behavior

JoVE Behavior documents observational and experimental techniques that seek to understand human and animal behavior. Work in this field includes analysis of genetic causes and physiological responses to internal or external stimuli as well as the implicated brain regions.

Comportamento

biochemistry

JoVE Biochemistry publishes experiments that investigate the structure, function, and interactions of biological molecules. The video articles in this section demonstrate methods to study nucleic acids, proteins, carbohydrates and lipids as well as enzyme function, protein folding, molecular interactions and biochemical reactions.

Biochimica

bioengineering

JoVE Bioengineering merges both physical and life sciences to understand and predict biological processes. Applying physical science tools to life science questions allow for the discovery of better technologies to measure, diagnose, and clinically treat disease.

Bioingegneria

biology

JoVE Biology welcomes all general biology research methodologies. Content in this section canvases all fields of cell, molecular, and organismal biology, ranging from new applications of standard techniques to novel approaches aimed at understanding the functions of life and living organisms. This diverse section includes, but is not limited to, techniques in physical biology, cellular biochemistry, genetics, physiology, systems biology and a combination of eukaryotic and prokaryotic model systems.

Biologia

cancer research

JoVE Cancer Research illustrates experimental approaches in biomedical research and clinical practice aimed at understanding, detecting, treating and preventing cancer. The video articles in this section demonstrate methods to study the cellular and molecular mechanisms of cancer, animal models of cancer research, development of new therapies and diagnostics, tumor imaging, chemical and radiation treatments, surgical procedures and improvements in therapy. These video articles offer researchers and clinical practitioners an efficient way to learn the technologies and experimental methods in cancer research.

Ricerca sul cancro

chemistry

JoVE Chemistry surveys a broad scope of chemical disciplines. This collection includes fundamental and applied research that investigates molecular interactions, properties, and structure spanning in the following core divisions: analytical, organic, organometallic, inorganic, bioinorganic, and biochemistry. This section also includes the design and preparation of materials and small molecules for advanced or unique applications.

Chimica

developmental biology

JoVE Developmental Biology publishes research methodologies in the field of developmental biology. This includes methods that are used to study biological development in vitro and in vivo at the molecular, cellular, tissue, organ and whole organism levels.

Biologia dello sviluppo

engineering

JoVE Engineering encompasses a broad range of experimental and instrumental techniques utilized in physics research. Investigations in this area strive to address and answer a broad range of scientific questions, such as device mechanisms and efficiencies, using physical tools. This approach often requires a combination of specialties, and research in this area tends to be interdisciplinary with contributions from mechanical, electrical, and chemical engineers.

Ingegneria

environment

JoVE Environment is dedicated to research methodologies that address environmental concerns and seek to better understand Earth's ecosystem.  Special consideration is given to experimental methodologies that assess society’s impact on the environment, suggest solutions for protecting Earth’s resources, and develop sustainable fuel sources. Moving towards an environmentally conscious planet is a collaborative effort with contributions from both life and physical scientists.

Ambiente

genetics

JoVE Genetics publishes video articles demonstrating experimental approaches to investigate gene function and regulation. This includes methods to study gene expression, mutagenesis, epigenetics, genetic determinants of development, genetic disorders, population genetics and evolution.

Genetica

immunology and infection

JoVE Immunology and Infection includes content from a variety of fields working toward a shared initiative of improving global health. Techniques evaluating the biological response to pathogens from the molecular to organismal level are presented as well as therapeutic agents and their efficacy in treating disease.

Immunologia e infezioni

medicine

JoVE Medicine serves as a connection between basic research and clinical applications. Articles may present medical procedures, case studies or clinical trial methodologies. Included studies are often modeled towards predictive, preventative, personalized, and practical approaches to treatment of disease. The goal of research methodologies in this section is to improve overall patient care and prognosis.

Medicina

neuroscience

JoVE Neuroscience is a multidisciplinary section devoted to investigations of the structure, function, physiology, and pathophysiology of the brain and nervous system. Included methodologies range from molecular and cellular level studies to full central and peripheral neural systems. Potential treatment platforms and surgical techniques for neurological diseases and disorders are also presented in this section.

Neuroscienze

jove journal

JoVE Journal

Video rivista scientifica. Peer reviewed. Multidisciplinare. Indicizzato su PubMed e Web of Science.

https://cloudfront.jove.com/files/banners/journal/journal_lander.jpg

Research

2D NMR: Heteronuclear Single-Quantum Correlation Spectroscopy (HSQC)

Heteronuclear single-quantum correlation spectroscopy (HSQC) is a 2D NMR technique that reveals one-bond correlations between hydrogen and a heteronucleus. The HSQC experiment is similar to the heteronuclear correlation experiment (HETCOR) but is more sensitive. In the HSQC spectrum, the proton chemical shift is plotted on the horizontal F2 axis, while the 13C chemical shift is plotted on the vertical F1 axis. The corresponding proton and 13C spectra are also shown. The HSQC contour plot does not have any diagonal peaks. All the cross-peaks indicate the protons directly attached to each carbon in a molecule. Similar to the COSY spectrum, imaginary lines are drawn in horizontal and vertical directions to identify the correlated peaks.
For example, the HSQC spectrum of 2-chlorobutane has a carbon signal at 11 ppm that can be assigned to the shielded methyl group and correlated to the proton multiplet at 1.01 ppm. The carbon signal at 24.8 ppm arises from the deshielded methyl group, which correlates to the proton multiplet at 1.52 ppm. Similarly, the methylene carbon signal at 33.3 ppm and methine carbon signal at 60.4 ppm correlate to the multiplets at 1.72 and 3.96 ppm, respectively.&#160; HSQC is a very useful technique for structurally elucidating complex molecules and is routinely used in protein NMR studies.

Heteronuclear single-quantum correlation spectroscopy (HSQC) is a 2D NMR technique that reveals one-bond correlations between hydrogen and a ...

2D NMR: Overview of Heteronuclear Correlation Techniques

Heteronuclear correlation spectroscopy is an analytical technique that investigates the coupling between different types of nuclei, often a proton and an X-nucleus, such as carbon-13 or nitrogen-15. This method is commonly used in nuclear magnetic resonance (NMR) spectroscopy to gain insights into complex chemical compounds&#39; structural and compositional aspects. A typical heteronuclear correlation spectrum displays X-nucleus chemical shifts on one axis and a proton spectrum on the other axis. Cross-peaks reveal the connection between specific protons and X-nuclei.
The main heteronuclear correlation experiments include HETCOR, HSQC, HMQC, and HMBC. HSQC, HMQC, and HMBC record the proton spectrum, whereas the X-nucleus spectrum is recorded by standard heteronuclear correlation spectroscopy or HETCOR. The HETCOR-HMQC-HSQC group of experiments cannot detect X-nuclei without an attached proton. HETCOR is used when ultrahigh peak resolution is required along the X-nucleus axis.
The heteronuclear single quantum correlation technique, or HSQC, investigates proton-to-X-nucleus single bond correlations. In an HSQC experiment, polarization is transferred from a proton nucleus to a neighboring X-nucleus and then back to the proton nucleus. The signal from the proton nuclei is then recorded. HSQC generates a spectrum like that of heteronuclear multiple-quantum correlation spectroscopy or HMQC but uses a different suppression method, providing better X-nucleus resolution along the axis.
Heteronuclear multiple-bond correlation, or HMBC, is a proton-detected experiment, like HMQC but with a longer initial delay time. This experiment is used to observe long-range proton to X-nucleus connectivity separated by 2-3 bonds, with some experiments going out to 4 or 5 bonds. In an HMBC experiment, direct one-bond correlations are suppressed as part of the sequence. The spectra of HMBC give rise to cross peaks that correlate which protons are coupled to what other specific X-nucleus is more than one bond away. The gradient HMBC (gHMBC) pulse sequence offers better suppression of interfering signals, making it particularly useful for analyzing complex molecules with overlapping signals.

