscopy started a bit earlier, in 1912, beginning with William Henry Bragg and William Lawrence Bragg, a father-and-son team of British physicists.
5. To research how X-ray radiation interacted with atoms inside crystals, they used spectroscopy.
6. By the following year, their method, called X-ray crystallography, had become the standard in the field, earning the Nobel Prize in physics in 1915.
The absorbed photon’s energy lifts an electron from a deeply bound state into unoccupied bound states in x-ray absorption spectroscopy (XAS), or it gains enough energy to exit the atom. Thus, the absorption spectrum provides extensive information on the density of empty states and makes it possible to conclude coordination, the state of oxidation, and much more about the local structure. If the photon’s energy is sufficient to surpass the electron’s binding potential, the likelihood of absorption is affected by the mechanism of electron dispersion from the local atmosphere of the surrounding atoms. This system, called EXAFS, can be used to determine the local structure around the absorbed atoms.
Main parts of this instrument include:
An X-ray source,
The sample holder,
An X-ray monochromator, and
An X-ray is produced from the source by bombarding a heavy metal target with high energy electrons. The spectrum of the energy of the released X-rays influences the option of heavy metal. For instance, a tungsten target generates X-rays of more incredible energy than a silver (Ag) target. Most of the energy that drives this bombardment process is lost as heat, so it is essential to cool the target electrode. More modern sources and other X-ray sources, such as the Stanford synchrotron beamline, are more effective.
a. Source for X-ray:
1. the X-ray source aims to supply the sample with X-ray radiation so that either X-ray Fluorescence or absorption experiments can be carried out.
2. Atoms absorb X-rays from the source, and the wavelength of the absorbed X-ray in X-ray absorbance and the strength of that absorbance provides the identity of that atom, and concentration is consumed.
3. This X-ray absorption causes the electron that absorbs the X-ray to be ionized.
4. The atomic orbital electrons that absorb this light, in their orbitals, are very similar to the nucleus.
5. Absorbed X-rays in X-ray fluorescence cause an atomic electron to be expelled, that is, atomic ionization, and the void is subsequently filled by an electron from an orbital further from the nucleus.
6. The outer electron must emit energy in order to drop down to fill the hole. This emitted light is fluorescence from X-rays.
b. Samples are exposed to X-rays:
1. The monochromator in an X-ray instrument is very different from a wavelength grating or prism monochromator that is visible (400 to 700 nm) or UV (200 to 400 nm).
3. Since the X-ray wavelengths are so short (say, 0.1 to 1 nm), it is not practical to scatter light using a prism or (grating) closely spaced grooves. Instead, contact with crystals of high purity is required; they function like a grating.
4. The crystals are mounted on a movable stage at which the angle at which the incoming X-rays hit the crystal can be continuously and smoothly varied.
5. The angle at which X-rays are detected to disperse from the crystal is also varied at twice the angle of entry.
6. The crystal stage has rotated by 80 degrees in the picture below, so the detector stage has rotated by 160 degrees at this moment.
7. By the collision of X-rays with high purity argon (Ar) gas, the detector mentioned here produces an electronic signal, an X-ray photon transducer.
8. The collision causes Ar ionization and the development of free electrons that flow to a positive electrode. The detector’s current flux between the electrodes is proportional to the incoming X-rays: a signal, Voilà.
c. Detector for X-ray:
1. this double-stage rotation generates the X-ray spectrum with a wavelength on the x-axis and absorbance on the y axis as the detector signal is captured.
2. Energy plotted for a fluorescence spectrum is on the x, and fluorescence emission is on the y axis.
3. An absorbance spectrum is given below. K-edges are considered the shortest wavelength (highest energy) absorbance of elements studied by X-ray.
4. Longer absorbances for wavelengths are L-edge, M-edge. The absorbance edge shape is very typical of the atoms involved in the absorbance when the function is closely examined.
5. To assess the oxidation state of heavy metal atoms and whether the heavy metal atom is bound to carbon or hydrogen, modern K-edge X-ray spectra can be used.
6. In other words, X-ray spectroscopy can be used to determine the chemical environment of heavy metal atoms in complex samples by spectral fitting to the available specifications.
7. The atmosphere here means the environment for atomic bonding.
One of the pioneers who helped in the production of X-ray emission spectroscopy was Karl Manne Georg Siegbahn from Uppsala, Sweden (1924 Nobel Prize). He painstakingly developed numerous diamond-ruled glass diffraction gratings for his spectrometers (also called X-ray fluorescence spectroscopy). He measured high precision X-ray wavelengths of several elements, using high-energy electrons as a source of excitation.
With synchrotrons, intense and wavelength-tunable X-rays are now usually produced. In a material, relative to the incoming beam, the X-rays can suffer a loss of energy. This energy loss of the re-emerging beam reflects the atomic system’s internal excitation, an analogous X-ray to the well-known Raman spectroscopy typically used in the optical field.
Highly accelerated electrons are bombarded with a piece of metal wire called an anticathode. The metal piece becomes a source of radiation from X-ray. With a crystal spectrometer, this radiation can be analyzed.
The spectrum of emissions is composed of two parts:
(a) Continuous spectrum
(b) Line spectrum
It consists of a line spectrum with a continuum of history. X-ray fluorescence generates X-radiation that only has a line spectrum without a continuous spectrum background.
(a). Continuous spectrum:
1. The continuous spectrum depends little on the metal used for the anticathode; with the increase of the metal’s Z, the curve’s height increases, but the curve’s form is independent of z. νmax is entirely independent of the anticathode metal used.