Heteronuclear correlation spectroscopy is an analytical technique that investigates the coupling between different types of nuclei, often a proton and an ...

2D NMR: Homonuclear Correlation Spectroscopy (COSY)

Homonuclear correlation spectroscopy, or COSY, is a 2-dimensional NMR technique that provides information about coupled protons. Typically, the geminal and vicinal coupling are observed. For example, consider the COSY spectrum of ethyl acetate, where its 1D proton NMR spectrum is plotted along the vertical and horizontal axes with their corresponding chemical shift scale. Three spots on the diagonal corresponding to the three peaks in the 1D proton spectrum are called diagonal peaks. The COSY spectrum is symmetric about the diagonal, with signals above and below the diagonal called cross-peaks or off-diagonal peaks. An imaginary line is drawn from diagonal peaks parallel to the horizontal axis, leading to a cross peak. A vertical line is then drawn from the same cross peak towards the diagonal peak to correlate the peaks. The off-diagonal peaks correlating the multiplets at 1.25 and 4.2 ppm indicate that the methyl protons are coupled to the methylene protons. The signal at 2 ppm is not associated with cross-peaks because the protons are not involved in homonuclear coupling. A COSY spectrum is useful when overlapping multiplets complicate the corresponding 1D spectrum.

Homonuclear correlation spectroscopy, or COSY, is a 2-dimensional NMR technique that provides information about coupled protons. Typically, the geminal ...

Insensitive Nuclei Enhanced by Polarization Transfer (INEPT)

Insensitive Nuclei Enhanced by Polarization Transfer (INEPT) is an advanced Nuclear Magnetic Resonance (NMR) technique specifically designed to detect and enhance the signals of low-abundance nuclei, such as carbon-13 and nitrogen-15, in small molecules. The fundamental principle behind INEPT is the transfer of polarization from a more abundant and highly polarizable nucleus, typically hydrogen-1, to the low-abundance nucleus of interest. This process effectively boosts the NMR signal of the low-abundance nuclei, making them more easily detectable.
The INEPT technique involves a series of carefully timed radiofrequency (RF) pulses applied to the sample. These RF pulses excite the more abundant nuclei, causing them to align their spins in a specific manner. Through the process of spin coupling, the polarization of the more abundant nuclei is then transferred to the low-abundance nuclei. As a result, the NMR signal of these low-abundance nuclei is significantly enhanced, allowing for a more detailed analysis of the molecular structure.
Various pulse sequences have been developed to improve the observability of these low-abundance nuclei when coupled to a high-receptivity nucleus. The INEPT pulse sequence combines a spin echo and selective population inversion, which includes 90&#176; and 180&#176; pulses on the high-sensitivity (the I spins) and low-sensitivity nuclei (the S spins), respectively. Coupling-constant-based delays between pulses facilitate magnetization transfer through space or bond interactions. Additional RF pulses and delays invert the low-gamma magnetization, producing an observable NMR signal. The net result is an enhanced NMR signal from low-gamma nuclei that would otherwise be challenging to detect.
Various pulse sequences have been developed to improve the detection of low-abundance nuclei when coupled with high-receptivity nuclei such as protons. The INEPT sequence yields enhanced signals for insensitive nuclei, with half of the CH group producing negative peaks and the other half positive peaks. This pattern allows for discrimination between different types of carbon-hydrogen bonds within the molecule.
Decoupling methods can be employed to enhance sensitivity and resolution in distinguishing various carbon-hydrogen bonds, making INEPT an invaluable tool in the chemical and pharmaceutical industries.

Insensitive Nuclei Enhanced by Polarization Transfer (INEPT) is an advanced Nuclear Magnetic Resonance (NMR) technique specifically designed to detect and ...

Two-Dimensional (2D) NMR: Overview

The 1D NMR spectrum of large and complex molecules like natural products has complicated splitting patterns and overlapping signals, which can be easily interpreted using 2-dimensional (2D) NMR. Unlike 1D NMR, 2D NMR has two frequency axes that provide the coupling information between the nucleus A and nucleus B in a molecule. The process from which 2D spectra are obtained has four steps.
The first step is the preparation period, during which nucleus A is excited with a radiofrequency pulse. The second step is the evolution or wait period t1, which is incremented systematically, and no data is observed during this period. The next step is the mixing period, in which a second radiofrequency pulse is introduced, which transfers the magnetization to the spins of nucleus B. In the last step, the magnetization of nuclear spins from B is detected during the acquisition time t2 for each t1. Time domains t1 and t2 are Fourier transformed into frequency domains f1 and f2, and these correspond to two dimensions of 2D spectra, while the third dimension is the intensity. In 2D experiments, coupling between the same kind of nucleus is called homonuclear 2D experiments, whereas coupling between two types of nucleus is called heteronuclear 2D experiments. 2D spectra can be displayed as stacked or contour plots. When presented as stacked plots, 2D spectra are difficult to interpret. The contour plot is the horizontal cross-section of a stacked plot where the size of the circles shows the signal intensity. Its interpretation is straightforward and it is preferred for data analysis.

The 1D NMR spectrum of large and complex molecules like natural products has complicated splitting patterns and overlapping signals, which can be easily ...

2D NMR: Overview of Homonuclear Correlation Techniques

Homonuclear correlation spectroscopy (COSY) is a powerful technique used in Nuclear Magnetic Resonance (NMR) spectroscopy to study the correlations between nuclei of the same type within a molecule. It provides information about scalar couplings between adjacent nuclei, which helps determine connectivity and structural information. There are several COSY variants, each with its unique strengths and experimental parameters.
COSY90 is the standard two-dimensional (2D) COSY experiment that utilizes a second 90-degree pulse to achieve high sensitivity and resolution. It excels in analyzing small to medium-sized molecules and provides information on directly coupled protons. COSY45, similar to COSY90, uses 45-degree pulses instead of 90-degree pulses. This results in reduced sensitivity but improved resolution.
Long-range COSY (LR-COSY) extends the COSY experiment to detect scalar couplings between nuclei further apart in the molecule. It achieves this by combining multiple pulse sequences and magnetization transfers.
Relayed COSY is a variant of COSY that adds extra pulses to the standard pulse sequence to produce cross-peaks between cross-peaks, showing specific molecular connections hidden by overlapping signals.
TOCSY (Total Correlation Spectroscopy), also known as HOHAHA (Homonuclear Hartmann-Hahn Spectroscopy), reveals all through-bond connectivity between all coupled nuclei. TOCSY is especially useful for analyzing molecules with overlapping signals and complex coupling patterns.
NOESY (Nuclear Overhauser Effect Spectroscopy) measures the nuclear overhauser effect, which arises from dipole-dipole interactions between nuclei. It provides information about spatial proximity and can be used to determine the three-dimensional structure of a molecule.
ROESY (Rotating-frame Overhauser Enhancement Spectroscopy) is similar to NOESY but employs a different mechanism to obtain through-space correlations. It provides information about spatial proximity and is particularly useful for studying molecules with weak NOE signals.
In conclusion, choosing a COSY variant depends on the molecule or reaction under investigation and the desired information. The right COSY variant can be determined based on molecular size, coupling complexity, and the need for spatial or long-range connectivity data.

Homonuclear correlation spectroscopy (COSY) is a powerful technique used in Nuclear Magnetic Resonance (NMR) spectroscopy to study the correlations ...