I (v) = constant Z (vmax– v)
2. The curve depends heavily on the voltage V used for electron acceleration.
3. With the voltage V, the maximum frequency increases proportionally.
eV = hvmax
4. Since the continuous spectrum is highly dependent on the velocity of the incident electron, it can be concluded that these electrons emit the corresponding X radiation.
1. They are subjected to intense electrostatic forces arising primarily from the nuclei of the constituent atoms as electrons traveling at high velocities enter the anticathode.
2. The electron is enormously accelerated, and the electrostatic charges emit electromagnetic waves, according to classical radiation theory, and the higher the acceleration, the higher the frequency.
3. It is the sudden slowing down of the electrons responsible for the continuous spectrum when they penetrate the anticathode; this can be defined as deacceleration radiation, but the German term Bremsstrahlung is also used.
1. The spectrum of the line primarily depends on the material from which the X-rays come, either the X-ray tube anticathode or the absorbing material used in a fluorescence experiment.
2. The spectral lines’ frequencies are independent of the electron-accelerating voltage and the incident radiation frequency. It only depends on the chemical components of which the substance is composed.
3. The frequencies are properties of the chemical elements’ atoms.
Several Applications :
In science and technology fields, including archaeology, astronomy, engineering, and health, X-ray spectroscopy is used today.
– By studying them with X-ray spectroscopy, anthropologists and archaeologists can reveal secret knowledge about the ancient artifacts and remains they discover. For example, to determine the sources of obsidian arrowheads produced by prehistoric people in the North American Southwest, Lee Sharpe, associate professor of chemistry at Grinnell College in Iowa, and his colleagues used a tool called X-ray fluorescence (XRF) spectroscopy.
– X-ray spectroscopy also allows astrophysicists to learn more about how space phenomena function.
– Researchers at Washington University, for instance, are preparing to observe X-rays that come from interstellar phenomena, such as black holes, for the future prospectus.
– The team, led by an experimental and theoretical astrophysicist, Henric Krawczynski, is preparing to launch a form of X-ray spectrometer called an X-ray polarimeter.
– The instrument will be suspended in the Earth’s atmosphere by a long-term, helium-filled balloon beginning in December 2018.
– Yury Gogotsi, a chemist and materials engineer at Drexel University in Pennsylvania, uses materials analyzed by X-ray spectroscopy to create spray-on antennas and water desalination membranes.
– The invisible spray-on antennas are only a few hundred nanometers thick but can relay radio waves and steer them.
A technique called X-ray absorption spectroscopy (XAS) ensures that the fragile material composition is right and helps assess the conductivity. To study the surface chemistry of complex membranes that desalinate water by filtering out particular ions, such as sodium, Gogotsi, and his colleagues also use X-ray spectroscopy.
– X-ray spectroscopy can also be used in various medical research and practice areas, such as modern CT scanning machines.
– According to Phuong-Anh T. Duong, Director of CT at Emory University Department of Radiology and Imaging Science, Phuong-Anh T. Duong, Director of CT at Emory University Department of Radiology and Imaging Science, Collecting X-ray absorption spectra during CT scans (via photon counting or spectral CT scanner) may provide more accurate information and contrast on what is going on inside the body, with lower radiation exposures from the X-rays and fewer or no need to use contrast materials (dyes)
b. most convenient and commonly used tool.
c. X-rays are not absorbed by air, so the specimen does not have to be in an evacuated chamber.
Disadvantages of X-rays:
With lighter elements, they do not interact very strongly.
It is a type of electromagnetic spectroscopy that analyses fluorescence from a sample. It is also known as fluorometry or spectrofluorometry. It requires the use of a light ray, usually, ultraviolet light, which excites the electrons of certain compounds in molecules and causes them to emit low-energy light, typically, but not always, visible light. Absorption spectroscopy is a complementary technique. Fluorometers or fluorimeters are called instruments that measure fluorescence.
Theory of Fluorescence spectroscopy:
Molecules have different states, referred to as levels of energy. Electronic and vibrational states are mainly concerned with fluorescence spectroscopy. In general, the species being studied would have an interest in the ground electronic state (a low energy state) and a higher energy excited electronic state. Various vibrational states are within any of these electronic states.
1. In fluorescence spectroscopy, from its ground electronic state to one of the different vibrational states in the excited electronic state, the species is first excited by absorbing a photon.
2. Collisions with other molecules cause vibrational energy to be lost by the excited molecule before the excited electronic state’s lowest vibrational state is reached.
3. The molecule then drops down, releasing a photon in the process to one of the ground electronic state’s different vibrational levels again.
4. The emitted photons have different energies, and therefore frequencies, as molecules can drop down into any of many vibrational levels in the ground state.
5. Therefore, the structure of the various vibrational levels can be calculated by studying the different wavelengths of light emitted in fluorescent spectroscopy and their relative intensity.
The various fluorescent light frequencies produced by a sample are calculated in a standard experiment, maintaining the excitation light at a constant wavelength. It is called the continuum of pollution. An excitation spectrum is measured using different wavelengths of excitation light by recording several emission spectra.
There are two general types of instrument:
a. In order to separate incident light and fluorescent light, filter fluorometers use filters.
b. In order to insulate the incident light and fluorescent light, spectrofluorometers use diffraction grating monochromators.
Both types use the following system:
1. The light passes through a monochromator or filter from an excitation source and strikes the sample.
2. The sample absorbs a proportion of the incident light, and some of the molecules fluoresce in the sample.
3. In all directions, fluorescent light is released.
4. To minimize the chance of emitted or reflected incident light hitting the detector, some fluorescent light passes through a monochromator or second filter and enters a detector usually positioned at 90° to the incident light beam.
5. Various light sources, including lasers, photodiodes, and lamps, can be used as excitation sources, xenon arcs, and mercury vapor lamps in particular.
6. At a very narrow wavelength interval, usually below 0.01 nm, a laser only emits high-irradiance light, making an excitation monochromator or filter unnecessary.
7. The drawback of this approach is, it is impossible to adjust a laser’s wavelength by much.
8. A mercury-vapor lamp is a line lamp, which means that it emits light near peak wavelengths.
9. The xenon arc, on the other hand, has a continuous emission spectrum of almost constant intensity in the 300-800 nm range and ample irradiance for measurements down to just over 200 nm.
10. Fluorimeter filters or monochromators can be used. With an adjustable tolerance, a monochromator transmits light at an adjustable wavelength.
11. A diffraction grating is the most common type of monochromator, i.e., collimated light illuminates a grating and exits depending on the wavelength at a different angle.
12. To choose which wavelengths to transmit, the monochromator can then be modified.
13. The addition of two polarization filters is required to enable anisotropic measurements: one before the emission monochromator or filter and one after the excitation monochromator or filter.
As stated before, the fluorescence relative to the excitation light is most commonly measured at a 90 ° angle. Instead of positioning the sensor at the excitation light line at an angle of 180 ° to prevent interference with the excitation light transmitted, this geometry is used. No monochromator is perfect and some stray light, that is, light with wavelengths other than the target, will be transmitted. An ideal monochromator can transmit only light in the specified range and have a high wavelength-independent transmission. Only the light scattered by the sample induces stray light when measured at a 90° angle and results in a higher signal-to-noise ratio relative to the 180 ° geometry, reducing the detection limit by about a factor of 10000. Besides, fluorescence from the front may also be measured, often done for turbid or opaque samples.
1. The detector can be either single- or multi-channeled.
2. The single-channel detector can only detect one wavelength’s intensity at a time, while the multi-channel detector simultaneously detects the intensity at all wavelengths, rendering the monochromator or filter of the emission unnecessary.
3. The most flexible fluorimetry can record both an excitation spectrum and a fluorescence spectrum with dual monochromators and a continuous excitation light source.
4. When measuring fluorescence spectra, the excitation light wavelength is kept constant, ideally at a high absorption wavelength, and the emission monochromator scans the spectrum.
5. The wavelength going through the emission filter or monochromator is kept constant for calculating excitation spectra, and the excitation monochromator is scanned.
6. As the fluorescence intensity is equal to the absorption, the excitation spectrum is usually similar to the absorption spectrum.
The fluorescence intensity usually is proportional to the concentration of the fluorophore at low concentrations.
1. To achieve ‘real,’ i.e., machine-independent spectra, multiple variables influence and distort the spectra, and corrections are required.
2. Here, the various forms of distortions will be categorized as either instrument or sample-related.
3. Firstly, it addresses the distortion that occurs from the instrument. During each experiment and between each experiment, the intensity of the light source and wavelength characteristics differ over time.
4. Besides, at all wavelengths, no lamp has constant power.
5. To correct this, following the excitation monochromator or filter, a beam splitter may be added to direct a portion of the light to the reference detector.
6. Furthermore, attention must be given to the transmission efficiency of monochromators and filters. These can alter over time as well.
7. Depending on the wavelength, the propagation efficacy of the monochromator often varies. This is the reason why the excitation monochromator or filter should be put after an optional reference detector.
8. The percentage of fluorescence that the detector collects depends on the device as well.
9. Besides, the detector’s quantum efficiency, that is, the percentage of photons detected, differs between different detectors, as the detector eventually deteriorates with wavelength and with time.
10. A tedious method is the adjustment of all these instrumental variables to achieve a ‘normal’ continuum, which is only implemented in practice when it is strictly necessary.
11. This is when the quantum yield is measured. For example, when the wavelength with the highest emission intensity is detected.
As noted earlier, distortions also emerge from the sample. Any elements of the sample must be taken into account as well. Firstly, over time, photodecomposition can decrease the fluorescence intensity. The dispersion of light must also be noticed. Rayleigh and Raman’s scattering is the primary form of scattering in this context. The light dispersed by Rayleigh dispersion has the same wavelength as the incident light, while the dispersed light typically shifts wavelengths to longer wavelengths in Raman dispersion. The dispersion of Raman is the result of a simulated electronic state caused by the light of excitation. The molecules can relax from this virtual state back to a vibrational level other than the vibrational ground state. It is often seen in fluorescence spectra at a constant wavenumber difference compared to the excitation wavenumber, e.g., the peak appears lower than the excitation light in the water at a wavenumber of 3600 cm-1.
The inner filter effects are other things to remember. Reabsorption involves these. Reabsorption occurs because at the wavelengths at which the fluorophore releases radiation, another molecule or part of a macromolecule absorbs it. Any or all of the photons released by the fluorophore may be absorbed again if this is the case. Due to large concentrations of absorbing molecules, including fluorophores, another inner filter effect occurs. The consequence is that in the solution, the excitation light’s strength is not constant, by which a small amount of the excitation light, which is apparent to the detection system, enters the fluorophores. Both the intensity and spectrum of the emitted light are modified by the internal filter effects and must be considered when examining the fluorescent light emission spectrum.
1. Tryptophan is an effective intrinsic fluorescent (amino acid) probe that can be used to estimate the tryptophan microenvironment’s existence.
2. The microenvironment of tryptophan growth changes when conducting experiments with denaturants, surfactants, or other amphiphilic molecules.