Double Resonance Techniques: Overview

Double resonance techniques in Nuclear Magnetic Resonance (NMR) spectroscopy involve the simultaneous application of two different frequencies or radiofrequency pulses to manipulate and observe two distinct nuclear spins. One important application of double resonance is spin decoupling, which selectively suppresses coupling with one type of nucleus while observing the NMR signal from another nucleus, simplifying the spectrum and enhancing resolution.
Spin decoupling is usually achieved by irradiating the sample atoms with a suitable radio frequency (rf) pulse sequence, effectively eliminating all the coupling with one nuclide. This simplifies the observed spectrum, making it easier to analyze and understand the relationships between different nuclei.
Double resonance experiments can be classified as heteronuclear or homonuclear, depending on whether the two sets of nuclei are of different isotopes or the same isotope, respectively. They can also be selective or nonselective, depending on whether the irradiating frequency covers only a portion or all of the resonance frequencies.
In nonselective heteronuclear decoupling, samples are exposed to an appropriate radiofrequency range to remove all coupling with one nuclide. However, increased external field strengths necessitate more powerful irradiation across broader frequency ranges. In certain instruments, continuous irradiation creates enough heat to cause damage to thermally sensitive samples.
To overcome these problems, modern methods using a series of pulses and exact timing delays, like J-modulated spin echo pulse sequences, can be employed to eliminate or adjust coupling effects within the spectrum.
For example, the attached proton transfer (APT) experiment applies a J-modulated spin echo pulse sequence to focus on the phase of detected carbon signals. Carbon atoms attached to an even number of protons exhibit positive signals in the spectrum, while those bonded to an odd number of protons appear as negative signals.
It utilizes a combination of a 180&#176; proton pulse and broadband decoupling to simplify spectral interpretation and assign multiplicities. The 180&#176; pulse is applied to the protons during the APT experiment. Its role is to refocus the spin coupling evolution that naturally occurs due to interactions between protons and carbons.
Specifically, after the 90&#176; pulse creates transverse magnetization, the 180&#176; pulse ensures that the effects of coupling are frozen during the initial period where the decoupler is turned off. This means the chemical shifts evolve only during the time 1/J&#8203; (the coupling constant period) after the first 180&#176; pulse.
By carefully timing this pulse sequence, the experiment isolates carbon multiplicities by utilizing the J-coupling between carbons and their attached protons. After the coupling evolution period (1/J&#8203;), broadband decoupling is turned on. This removes the J-coupling signals, collapsing multiplets into single peaks for each carbon resonance.
The decoupling ensures that carbon signals are free of splitting, allowing for a clear differentiation of signals based on phase (positive or negative) rather than splitting patterns.
The APT technique selectively enhances carbon signals, providing valuable information about carbon-proton connectivity, the number of attached hydrogen atoms, and the overall molecular structure.

Double resonance techniques in Nuclear Magnetic Resonance (NMR) spectroscopy involve the simultaneous application of two different frequencies or ...

&sup1;H NMR of Labile Protons:  Deuterium (&sup2;H) Substitution

This lesson illustrates the role of deuterium substitution in simplifying the NMR spectrum of compounds comprising labile protons. One method employed is the use of deuterium. Amongst the three isotopes of hydrogen, deuterium (2H) has a nucleus composed of one proton and one neutron. When the D2O solvent is added to a pure dry ethanol solution, its labile proton is substituted with deuterium.
<img alt="Figure1" src="/files/ftp_upload/12280/12280-PT_Figure_1.svg" style="text-align: center; height: 700px;" />
Figure 1. The comparison of the proton NMR spectra of pure dry ethanol and the same in D2O solvent.
The signals from deuterium are not observed in a proton NMR spectrum, as deuterium&#8211;proton coupling is negligible, and no spin-spin splitting occurs between them. As shown in Figure 1, the NMR spectra of pure ethanol and ethanol in deuterium, the disappearance of the triplet in the latter, confirms that it corresponds to the labile &#8211;OH proton. Furthermore, the methylene protons that initially showed up as a multiplet transform to a quartet, proving that the multiplet is due to the coupling with the &#8211;OH proton and the methyl protons.

This lesson illustrates the role of deuterium substitution in simplifying the NMR spectrum of compounds comprising labile protons. One method employed is ...

Nuclear Overhauser Enhancement (NOE)

Irradiation of a spin-active nucleus causes an increase or decrease in the signal intensity of neighboring nuclei that are not necessarily chemically bonded or involved in J-coupling. &#160;This phenomenon, called the Nuclear Overhauser Enhancement (NOE), results from through-space interactions between the nuclear spins. The NOE effect decreases with increasing internuclear distance and is generally not observed beyond 4 angstroms. In NOE, dipole-dipole interactions between neighboring spin-active nuclei result in the transfer of nuclear spin polarization. These interactions can cause an increase or decrease in the signal intensity, which results in a positive or negative NOE, respectively.
NOE can be applied to improve the signal intensity of less sensitive nuclei such as carbon-13. Proton-decoupled carbon-13 spectra show a positive NOE effect, where the carbon signal intensities are substantially increased compared to those in proton-coupled spectra.
Targeted irradiation of specific nuclei can also reveal through-space proximity, which helps confirm stereochemistry and verify spectral peak assignments. The NOE effect is also applied in 2D NMR experiments, such as Nuclear Overhauser Effect Spectroscopy (NOESY), to determine the 3D structure of proteins.

Irradiation of a spin-active nucleus causes an increase or decrease in the signal intensity of neighboring nuclei that are not necessarily chemically ...

&sup1;H NMR of Labile Protons: Temporal Resolution

Protons bonded to heteroatoms such as nitrogen and oxygen exhibit a range of chemical shift values. This is due to the varying degree of hydrogen bonding between the proton and the heteroatom in other molecules. The extent of hydrogen bonding affects the electron density around the proton, thereby giving different chemical shift values for the protons in the proton NMR spectrum.
The &#8211;OH proton in alcohols typically appears in the range of &#948; 2 to 5 ppm but can vary depending on the specific alcohol and conditions. This &#8211;OH proton undergoes rapid proton exchange with other molecules and is referred to as a labile proton. So, the exact &#948; value of the &#8211;OH proton and the spin-spin coupling involving the &#8211;OH proton and the other protons in the alcohol depends on the rate of the &#8211;OH proton exchange. The proton exchange rate, in turn, depends on multiple factors, including the purity of the alcohol sample, temperature, and solvent.
<img alt="Figure1" src="/files/ftp_upload/12279/12279-PT_Figure_1.svg" style="text-align: center; height: 350px;" />
Figure 1. The triplet of a &#8211;OH proton in the proton NMR spectrum of neat ethanol.
For example, the &#8211;OH proton exchange in a pure dry ethanol sample is very slow on the NMR time scale, giving enough time for the spin-spin coupling. Consequently, as depicted in Figure 1, the proton NMR spectrum of pure dry ethanol shows a triplet for the &#8211;OH proton due to the spin-spin coupling with adjacent methylene protons. The methylene protons are seen as a multiplet due to the coupling with the &#8211;OH proton and the methyl protons.
The presence of trace amounts of an acid or base increases the proton exchange rate. The proton exchange becomes rapid, and the NMR spectrometer only records an average of all the possible environments for the &#8211;OH proton.
<img alt="Figure2" src="/files/ftp_upload/12279/12279-PT_Figure_2.svg" style="text-align: center; height: 325px;" />
Figure 2. The broad singlet of a &#8211;OH proton in the proton NMR spectrum of neat ethanol.
As a result, as recorded in Figure 2, the &#8211;OH proton exhibits a broad singlet. The protons from the adjacent methylene group present a quartet where the splitting is caused only by the adjoining methyl group.

Protons bonded to heteroatoms such as nitrogen and oxygen exhibit a range of chemical shift values. This is due to the varying degree of hydrogen bonding ...

&sup1;H NMR of Conformationally Flexible Molecules: Variable-Temperature NMR

The axial and equatorial protons in cyclohexane can be distinguished by performing a variable-temperature NMR experiment. In this process, except for one proton, the remaining eleven protons are replaced by deuterium. The deuterium substitution avoids the possible peak splitting caused by the spin-spin coupling between the adjacent protons. The remaining proton flips between the axial and equatorial positions.
<img alt="Figure1" src="/files/ftp_upload/13016/13016-PT_Figure_1.svg" style="text-align: center; height: 480px;" />
Figure 1. The temperature-dependent proton NMR spectra of cyclohexane.
Figure 1 depicts the proton NMR spectrum of the deuterium substituted sample, recorded at various temperatures. At room temperature, the rapid ring flipping of cyclohexane results in a single sharp peak. As the temperature is lowered to &#8722;60&#176;C, the rate of chair-chair interconversion reduces, resulting in the broadening of the peak.
Further lowering of the temperature broadens the peak, forming a saddle that splits into two peaks. On reaching a temperature lower than &#8722;89&#176;C, two sharp, well-resolved peaks corresponding to the equatorial and axial protons are formed. The peaks corresponding to the equatorial and axial protons occur at &#948; 1.62 and 1.14, respectively. At such low temperatures, the rate of chair-chair interconversion is reduced significantly, enabling the detection of the two distinct sets of protons on the NMR time scale.

The axial and equatorial protons in cyclohexane can be distinguished by performing a variable-temperature NMR experiment. In this process, except for one ...

&sup1;H NMR of Conformationally Flexible Molecules: Temporal Resolution

At room temperature, the chair conformer of cyclohexane undergoes rapid ring flipping between two equivalent chair conformers at a rate of approximately 105 times per second. These two chair conformers are in equilibrium. The rapid ring flipping results in the interconversion of the axial proton to an equatorial proton and an equatorial to the axial proton. Such interconversions are too rapid and cannot be detected on the NMR timescale. Hence, the NMR spectrometer cannot distinguish between the axial and equatorial protons, resulting in a single peak at &#948; 1.4.
Similarly, NMR averages all the conformations of any molecule with rapid conformational equilibria. For example, the CH3 protons in bromoethane have a single resonance and coupling constant for splitting by the CH2 protons due to the rapid internal rotation of the carbon-carbon bond. Lastly, besides conformational equilibria, the time-averaging effect of NMR spectroscopy is also observed in some chemical reactions.

At room temperature, the chair conformer of cyclohexane undergoes rapid ring flipping between two equivalent chair conformers at a rate of approximately ...

Mass Spectrometry: Carboxylic Acid, Ester, and Amide Fragmentation

The fragmentation patterns observed for compounds such as carboxylic acids, esters, and amides in the mass spectra include &#9082;-cleavage and McLafferty rearrangement. Fragmentation by &#9082;-cleavage preferentially occurs at the carbon-carbon bond at the &#9082;-position next to the carboxylic group to generate a neutral radical and a cation. Long chain compounds with hydrogen at their &#947;-carbon undergo McLafferty rearrangement to give a radical cation and a neutral alkene.
For example, the fragmentation of butyramide is demonstrated in Figure 1. The molecular ion peak of butyramide, which contains one nitrogen atom, shows an odd mass-to-charge ratio of 87. Fragmentation of butyramide molecular ion by &#9082;-cleavage produces a cation at a mass-to-charge ratio 44. In addition to &#9082;-cleavage, butyramide molecular ion also undergoes fragmentation via McLafferty rearrangement. The base peak at the mass-to-charge ratio 59 arises due to McLafferty rearrangement.
<img alt="Figure1" src="/files/ftp_upload/13051/13051_PT_Figure_1.svg" style="text-align: center; height: 250px;" />

The fragmentation patterns observed for compounds such as carboxylic acids, esters, and amides in the mass spectra include &#9082;-cleavage and McLafferty ...

Chemical Ionization (CI) Mass Spectrometry

The molecular ion peak of a molecule in the mass spectrum provides vital information for molecular identification. However, conventional electron impact ionization can lead to the rapid dissociation of some molecular ions before they reach the detector. A milder ionization method is required to increase the lifetime of such ionized analyte molecules. Chemical ionization (CI) is a gas-phase protonation reaction useful for mass-analyzing analyte molecules that are easily protonated to yield the corresponding conjugate acid. In this process, the sample is mixed with an excess reagent gas, which ensures the electron impact occurs primarily on the reagent gas. The charged species formed from the reagent protonates the analyte molecule, producing a relatively stable protonated analyte (conjugate acid) compared to the molecular ion. This results in an M+1 peak in the mass spectrum. The conjugate acid may then undergo fragmentation that generates additional signals.
For example, the CI of di-sec-butyl ether in methane gas illustrates this process. When the ether is mixed with excess methane gas as a reagent, electron impact occurs on methane rather than the ether. The resulting methane radical cation can react with another methane molecule to generate a methane radical and a methanium ion. The methanium ion is a source of gas-phase protons, which can protonate the ether to form its conjugate acid. The sequential reactions during the chemical ionization of di-sec-butyl ether are depicted in Figure 1.
<img alt="Figure1" src="/files/ftp_upload/12300/12300_PT_Figure_1.svg" style="text-align: center; height: 175px;" />
Figure 1: Chemical ionization of di-sec-butyl ether methane mixture.
This conjugate acid of the ether (m/z = 131) is relatively more stable than the molecular ion (m/z = 130) of ether formed via the conventional electron impact ionization. In the traditional route, the molecular ion undergoes fragmentation via &#945; cleavage, giving a signal at m/z = 101. Figure 2 illustrates the reactions that occur during electron impact ionization directly on di-sec-butyl ether.
<img alt="Figure2" src="/files/ftp_upload/12300/12300_PT_Figure_2.svg" style="text-align: center; height: 175px;" />
Figure 2: Electron impact ionization of di-sec-butyl ether and fragmentation of the molecular ion.
So, the mass spectra of di-sec-butyl ether ionized via CI feature an M+1 peak at m/z = 131. On the other hand, mass spectra of di-sec-butyl ether ionized via electron impact ionization do not show any peak at the m/z value of its molecular weight. Figures 3a and 3b show the mass spectra of di-sec-butyl ether ionized via the electron impact and chemical ionization methods, respectively.
<img alt="Figure3" src="/files/ftp_upload/12300/12300_PT_Figure_3.svg" style="text-align: center; height: 225px;" />
Figure 3:&#160;a) No m/z = 130 peak is visible in the mass spectrum obtained via electron impact ionization of di-sec-butyl ether. b) The m/z = 131 peak is clearly visible in the mass spectrum obtained via chemical ionization of di-sec-butyl ether.

The molecular ion peak of a molecule in the mass spectrum provides vital information for molecular identification. However, conventional electron impact ...

Mass Spectrometry: Alkyl Halide Fragmentation

Chlorine isotopes exist as 35Cl and 37Cl in a 3:1 ratio, while bromine isotopes exist as 79Br and 81Br in a 1:1 ratio. The mass spectrum of alkyl halides typically produces two distinct molecular ion peaks, the molecular ion peak, [M], and the molecular ion plus two, [M + 2] peak. The relative heights of these two peaks are proportional to the isotopic abundance ratios of the halide. For example, 2&#8208;chloropropane and 1&#8208;bromopropane display two peaks with relative peak heights in a 3:1 and 1:1 ratio, respectively.
Alkyl halides typically lose a non-bonding electron from the halogen to form molecular ions. The base peak results from the heterolytic cleavage of the carbon&#8211;halogen bond, where the bonding electrons go to the most electronegative atom, forming a cation and halogen atom. Interestingly, the &#9082;-cleavage is observed only in alkyl chlorides, not alkyl bromides. In alkyl chlorides, the&#160; C&#8211;Cl and C&#8211;C bonds exhibit similar strengths, while in alkyl bromides, the C&#8211;Br bond is weaker than the C&#8211;C bond. Consequently, the alkyl chlorides form a stable cation, where the positive charge is shared between the two atoms.
While less commonly discussed, fluoroalkanes and iodoalkanes exhibit unique fragmentation behavior. Fluoroalkanes, due to the strength of the C&#8211;F bond and the presence of only one isotope (19F), show limited fragmentation and lack isotopic peaks. Iodoalkanes, with their weak C&#8211;I bond and a single isotope (127I), predominantly fragment by losing iodine, resulting in simpler spectra with less isotopic splitting.