3. E.g., if a protein containing a single tryptophan is denatured at a growing temperature in its ‘hydrophobic’ center, a red-shift emission spectrum will appear.
4. Compared to a hydrophobic protein interior, this is due to the proximity of the tryptophan to an aqueous environment.
5. In comparison, if the tryptophan is incorporated in the surfactant vesicle or micelle, the addition of a surfactant to a protein containing a tryptophan exposed to the aqueous solvent can produce a blue-shifted emission spectrum.
6. A fluorophore can be bound to proteins that lack tryptophan.
7. The tryptophan emission spectrum at 295 nm is dominant over tyrosine and phenylalanine’s weaker fluorescence.
Applications of fluorescence spectroscopy:
For the study of organic compounds, fluorescence spectroscopy is used in biochemical, medical, and chemical research fields, among others.
It is used in differentiating malignant, bashful skin tumors from benign tumors has also been documented.
Atomic absorption spectroscopy (AAS) is a method in analytical chemistry for determining the concentration of a specific metal element in a sample. The process can be used in a solution to analyze the concentration of over 70 different metals. While atomic absorption spectroscopy dates from the nineteenth century, a team of Australian chemists primarily developed the modern form during the 1950s. They were headed by Alan Walsh and served in the Chemical Physics Division of the CSIRO (Commonwealth Science and Industry Research Organisation) in Melbourne, Australia.
By applying characteristic wavelengths of electromagnetic radiation from a light source, atomic absorption spectrometry detects elements in either liquid or solid samples. Wavelengths can be absorbed differently by individual components, and these absorbances are calculated against expectations. In effect, AAS takes advantage of the various wavelengths of radiation that different atoms absorb. In AAS, analytes are first atomized so that their characteristic wavelengths are emitted and registered. When those atoms consume particular energy during excitation, electrons go up one energy level in their respective atoms.
These atoms emit energy in the form of light as electrons return to their original energy state. There is a wavelength of this light that is characteristic of the element. According to the light wavelength and intensity, relevant elements can be detected, and their concentrations determined according to the light wavelength.
The approach uses absorption spectrometry to determine an analyte’s concentration in a sample. Thus it relies heavily on the Beer-Lambert rule. In short, by consuming a given amount of energy, the atoms’ electrons in the atomizer can be promoted to higher orbitals for a short period. This quantity of energy is unique to a specific transformation of electrons in a particular element, and each wavelength corresponds to only one element in general. This gives its elemental selectivity to the process.
Since the amount of energy placed into the flame is known and it is possible to calculate the amount remaining on the other side of the detector, it is possible to estimate from the Beer-Lambert law how many of these transitions have occurred and thus obtain a signal proportional to the concentration of the measured product.
There are four components of the standard AAS instrument: the sample introduction region, the source of light (radiation), the monochromator or polychromator, and the detector.
It needs to be atomized in order to test a sample for its atomic constituents. The light could then illuminate the sample. Finally, the light emitted is measured through a detector. A spectrometer is usually used between the atomizer and the detector to minimize the effect of the atomizer’s emission (e.g., black body radiation) or from the atmosphere.
Types of Atomizer:
Usually, the method uses a flame to atomize the sample, but other atomizers are also used, such as a graphite furnace or plasmas, particularly inductively coupled plasmas.
It is side-long (usually 10 cm) and not deep when a flame is used. The flame’s height above the burner head can be adjusted by changing the fuel mixture’s flow. At its longest axis (the lateral axis), a ray of light passes through this flame and reaches a detector.
A liquid sample is usually converted in three stages into an atomic gas:
1. The liquid solvent is evaporated (Drying), and the dry sample remains
2. Vaporization (Ashing)-the solid specimen vaporizes into a gas
3. Atomization is divided into free atoms by the compounds that make up the sample.
Sources of Radiation
The chosen radiation source has a narrower spectral range than that of the atomic transitions.
Cathode Hollow Lamps
The most common source of radiation in atomic absorption spectroscopy is hollow cathode lamps. A cylindrical metal cathode holding the metal for excitation and an anode is inside the lamp, filled with argon or neon gas. Gas particles are ionized when a high voltage is applied to the anode and cathode. Gaseous ions gain sufficient energy to eject metal atoms from the cathode as the voltage increases. Some of these atoms are excited, releasing light with the characteristic frequency of the metal. Various modern hollow cathode lamps are selective for several metals.
Lasers with diodes
Lasers, especially diode lasers because of their strong properties for laser absorption spectrometry, can also conduct atomic absorption spectroscopy. The method is then either referred to as diode laser atomic absorption spectrometry (DLAAS or DLAS) or, since wavelength modulation is most commonly used, spectrometry of absorption of wavelength modulation.
Context Methods of Correction:
The spectral overlap is unusual due to the limited bandwidth of hollow cathode lamps. That is, an absorption line from one element is unlikely to overlap with another. Molecular emissions are much larger, so a specific molecular absorption band is more likely to overlap with an atomic line. This can lead to artificially high absorption and an improperly high measurement of the solution concentration. In order to correct this, three methods are usually used:
Zeeman correction: A magnetic field is used to break the atomic line into two sidebands. To still overlap with molecular bands, these sidebands are close enough to the initial wavelength, but far enough, they do not overlap with the atomic bonds. It is possible to equate the absorption in the presence and absence of a magnetic field, the difference being the absorption of interest atomically.
Correction to Smith-Hieftje: This was invented by Stanley B. Smith and Gary M. Hieftje. The high current pulses the hollow cathode lamp, creating more significant atoms and self-absorption population during the pulses. This self-absorption allows the line to be broadened, and the line intensity decreases at the original wavelength.