Chlorine isotopes exist as 35Cl and 37Cl in a 3:1 ratio, while bromine isotopes exist as 79Br and 81Br in a 1:1 ratio. The mass spectrum of alkyl halides ...

Electrospray Ionization (ESI) Mass Spectrometry

Higher molecular weight biomolecules are nonvolatile compounds that may decompose before ionizing or vaporizing during mass analysis with conventional electron impact ionization methods. Accordingly, electrospray ionization (ESI) is the favored method for vaporizing and ionizing biomolecules as it circumvents rapid fragmentation and enables the recording of mass signals for the entire biomolecule.
ESI utilizes electrical energy to transfer ions from the liquid phase of the sample into the gaseous phase. The analyte biomolecule is premixed with an ionic liquid&#8212;a nonmolecular compound serving as the ionization source. The solution is sprayed through a high-voltage capillary to generate a fine aerosol. The voltage difference applied between the capillary and the entrance of the analysis chamber causes the movement of charged droplets toward the analysis chamber. The ionic liquid from the aerosol evaporates during this movement, leaving behind a variably protonated or ionized analyte molecule. These charged species are detected in the mass spectrum at an m/z value equivalent to the sum of the analyte molecule&#39;s molecular mass and the ions&#39; molecular weight from the ionic liquid associated with the analyte molecule.
The position of mass signals varies depending on the type of cations present in the ionic liquid. For example, the presence of sodium ions in the ionic liquid leads to an (M+23) peak in the mass spectra, while the presence of a proton results in an (M+1) peak.

Higher molecular weight biomolecules are nonvolatile compounds that may decompose before ionizing or vaporizing during mass analysis with conventional ...

Matrix-Assisted Laser Desorption Ionization (MALDI)

Matrix-assisted laser desorption ionization (MALDI) is a powerful analytical technique used in mass spectrometry. It enables the identification and characterization of various biomolecules, including proteins, peptides, nucleic acids, and carbohydrates. MALDI spectrometry is widely employed in biological and medical research, as well as in fields like pharmacology and biochemistry.
The analyte of interest, a biomolecule or a mixture of biomolecules, is mixed with a suitable matrix material. The matrix is usually an organic compound, such as a small organic acid or a crystalline substance, that facilitates ionization. This analyte-matrix mixture is then deposited onto a target surface, inserted into the mass spectrometer, and dried. During drying, the matrix forms small crystals that incorporate the analyte molecules within their structure.
Once the sample is prepared, a pulsed laser is directed onto the target surface. The matrix molecules absorb the laser beam energy, causing them to heat up and vaporize rapidly. This process is known as laser desorption. As the matrix molecules vaporize, they carry the embedded analyte molecules. The vaporized analyte molecules become ionized through a process called proton transfer. The proton transfer occurs between the matrix and the analyte molecule. The matrix molecule donates a proton to the analyte molecule, resulting in the formation of protonated ions. The analyte ions are then accelerated by an electric field and further separated based on their mass-to-charge ratio in a time-of-flight mass analyzer.
The mass spectrum obtained from MALDI spectrometry provides valuable information about the mass and relative abundance of the analyzed biomolecules.

Matrix-assisted laser desorption ionization (MALDI) is a powerful analytical technique used in mass spectrometry. It enables the identification and ...

Mass Spectrometry: Aldehyde and Ketone Fragmentation

In mass spectrometry, the fragmentation of aliphatic aldehydes and ketones generally occurs through three key mechanisms: &#945;-cleavage, inductive cleavage, and the McLafferty rearrangement.
<ol>
	<li>&#945;-Cleavage 
	&#945;-Cleavage is a common fragmentation in carbonyl compounds where the bond adjacent to the carbonyl group is broken. This process produces an alkyl radical and an acylium cation, which is often detected in the mass spectrum due to its stability.</li>
	<li>Inductive Cleavage 
	In inductive cleavage, fragmentation yields an acyl radical and an alkyl cation. This occurs when electrons are drawn toward the carbonyl, weakening adjacent bonds and resulting in a distinctive fragmentation pattern. For example, in the mass spectrum of 5-methyl-2-hexanone, inductive cleavage produces an alkyl cation at a mass-to-charge (m/z) ratio of 71.</li>
	<li>McLafferty Rearrangement 
	The McLafferty rearrangement is a specific fragmentation pattern for carbonyl compounds with &#947;-hydrogen (hydrogen on the third carbon from the carbonyl group). The molecule undergoes a six-membered cyclic transition in this rearrangement, yielding a radical cation and a neutral alkene. For 5-methyl-2-hexanone, this rearrangement results in a peak at m/z 58.</li>
</ol>
Example: Fragmentation of 5-methyl-2-hexanone 
In the mass spectrum of 5-methyl-2-hexanone, the molecular ion undergoes these fragmentation patterns:
<ol style="list-style-type: circle;">
	<li>Inductive cleavage produces an alkyl cation at m/z 71.</li>
	<li>&#945;-Cleavage results in the base peak at m/z 43.</li>
	<li>McLafferty rearrangement creates a distinct peak at m/z 58.</li>
</ol>
An additional diagnostic feature in aldehydes is the M&#8722;1 peak, which arises due to the &#945;-cleavage of the aldehyde proton. In mass spectrometric analysis, this peak can help identify aldehydes, specifically among other carbonyl compounds.
Understanding these fragmentation pathways is crucial for interpreting mass spectra, particularly in identifying functional groups and distinguishing between similar compounds.

In mass spectrometry, the fragmentation of aliphatic aldehydes and ketones generally occurs through three key mechanisms: &#945;-cleavage, inductive ...

Mass Spectrometry: Alcohol Fragmentation

Alcohols (R-OH) ionize to lose one non-bonded electron from the oxygen atom, forming molecular ions. Due to their tendency to fragment rapidly, the intensity of the molecular ion peak in the mass spectrum is weak or sometimes absent. The fragmentation patterns for alcohols occur in two ways, i.e. &#9082;-cleavage and dehydration. During &#9082;-cleavage, the bond at the &#9082;-position adjacent to the hydroxyl group cleaves to give a resonance-stabilized cation and a radical. However, intramolecular dehydration involves losing a water molecule from an alcohol, forming an alkene cation, which shows a peak at M&#8722;18. Consider the fragmentation of butanol as shown below. &#9082;-cleavage produces a base peak at a mass-to-charge ratio of 31. In the case of dehydration, the fragmentation forms an alkene radical cation at a mass-to-charge ratio of 56.
<img alt="Figure1" src="/files/ftp_upload/13046/13046-PT_Figure_1.svg" style="text-align: center; height: 175px;" />

Alcohols (R-OH) ionize to lose one non-bonded electron from the oxygen atom, forming molecular ions. Due to their tendency to fragment rapidly, the ...

Mass Spectrometry: Alkyne Fragmentation

The fragmentation of alkynes preferentially occurs at the carbon&#8211;carbon bond between the &#945; and &#946; carbon of the alkyne bond to generate a 3-propynyl cation (or propargyl cation). In terminal alkynes, there is the only type of fragmentation that yields the 3-propynyl cation. The unsubstituted 3-propynyl cation exhibits a peak at a mass-to-charge ratio of 39. In internal alkynes, the 3-propynyl cation is substituted. For example, 2-pentyne fragments into methyl-substituted 3-propynyl cation, which subsequently features as a mass signal at the mass-to-charge ratio of 56. The 3-propynyl cation is resonance-stabilized, and the corresponding mass signal is intense in the mass spectrum of alkynes. Figure 1 shows the fragmentation of 1-pentyne into an ethyl radical and a resonance stabilized 3-propynyl cation.
<img alt="Figure1" src="/files/ftp_upload/13045/13045-PT_Figure_1.svg" style="text-align: center; height: 115px;" />
Figure 1: Fragmentation of 1-pentyne molecular ion to yield the 3-propynyl cation.
Another fragmentation pathway observed in terminal alkynes is the loss of hydrogen from the terminal sp carbon. This results in a mass signal with a mass-to-charge ratio of one unit less than the molecular ion. This M&#8211;1 peak is very intense and becomes the base peak in the mass spectra of many alkynes, so it can be used to identify alkynes.
<img alt="Figure2" src="/files/ftp_upload/13045/13045-PT_Figure_2.svg" style="text-align: center; height: 275px;" />
Figure 2: The mass spectrum of 1-pentyne.

The fragmentation of alkynes preferentially occurs at the carbon&#8211;carbon bond between the &#945; and &#946; carbon of the alkyne bond to generate a ...

Mass Spectrometry: Aromatic Compound Fragmentation

Upon ionization, aromatic compounds generate a molecular ion that is observed as a prominent peak in their mass spectra. For example, the molecular ion peak for benzene appears at a mass-to-charge ratio of 78, while toluene is observed at a mass-to-charge ratio of 92. The molecular ion benzene is highly stable and does not readily undergo further fragmentation due to the significant amount of energy required to disrupt the aromatic stability of the benzene ring. In contrast, the molecular ion of alkyl&#8208;substituted benzenes, such as toluene, can undergo fragmentation by losing a hydrogen atom, forming a benzylic carbocation. This carbocation can rearrange to form the resonance-stabilized tropylium cation, which appears in the mass spectra as a strong peak. In the case of toluene, this strong peak is observed at a mass-to-charge ratio of 91.

Upon ionization, aromatic compounds generate a molecular ion that is observed as a prominent peak in their mass spectra. For example, the molecular ion ...

Mass Spectrometry: Cycloalkene Fragmentation

The molecular ions of cycloalkenes undergo fragmentation via a retro-Diels&#8211;Alder reaction.

The reaction proceeds via the cleavage of two carbon-carbon bonds in the cycloalkene to yield ethene. The remaining part of the cycloalkene structure is a dienyl radical cation with a molecular weight of 28 u lower than the molecular ion. This fragmentation pathway is similar to the fragmentation of cycloalkanes releasing ethene, differing only in the resultant radical cation. An alkyl species forms from the cycloalkane, whereas a dienyl species originates from the cycloalkene.

Retro-Diels&#8211;Alder reactions of cycloalkenes are also similar to the McLafferty-rearrangement-type fragmentation observed in acyclic alkenes with hydrogen on the &#947; carbon. In both cases, cleavage occurs at two positions, yielding an ethene molecule and an alkenyl species. In acyclic alkenes, the cleavage occurs at one carbon-carbon bond and a carbon-hydrogen bond, along with rearranging hydrogen from one carbon to another. In cyclic alkenes, cleavage occurs at two carbon&#8211;carbon bonds without any atomic rearrangement. Figure 1 depicts the fragmentation of cyclohexene via a retro-Diels&#8211;Alder reaction.
<img alt="Figure1" src="/files/ftp_upload/13044/13044-PT_Figure_1.svg" style="text-align: center; height: 90px;" />
Figure 1: Fragmentation of cyclohexene via a retro-Diels&#8211;Alder reaction.
In addition to the retro-Diels&#8211;Alder reaction, branched cycloalkenes exhibit fragmentation via cleavage of the side chain to form a cycloalkene cation. For example, the fragmentation of 1-methyl-1-cyclohexene via a retro-Diels&#8211;Alder reaction and side-chain cleavage are shown in Figure 2.
<img alt="Figure2" src="/files/ftp_upload/13044/13044-PT_Figure_2.svg" style="text-align: center; height: 225px;" />
Figure 2: Fragmentation of 1-methyl-1-cyclohexene via (top) a retro-Diels&#8211;Alder reaction; (bottom) side-chain cleavage.
The corresponding signals of the isoprenyl radical cation and the cyclohexenyl cation are evident in the mass spectrum of 1-methyl-1-cyclohexene shown in Figure 3.
<img alt="Figure3" src="/files/ftp_upload/13044/13044-PT_Figure_3.svg" style="text-align: center; height: 250px;" />
Figure 3: Mass spectrum of 1-methyl-1-cyclohexene.

The molecular ions of cycloalkenes undergo fragmentation via a retro-Diels&#8211;Alder reaction.

The reaction proceeds via the cleavage of two ...

Mass Spectrometry: Amine Fragmentation

Amines can be identified using mass spectroscopy based on their characteristic fragmentation patterns. The molecular ions of amines undergo fragmentation via &#9082;-cleavage. The &#9082;-cleavage of the carbon-carbon bonds in amines generates an alkyl radical and resonance-stabilized nitrogen-containing cation.
In amines, the number of nitrogen atoms affects the mass of the molecular ion, which is described by the nitrogen rule of mass spectrometry. This rule states that a compound containing a single or an odd number of nitrogen atoms produces a molecular ion with an odd molecular weight, while a compound with no or an even number of nitrogen atoms generates a molecular ion with an even molecular weight.
For example, the mass spectrum of 3-methyl-1-butanamine, which contains one nitrogen atom, displays a molecular ion having an odd mass-to-charge ratio of 87. Fragmentation of this molecular ion via &#9082;-cleavage produces a base peak, which features a mass signal at a mass-to-charge ratio of 30, corresponding with the loss of the 2-methyl propyl radical.
Similarly, the fragmentation of triethylamine via &#9082;-cleavage generates a cation at a mass-to-charge ratio of 86. The molecular ion peak shows an odd mass-to-charge ratio of 101, as shown below.
<img alt="Figure1" src="/files/ftp_upload/13048/13048_PT_Figure_1.svg" style="text-align: center; height: 105px;" />

Amines can be identified using mass spectroscopy based on their characteristic fragmentation patterns. The molecular ions of amines undergo fragmentation ...

Mass Spectrometry: Long-Chain Alkane Fragmentation

The molecular ions of linear alkanes prefer to fragment at the carbon-carbon bond away from the end of the chain since the cleavage of an inner bond creates a stable carbocation and a stable radical. Consequently, the mass signals of linear alkanes feature intense peaks in the middle of the mass-to-charge ratio plot with weaker peaks on either end. The fragmentation of each carbon-carbon bond with the release of a methyl group in each splitting leads to prominent peaks in the mass spectra separated by 14 u. Smaller mass signals near each prominent peak result arise from secondary fragmentation at the carbon-hydrogen bond.
For example, consider the fragmentation pattern of n-hexane shown in Figure 1 and the corresponding mass spectra in Figure 2.
<img alt="Figure1" src="/files/ftp_upload/12299/12299-PT_Figure_1.svg" style="text-align: center; height: 500px;" />
Figure 1: Fragmentation of n-hexane molecular ion.
<img alt="Figure2" src="/files/ftp_upload/12299/12299-PT_Figure_2.svg" style="text-align: center; height: 275px;" />
Figure 2: Mass spectrum of n-hexane.
The signals at a mass-to-charge ratio of 71, 57, 43, 29, and 15 indicate that the cleavage can occur at all carbon-carbon bonds in the molecule. However, the relative abundance of these peaks varies. The difference between molecular ion signal and signal at 71 indicates the separation of methyl groups (M.W. = 15 u) group from molecular ions. Further release of a CH2 group from that fragment (M.W. = 14 u) results in a signal at 57. The signals at 43, 29, and 15 are due to the subsequent releases of CH2 groups. The abundance of signals at 15 and 71 is minimal due to the least probability for the formation of unstable methyl carbocation or a methyl radical, respectively. The base peak at 43 indicates the most preferred fragmentation, which yields a pair of stable carbocation and radical.

The molecular ions of linear alkanes prefer to fragment at the carbon-carbon bond away from the end of the chain since the cleavage of an inner bond ...