Deuterium lamp correction: In this case, for the calculation of background emissions, a different source known as a broad-emission deuterium lamp is used. The use of a specific lamp makes this method the least reliable, but this method is most widely used because of its relative simplicity and the fact that it is the oldest of the three.
Advantages of AAS are given below:
Strong throughput of samples
Simple to make use of
Disadvantages/drawbacks of AAS are as follows:
It is only possible to evaluate solutions.
Less sensitivity compared to the furnace with graphite
Relatively large quantities of samples are needed (1-3 ml)
Absorption spectroscopy is a technique that compares the power of a beam of light determined before and after a sample contact. It is also referred to as Tunable Diode Laser Absorption Spectroscopy (TDLAS) when done with a tunable diode laser. To decrease the device’s noise, it is most often paired with a modulation technique, most often wavelength modulation spectrometry (WMS) and sometimes frequency modulation spectrometry (FMS).
To excite a sample, fluorescence spectroscopy uses higher-energy photons, which will then release lower energy photons. This method is known for its biochemical and medical applications and can be used for confocal microscopy, energy transfer of fluorescence resonance, and lifetime imaging of fluorescence.
When X-rays with appropriate frequency interact with a material, the atom’s inner shell electrons are excited into empty outer orbitals, or they can be entirely expelled, ionizing the atom. Then electrons from the outer orbitals would fill the inner shell “hole.” In this de-excitation process, the energy available is released as radiation (fluorescence), or other less-bound electrons are extracted from the atom (known as Auger effect). The frequencies (energies) of absorption or emission are characteristic of the individual atom. Moreover, there are minor frequency variations for a single atom that is typical of chemical bonding. These specific X-ray frequencies or Auger electron energies can be determined with an appropriate instrument. In chemistry and material sciences, X-ray absorption and emission spectroscopy are used for determining the elemental composition and chemical bonding. X-ray crystallography is a method of scattering; X-rays are dispersed at well-defined angles by crystalline materials. If the incident X-ray wavelength is known, the distances between the atoms’ planes inside the crystal can be measured. The scattered X-ray intensities provide information about the atomic positions and measure the atoms’ arrangement within the crystal structure.
Samples of liquid solution are aspirated into a combination of a burner or nebulizer/burner, dissolved, atomized, and often excited to a higher electronic state of energy. During analysis, the use of a flame includes fuel and oxidant, usually in gases. Gases such as acetylene (ethyne) or hydrogen are used as typical fuel gases. Oxygen, air, or nitrous oxide are common oxidant gases used. These methods can also analyze metallic element analytes in the concentration ranges of part per million, billion, or probably lower. In order to identify light with the analysis data coming from the flame, light detectors are required.
Atomic Emission Spectroscopy: This technique uses the flame’s excitation; atoms are excited to emit light from the flame’s heat. The total consumption burner with a round burning outlet is usually used in this technique. A more significant temperature flame is usually used to induce analyte atoms’ excitation than atomic absorption spectroscopy (AA). Since the flame’s heat excites the analyte atoms, no particular elemental lamps must shine into the flame. A high-resolution polychromator can be used to generate an emission intensity vs. wavelength spectrum over a range of wavelengths exhibiting multiple-element excitation lines, meaning multiple elements can be detected in one run. Alternatively, a single wavelength monochromator may be set to focus on studying a single element at a specific emission line. A more advanced variant of this process is plasma emission spectroscopy.
Atomic absorption spectroscopy (often referred to as AA) – A pre-burner nebulizer (or nebulizing chamber) is widely used to produce a sample mist and a slot-shaped burner that gives a longer flame pathlength. The flame temperature is low enough that sample atoms are not excited from their ground state by the flame itself. The nebulizer and flame are used to dissolve and atomize the sample, but for each type of analyte, the analyte atoms’ excitation is achieved by using lamps that glow through the flame at different wavelengths. The amount of light absorbed after passing through the flame defines the analyte quantity in the sample in AA. For greater sensitivity, a graphite furnace is typically used to heat the sample for desolvation and atomization. The graphite furnace process can also analyze any substantial or slurry samples. It is still a widely used analysis method for some trace elements in aqueous (and other liquid samples, due to its strong sensitivity and selectivity.
Atomic Fluorescence Spectroscopy: A burner with a circular burning outlet is widely used in this technique. To solve and atomize the sample, the flame is used. However, a lamp shines a light into the flame at a particular wavelength to excite its analyte atoms. Then the atoms of some components will fluoresce, emitting light in another direction. For quantifying the amount of analyte component in the sample, this fluorescent light’s strength is used. A graphite furnace is also used for atomic fluorescence spectroscopy. This technique is not as widely used as spectroscopy of atomic absorption or plasma emission.
Plasma Emission Spectroscopy:
It has virtually replaced in several respects similar to flame atomic emission spectroscopy.
Direct-current plasma (DCP) An electrical discharge between two electrodes creates a direct-current plasma (DCP). It needs a plasma support gas, and Ar is standard. Samples could be deposited on one of the electrodes, or one electrode can be built up by conducting them.
Glow discharge-spectrometry of optical pollutants (GD-OES)
Laser-Induced Breakdown Spectroscopy (LIBS) (LIBS), also called plasma spectrometry induced by laser (LIPS)
Plasma caused by microwave (MIP)
Spark or arc/emission spectroscopy – used in solid samples for the study of metallic elements. In order to make it conductive, a sample is ground with graphite powder for non-conductive materials. A sample of the solid was usually ground up and damaged during research in conventional arc spectroscopy methods. The spark or electric arc is passed through the sample to excite the atoms, heating the sample to a high temperature. The excited analyte atoms glow at different wavelengths, producing light that can be detected by standard spectroscopic methods. Since the conditions generating the arc emission are usually not quantitatively regulated, the study is qualitative for the components. Nowadays, under an argon atmosphere, spark sources with controlled discharges allow this method to be considered eminently quantitative, and its use is widely applied worldwide through the production control laboratories of foundries and steel mills.
Many atoms emit visible light or absorb it. In order to achieve a continuum of fine lines, the atoms must be in the gas phase. It suggests the material has to be vaporized. In absorption or emission, the spectrum is studied. In UV/Vis spectroscopy, visible absorption spectroscopy is mostly paired with UV absorption spectroscopy. While this type may be unusual as a similar indicator is a human eye, it still helps identify colors.
In the Ultraviolet (UV) field, all atoms are absorbed because these photons are energetic enough to excite outer electrons. Photoionization takes place if the frequency is high enough. In quantifying protein and DNA concentration and protein ratio to DNA concentration in a solution, UV spectroscopy is also used. Several amino acids, such as tryptophan, usually present in proteins, absorb light in the range of 280 nm, and DNA absorbs light in the 260 nm range. For this reason, in terms of these two macromolecules, the 260/280 nm absorption ratio is a good general measure of the relative purity of a solution. It is also possible to make fair estimates of protein or DNA concentration using Beer’s law.
The IR absorption spectrum analysis shows what kind of bonds are present in the sample, especially in organic chemistry. The study of polymers and components such as fillers, pigments, and plasticizers is also necessary.
To study the vibrational and rotational modes of molecules, Raman spectroscopy uses the inelastic scattering of light. An interpretation help is the resulting ‘fingerprints.’
Coherent anti-Stokes Raman spectroscopy (CARS) is a recent technique for in vivo spectroscopy and imaging with high sensitivity and robust applications.
Nuclear Magnetic Resonance Spectroscopy (NMR):
To determine the various electronic local environments of hydrogen, carbon, or other atoms in an organic compound or other compounds, nuclear magnetic resonance spectroscopy analyses such as atomic nuclei’s magnetic properties. This is used to assist in assessing the compound structure.
Mossbauer spectroscopy modes of transmission or conversion-electron (CEMS) probe individual isotope nuclei’s properties in various atomic environments by studying the resonant absorption of characteristic gamma-ray energy as the Mossbauer effect.
In the next chapter we will discuss in detail about each spectroscopic methods.
Originally, spectroscopy studied the interaction between radiation and matter as a wavelength (λ) feature. Historically, spectroscopy has been applied to the use of visible light, e.g., by a prism, distributed according to its wavelength. The idea later extended to include any calculation of a quantity as a function of either wavelength or frequency. It may thus also apply to an alternating field or changing frequency response (v).
When the very similar relationship E = h𝛎 for photons was realized, a further extension of the concept’s scope added energy (E) as a variable (h is the Planck constant). A response plot is referred to as a continuum as a function of wavelength, or more generally, frequency.
The spectroscopic method used to determine the concentration or quantity of a given species is spectrometry. In such cases, a spectrometer or spectrograph is the device that conducts such measurements. Spectroscopy/spectrometry can also detect compounds across the spectrum released from or absorbed by them in physical and analytical chemistry. In astronomy and remote sensing, spectroscopy/spectrometry is also extensively used. Many large telescopes have spectrometers used to calculate the chemical composition and physical characteristics of astronomical objects or measure their spectral lines’ speeds from the Doppler shift.
Methods of Classification:
The spectroscopy type depends on the calculated physical quantity. The quantity that is measured is usually an intensity, either absorbed or created by energy.
Electromagnetic spectroscopy requires material interactions, such as light, with electromagnetic radiation.
Spectroscopy of electrons requires interactions with electron beams. Auger spectroscopy involves inducing, with an electron beam, the Auger effect. In this case, the calculation usually requires the electron’s kinetic energy as a variable.
The interaction of charged species with electric and magnetic fields requires mass spectrometry, giving rise to a mass spectrum.
The word “mass spectroscopy” is deprecated since it is primarily a measurement method, although a spectrum for observation is created.
Mass ‘m’ is a variable in this spectrum, but the calculation is one of the particle’s kinetic energy.
Acoustic spectroscopy requires sound frequency.
Dielectric spectroscopy requires an external electrical field frequency.
Mechanical spectroscopy requires the frequency of external mechanical stress, e.g., torsion applied to a material object.
Method of Measurement:
Whether or not they refer to atoms or molecules, most spectroscopic methods are differentiated as either atomic or molecular. Along with that distinction, they can be categorized according to the form of their communication:
Absorption spectroscopy uses the range in which a material absorbs the electromagnetic spectrum. It involves atomic absorption spectroscopy and different molecular techniques in that area, such as infrared spectroscopy and radio region nuclear magnetic resonance (NMR) spectroscopy.
Emission spectroscopyuses the electromagnetic spectrum range in which a material radiates (emits). The material must consume energy first. This energy may come from several sources, such as luminescence, which defines the subsequent emission. Spectrofluorimetry includes molecular luminescence techniques.
Scattering spectroscopy tests the amount of light at specific wavelengths, incident angles, and polarization angles that a material scatters. The method of scattering is much quicker than the process of absorption/emission. Raman spectroscopy is one of the most beneficial applications of light scattering spectroscopy.