Eye Exam

Source: Richard Glickman-Simon, MD, Assistant Professor, Department of Public Health and Community Medicine, Tufts University School of Medicine, MA
Proper evaluation of the eyes in a general practice setting involves vision testing, orbit inspection, and ophthalmoscopic examination. Before beginning the exam, it is crucial to be familiar with the anatomy and physiology of the eye. The upper eyelid should be slightly over the iris, but it shouldn&#39;t cover the pupil when open; the lower lid lies below the iris. The sclera normally appears white or slightly buff in color. The appearance of conjunctiva, a transparent membrane covering the anterior sclera and the inner eyelids, is a sensitive indicator of ocular disorders, such as infections and inflammation. The tear-producing lacrimal gland lies above and lateral to the eyeball. Tears spread down and across the eye to drain medially into two lacrimal puncta before passing into the lacrimal sac and nasolacrimal duct to the nose.
The iris divides the anterior from the posterior chamber. Muscles of the iris control the size of the pupil, and muscles of the ciliary body behind it control the focal length of the lens. The ciliary body also produces aqueous humor, which largely determines intraocular pressure (Figure 1). Cranial nerves II and III control pupillary reaction and lens accommodation; cranial nerve III controls upper lid elevation; cranial nerves III, IV, and VI control eye movement. The six cardinal directions of gaze are controlled by six extraocular muscles (Figure 2) innervated by cranial nerves III, IV, and VI.
Visual testing is an essential part of the ophthalmological exam and is also performed as a part of cranial nerve II assessment during the neurological exam. A focused image is projected onto the retina after its light passes through the cornea, pupil, lens, and vitreous body. The projection is upside down and reversed right to left, which means that light entering from the lower temporal field of vision strikes the upper nasal quadrant of the retina. Photosensitive cells of the retina respond by generating electrical impulses, which are relayed to the optic nerve and passed to the visual cortex through the optic tracts. The right and left visual cortices process images entering from the left and right visual fields, respectively.
<img alt="Figure 1" src="/files/ftp_upload/10149/10149fig1.jpg" /> 
Figure 1. Anatomy of the Eye. A diagram showing a sagittal view of the human eye with the structures labeled.
<img alt="Figure 2" src="/files/ftp_upload/10149/10149fig2.jpg" />
Figure 2. Muscles of the Eye. A cartoon showing a frontal view of the human eye and the extraocular muscles (labeled).

Anatomy and Physiology of Eye; Acuity and Vision Testing

Esame obiettivo dell'occhio

Source: Richard Glickman-Simon, MD, Assistant Professor, Department of Public Health and Community Medicine, Tufts University School of Medicine, MA
...

Ophthalmoscopic Examination

Source: Richard Glickman-Simon, MD, Assistant Professor, Department of Public Health and Community Medicine, Tufts University School of Medicine, MA
The simplest ophthalmoscopes consist of an aperture to look through, a diopter indicator, and a disc for selecting lenses. The ophthalmoscope is primarily used to examine the fundus, or the inner wall of the posterior eye, which consists of the choroid, retina, fovea, macula, optic disc, and retinal vessels (Figure 1). The spherical eyeball collects and focuses light on the neurosensory cells of the retina. Light is refracted as it passes sequentially through the cornea, the lens, and the vitreous body.
The first landmark observed during the funduscopic exam is the optic disc, which is where the optic nerve and retinal vessels enter the back of the eye (Figure 2). The disc usually contains a central whitish physiologic cup where the vessels enter; it normally occupies less than half the diameter of the entire disc. Just lateral and slightly inferior is the fovea, a darkened circular area that demarcates the point of central vision. Around this is the macula. A blind spot approximately 15&#176; temporal to the line of gaze results from a lack of photoreceptor cells at the optic disc.
<img alt="Figure 1" src="/files/ftp_upload/10146/10146fig1.jpg" /> 
Figure 1. Anatomy of the eye. A diagram showing a sagittal view of the human eye with the structures labeled.
<img alt="Figure 2" src="/files/ftp_upload/10146/10146fig2.jpg" /> 
Figure 2: Normal retina. A photograph showing an ophthalmoscopic view on the normal retina.

Ophthalmoscopic Functioning and Examination of the Fundus

Esame oftalmoscopico

Source: Richard Glickman-Simon, MD, Assistant Professor, Department of Public Health and Community Medicine, Tufts University School of Medicine, MA
The ...

Ear Exam

Source: Richard Glickman-Simon, MD, Assistant Professor, Department of Public Health and Community Medicine, Tufts University School of Medicine, MA
This video describes the examination of the ear, beginning with a review of its surface and interior anatomy (Figure 1). The cartilaginous auricle consists of the helix, antihelix, earlobe, and tragus. The mastoid process is positioned just behind the earlobe. The slightly curving auditory canal ends at the tympanic membrane, which transmits sound waves collected by the external ear to the air-filled middle ear. The Eustachian tube connects to the middle ear with the nasopharynx. Vibrations of the tympanic membrane transmit to the three connected ossicles of the middle ear (the malleus, incus, and stapes). The vibrations are transformed into electrical signals in the inner ear, and then carried to the brain by the cochlear nerve. Hearing, therefore, comprises a conductive phase that involves the external and middle ear, and a sensorineural phase that involves the inner ear and cochlear nerve.
The auditory canal and the tympanic membrane are examined with the otoscope, a handheld instrument with a light source, a magnifier, and a disposable cone-shaped speculum. It is important to be familiar with the tympanic membrane landmarks (Figure 2). Only two of the three ossicles - the malleus and incus - can normally be seen; the malleus is near the center, and the uncus is just posterior. A cone of light can be seen emanating downward and anteriorly from the umbo, or point of contact between the membrane and the tip of the malleus. The short process roughly demarcates the boundary between the two regions of the tympanic membrane: the pars flaccida, lying superior and posterior, and the far larger pars tensa, lying anterior and inferior. Normally, the tympanic membrane is pink-gray in color and readily reflects the light of the otoscope.
<img alt="Figure 1" src="/files/ftp_upload/10148/10148fig1.jpg" /> 
Figure 1. Anatomy of the Ear. A schematic drawing of the human ear in frontal section with outer, middle, and inner ear structures labeled.

Anatomy of the Ear, External Ear Exam and Hearing Tests

Esame obiettivo dell'orecchio

Source: Richard Glickman-Simon, MD, Assistant Professor, Department of Public Health and Community Medicine, Tufts University School of Medicine, MA
This ...

Nose, Sinuses, Oral Cavity and Pharynx Exam

Source: Richard Glickman-Simon, MD, Assistant Professor, Department of Public Health and Community Medicine, Tufts University School of Medicine, MA
This video provides an overview of sinus, nose, and throat examinations. The demonstration begins with a brief overview of the anatomy of the region. The upper third of the nose is bony, and the bottom two-thirds are cartilaginous. Air entering the nares passes through the nasal vestibules and into the narrow passageway between the nasal septum medially and the bony turbinates laterally. Beneath each curving turbinate is a groove or meatus. The nasolacrimal duct and most of the air-filled paranasal sinuses drain into the inferior and middle meatuses, respectively. Of the three sets of paranasal sinuses, only the maxillary and frontal can be readily examined. A continuous, highly vascular mucosa lines the entire nasal cavity and sinuses.
<img alt="Figure 1" src="/files/ftp_upload/10152/10152fig1.jpg" /> 
Figure 1. Anatomy of the Nose. 
<img alt="Figure 2" src="/files/ftp_upload/10152/10152fig2.jpg" /> 
Figure 2. Location of the Major Sinuses.
Muscular folds of the lips mark the entrance to the oral cavity. The pinkish gingiva, or gums, attach firmly to the teeth and adjacent bone into which the teeth embed. The buccal mucosa lines the inner cheeks, where the Stensen duct drains the parotid glands into small papillae near the upper second molars. Small, red papillae cover the dorsal surface of the tongue along with a whitish coat of varying thickness. The midline lingual frenulum connects the undersurface of the tongue to the floor of the mouth, where the Wharton duct drains the submandibular glands into papillae on either side. Arching over the tongue are the anterior and posterior pillars of the soft palate and midline uvula. If present, the tonsils can be seen protruding bilaterally between the pillars. The bony hard palate rises anteriorly above the soft palate. The pharynx lies posteriorly behind the soft palate and tongue.
<img alt="Figure 3" src="/files/ftp_upload/10152/10152fig3.jpg" /> 
Figure 3. Anatomy of the Oral Cavity.
<img alt="Figure 4" src="/files/ftp_upload/10152/10152fig4.jpg" /> 
Figure 4. Pharyngeal Regions.