A single lens, historically called a loupe, consists of a simple microscope. A reading or magnifying glass is the most familiar example nowadays. Higher-magnification lenses are often made of two glass elements that create a color-corrected image. They can be worn in a cylindrical shape around the neck, which can be kept immediately in front of the eye. These are commonly referred to as eye loupes or lenses for jewelers. A single magnifying lens was used to create the standard simple microscope, which was often of good optical quality to enable the study of microscopic species, including Hydra and protists.
1. When one wants to study an object’s information, it is instinctive to put it as close to the eye as possible.
2. The closest the object is to the eye, the greater the angle it subtends to the eye, and thus the more extensive the object appears.
3. However, the human eye can no longer form a clear picture of an object brought too close.
4. The magnifying lens between the viewer and the object makes it possible to construct a “virtual image” that can be viewed comfortably.
5. The magnifier should be positioned in front of the eye to obtain the best possible image. To make out the object of interest, one should position the object at the microscope’s lens’s focus.
6. The highest magnification possible without lenses is when the object is carried to the closest location where a clear virtual image is observed.
7. This distance from the picture is around 25 cm for many people. The closest point of separate vision recedes to greater distances as individuals age, making a magnifier a valuable adjunct to a vision for older people.
8. The optical system’s geometry is related to magnifying force, or the degree to which the object is seen appears to be expanded, and the field of view, or the scale of the object that can be seen.
9. The working value of the magnifying power of the lens can be measured by dividing the minimum distance of separate vision by the focal length of the lens, which is the distance from the lens to the plane where the incoming light is centered
10. A lens with a minimum different vision distance of 25 cm and a focal length of 5 cms would also have a magnifying power of around 5 percent, for example.
11. If the magnifying lens diameter is adequate to fill or exceed the eye’s pupil diameter, the viewed virtual image will appear to be of significantly the same brightness as the original object.
12. As the focal length of the magnifier is increased. the field of view will be determined by the degree to which the working diameter is exceeded by the lens and the distance between the lens and the eye. The clearer the virtual picture, the more dependent it will be on the irregularities of the lens, its contours, and the conditions of its use.
Various aberrations affect the picture’s sharpness or consistency.
1. Chromatic aberrations create colored fringes around the image’s high-contrast regions since longer light wavelengths (such as red) are brought to focus slightly further from the lens than shorter wavelengths in a plane (such as blue).
2. Spherical aberration creates a picture in which, while the periphery may not be, the center of the field of view is in focus and uses lenses with spherical (rather than nonspherical or aspherical) surfaces.
3. The distortion produces curved images from straight lines in the object. The apparent shape and degree of distortion are closely related to the magnifier’s possible spherical aberration and is usually the most severe in high-powered lenses.
4. As relative aperture, i.e., the working diameter divided by the lens’s focal length, increases, the aberrations of a lens increase.
5. The aberration of the lens with a smaller diameter than the focal length would be more important than that of the other lens with a greater diameter.
6. Thus, there is a conflict between a short focal length that allows for high magnifying power but a narrow field of view and a longer focal length that offers a lower magnifying power but a wider linear field of view.
7. The 1670s high-powered lenses of Leeuwenhoek had a focal length and a few millimeters’ working size. This made it hard to use them, but they produced remarkable pictures that have not been changed for a century.
Several kinds of magnifiers are available. The option of an optical design for a magnifier depends on the power needed and the magnifier’s intended use.
For low powers:
a. A simple double convex lens is applicable for low forces, around 2-10x. Early simple microscopes, such as the microscope of Leeuwenhoek, magnified up to 300x.
b. If the lens has unique aspherical surfaces, as can be easily obtained in a plastic molded lens, the image can be enhanced.
c. When an aspheric lens is used, a reduction in distortion is noted, and the manufacture of such low-power aspheric plastic magnifiers is an important industry.
For high powers:
There are various types of magnifiers in which the basic magnifier is replaced by a compound lens consisting of multiple lenses mounted together for higher powers of 10-50x.
Using two simple lenses, usually plano-convex, which are flat on one side, angled outward on the other side, with curved surfaces facing each other, would automatically increase the distortion expected from a loupe. This form of magnifier is based on the Huygenian telescope eyepiece, in which the chromatic lateral aberration is corrected by removing the elements from the focal length. Since two components provide and share the image properties, the magnifier’s spherical aberration and distortion are substantially reduced compared to those of a single lens of the same strength.
To choose the imaging light portion with the lowest aberrations, a Coddington lens incorporates two lens components into a single thick element, with a groove cut in the center of the element. This is a simple and inexpensive system that suffers from the requirement that the distance between the optic beam and the target of imaging is small.
More complex magnifiers:
Three or more components are used by more advanced magnifiers, such as the Steinheil or Hastings types, to achieve better correction for chromatic aberrations and distortion. In general, the use of aspherical surfaces and fewer components is a safer solution.
– Often, mirrors are used. The British physicist C.R. brought reflecting microscopes, in which the image is magnified by concave mirrors rather than convex lenses, to their height of excellence in 1947.
– Burch, who made a set of giant tools that used ultraviolet rays.
– Using a magnifying mirror, chromatic aberration will not be minimized, although, with the use of an aspheric mirror carefully contoured, distortion and spherical aberration are reduced.
– The reflecting microscopes of today are limited to analytical instruments that use infrared rays.
The microscope is an instrument that creates enlarged photographs of small objects, enabling the viewer to display minute structures in a too near manner at a scale that is convenient for observation and study. Although optical microscopes are the focus of this article, many other types of wave, like acoustic, X-ray, or electron beam, can also enlarge the image and receive it by direct or digital imaging or a combination of these methods. The microscope can give a dynamic picture (as with traditional optical instruments) or a static one (as with conventional scanning electron microscopes).
The Resolution (or) Magnification:
1. The magnification of a microscope tests the amount of times the object tends to increase in size and the magnification ratio.
2. It is typically expressed in the 10-fold form (for a 10-fold magnified image), often misrepresented as “ten eks,” as though the algebraic symbol were an algebraic symbol, rather than the correct “ten times.” form.
3. A microscope’s resolution is a measure of the smallest object detail that can be observed. Resolution is expressed in linear units, which usually are micrometers.
Different types of microscopes:
Optical Microscope or Light Microscope:
The optical, or light microscope, consists of glass lenses used to shape the image, which is the most familiar microscope type. Optical microscopes, consisting of a single lens or compound consisting of many in-line optical components, may be straightforward.
a. The Resolution:
The hand magnifying glass will magnify approximately 3 to 20 points. Single-lensed simple microscopes, though compound microscopes can magnify up to 2,000, can magnify up to 300, and can expose bacteria. Under 1 micrometer (μm; one-millionth of a meter), a simple microscope can resolve; a compound microscope can resolve down to around 0.2 μm.
b. Image of Interest:
1. By photography through a microscope, a process is known as photomicrography, photographs of interest may be captured.
2. This has been done with film since the 19th century, but digital photography is now used widely instead. Some optical microscopes have dispensed with an eyepiece and view images on the computer screen directly.
3. A modern series of low-cost digital microscopes with a wide variety of imaging possibilities, including time-lapse microscopy, has made previously challenging and costly tasks much easier for the beginner or amateur microscopist.
Other types of microscopes use the wave nature of different physical processes. The electron microscope uses an electron beam in its image creation, which is the most important. There are magnifying powers of more than 1,000,000 in the transmission electron microscope (TEM). In a near-vacuum, TEMs shape images of thin specimens, usually parts. A scanning electron microscope typically has a lower resolution than a TEM, which produces a mirrored image of relief in a contoured specimen, but can display concrete surfaces in a way that the traditional electron microscope cannot. Microscopes that use lasers, sound, or X-rays are also available. The Scanning Tunnelling Microscope (STM) can generate atoms, and the Environmental Scanning Electron Microscope (ESEM), which produces images of specimens using electrons in a gaseous atmosphere, use other physical effects to broaden further the types of objects that can be examined.
History of Microscopes:
1. The theory of magnification has been established for a long time. “About 1267 In Perspectiva, English philosopher Roger Bacon wrote, “Because of the greatness of the angle under which we can see them, we can number the smallest particles of dust and sand,” and in 1538 in Homocentrica, Italian physician Girolamo Fracastoro wrote, “If someone can look through two spectacle lenses, one superimposed on the other, the image seems something much larger”.
2. Hans Jansen and his sons, Zacharias Jansen and Hans Lippershey, three Dutch spectacle makers, received credit for inventing the compound microscope around 1590.
3. The first illustration of a microscope was drawn in the Netherlands around 1631. It was a compound microscope with an oculus and an objective lens. In the mid-17th century, this kind of instrument, which came to be made of wood and cardboard, often decorated with polished fish skin, became increasingly popular and was used by the English natural philosopher Robert Hooke to provide the new Royal Society with frequent demonstrations.
4. These demonstrations began in 1663, and Hooke published a folio volume titled Micrographia two years later, which offered a wide variety of microscopic views of recognizable objects such as fleas, lice, and nettles. He coined the word cell in this book.
5. The description of how a single high-powered lens could be made into a serviceable microscope is concealed in the unnumbered pages of Micrographia’s preface. Using this template, the Dutch civil servant Antonie van Leeuwenhoek began his pioneering observations of freshwater microorganisms in the 1670s.
6. He made his postage-stamp-sized microscopes by hand, and details of about 0.7 μm could be resolved by the best of them. More than three centuries later, his fine specimens found at the Royal Society in excellent condition show what a great technician he was.
7. Leeuwenhoek’s simple microscopes launched microbiology in 1674, and single-lensed microscopes remained popular until the 1850s.
8. The Scottish botanist Robert Brown used them in 1827 to illustrate the ubiquity of the cell nucleus, a phrase he invented in 1831.
9. Using single lenses, simple microscopes can produce fine images; but they can also produce spurious colors in which various wavelengths of light do not come to the same focus due to chromatic aberration.
10. In the compound microscopes of the time, the aberrations were worse than magnifying the images.
11. They produced inferior images, although the compound microscopes were beautiful objects that bestowed status on their owners.
12. In 1733, by trial and error, the amateur English optician Chester Moor Hall discovered that a combination of a convex crown-glass lens and a concave flint-glass lens could help correct chromatic aberration in a telescope.
13. In 1774, Benjamin Martin of London designed an important set of color-corrected lenses for a microscope.
14. In the 19th century, the advent of new optical glasses stimulated continued microscope growth, and substantial advances were made to understand image forming’s geometric optics.
15. In 1791, Dutch optician Francois Beeldsnijder eventually introduced the idea of an achromatic (non-color-distorting) microscope target, and in 1830 the English scientist Joseph Jackson Lister published a work outlining a theoretical approach to the complete design of microscope goals.
16. The German physicist Ernst Abbe studied the physics of lens design. He developed an apochromatic lens system in 1868, which had even better color correction than achromatic lenses, and he published a detailed lens theory study in 1873.
17. The successful limits of optical microscopy were reached by light microscopes developed in the closing quarter of the 19th century.
18. Subsequent methods, such as phase-contrast microscopes, confocal microscopes, and interference microscopes, solved particular problems while examining specimens such as living cells.