Esame di naso, seni, cavità orale e faringe

Thyroid Exam

Source: Richard Glickman-Simon, MD, Assistant Professor, Department of Public Health and Community Medicine, Tufts University School of Medicine, MA
The thyroid gland is located in the neck anterior trachea between the cricoid cartilage (above) and the suprasternal notch (below) (Figure 1). It consists of a right and left lobe connected by an isthmus. The isthmus covers the second, third, and fourth tracheal rings, and the lobes curve posteriorly around the sides of the trachea and esophagus. The normal gland, weighing 10 - 25 g, is usually invisible on inspection and often difficult to palpate. A goiter is an enlarged thyroid from any cause. In addition to assessing its size, it is important to palpate the thyroid for its shape, mobility, consistency, and tenderness. A normal thyroid is soft, smooth, symmetrical, and non-tender, and it slides upward slightly when swallowing. Symmetrical enlargement of a soft, smooth thyroid suggests endemic hypothyroidism due to iodine deficiency or one of two prevalent autoimmune disorders: Graves&#39; disease or Hashimoto&#39;s thyroiditis. Thyroid nodules are common and usually incidental; however, 10% of thyroid nodules turn out to be malignant. They may be single or multiple, and are most often firm and non-tender. A tender, symmetrical goiter typically indicates thyroiditis.
<img alt="Figure 1" src="/files/ftp_upload/10098/10098fig1.jpg" /> 
Figure 1. Anatomy of the thyroid gland. Illustration of the location and anatomy of the thyroid gland with respect to the neck structures.
Thyroid disease rarely manifests as a palpable goiter in isolation. Thyroid hormones serve to maintain homeostasis primarily by stimulating cellular metabolism throughout the body. Thus, hypo and hyperthyroidism are associated with a range of symptoms and physical findings (Table 1). It is important to note that goiters may be euthyroid (normal thyroid hormone levels), hyperthyroid, or hypothyroid. Headaches or visual disturbances may suggest a secondary thyroid disorder due to a pituitary adenoma
<img alt="Table 1" src="/files/ftp_upload/10098/10098table1.jpg" />
Table 1. Symptoms and physical findings for hypo- and hyper-thyroidism.&#160;

Thyroid Anatomy, Physiology and Palpation Examination

Esame obiettivo della tiroide

Lymph Node Exam

Source: Richard Glickman-Simon, MD, Assistant Professor, Department of Public Health and Community Medicine, Tufts University School of Medicine, MA
The lymphatic system has two main functions: to return extracellular fluid back to the venous circulation and to expose antigenic substances to the immune system. As the collected fluid passes through lymphatic channels on its way back to the systemic circulation, it encounters multiple nodes consisting of highly concentrated clusters of lymphocytes. Most lymph channels and nodes reside deep within the body and, therefore, are not accessible to physical exam (Figure 1). Only nodes near the surface can be inspected or palpated. Lymph nodes are normally invisible, and smaller nodes are also non-palpable. However, larger nodes (&#62;1 cm) in the neck, axillae, and inguinal areas are often detectable as soft, smooth, movable, non-tender, bean-shaped masses imbedded in subcutaneous tissue.
Lymphadenopathy usually indicates an infection or, less commonly, a cancer in the area of lymph drainage. Nodes may become enlarged, fixed, firm, and/or tender depending on the pathology present. For example, a soft, tender lymph node palpable near the angle of the mandible may indicate an infected tonsil, whereas a firm, enlarged, non-tender lymph node palpable in the axilla of a female patient may be a sign of breast cancer.
Regional lymph nodes draining the area of a localized infection typically remain invisible but may become enlarged and tender on palpation. An infected wound or cellulitis may also result in lymphangitis or lymphadenitis, a condition in which the infection spreads along the chain of lymph channels and nodes. This may be accompanied by the appearance of red streaks and systemic symptoms such as fever, chills, and malaise. In rare cases, the intensity of the inflammatory reaction may cause the nodes to adhere to the surrounding soft tissue, fixing them in place.
Many metastatic cancers spread to regional lymph nodes first. Unlike infections, malignant cells invading lymph nodes may cause them to feel irregular and firm (even rock hard) but remain non-tender. If the cancer invades the outer capsules, nodes may become fixed to the surrounding soft tissue or matted together. Lymphoma, a primary cancer intrinsic to the lymphatic system, may be present anywhere in the body as single or multiple enlarged lymph nodes, which may become big enough to see on inspection, and are generally hard and non-tender on palpation. In addition to lymphoma, diffuse lymphadenopathy may be an indication of generalized infectious or inflammatory disorders such as HIV, mononucleosis, or sarcoidosis.
<img alt="Figure 1" src="/files/ftp_upload/10061/10061fig1.jpg" /> 
Figure 1. The Lymphatic System.

Lymph Nodes: Head &amp; Neck, Axillae, Upper &amp; Lower Extremities

Esame obiettivo dei linfonodi

Abdominal Exam I: Inspection and Auscultation

Source: Alexander Goldfarb, MD, Assistant Professor of Medicine, Beth Israel Deaconess Medical Center, MA
Gastrointestinal disease accounts for millions of office visits and hospital admissions annually. Physical examination of the abdomen is a crucial tool in diagnosing diseases of the gastrointestinal tract; in addition, it can help identify pathological processes in cardiovascular, urinary, and other systems. As physical examination in general, the examination of the abdominal region is important for establishing physician-patient contact, for reaching the preliminary diagnosis and selecting subsequent laboratory and imaging tests, and determining the urgency of care.
As with the other parts of a physical examination, visual inspection and auscultation of the abdomen are done in a systematic fashion so that no potential findings are missed. Special attention should be paid to potential problems already identified by the patient&#39;s history. Here we assume that the patient has already been identified, and has had history taken, symptoms discussed, and areas of potential concern identified. In this video we will not review the patient&#39;s history; instead, we will go directly to the physical examination.
Before we get to the examination, let&#39;s briefly review surface landmarks of the abdominal region, abdominal anatomy, and topography. Here is a list of useful landmarks: costal margins, xiphoid process, rectus abdominal muscle, linea alba, umbilicus, iliac crest, inguinal ligament, and symphysis pubis. The abdominal exam covers the area down from the xiphoid and costal margins superiorly to the symphysis pubis inferiorly.
For diagnostic and descriptive purposes, the abdomen is subdivided into four quadrants: right upper quadrant (often designated as RUQ), left upper quadrant (LUQ), right lower quadrant (RLQ), and left lower quadrant (LLQ) (Figure 1). The more detailed topography of the abdomen divides it into 9 regions: right and left hypochondriac, right and left lumbar, right and left iliac, and also epigastric, umbilical, and hypogastric regions in the middle (Figure 2).
Remember which organs typically project into each abdominal region (Figure 3). It is essential to know the region&#39;s anatomy and topography well to adequately document and interpret a patient&#39;s complaints and symptoms, as well as physical findings during the examination.
<img alt="Figure 1" src="/files/ftp_upload/10088/10088fig1.jpg" /> 
Figure 1. Four abdominal quadrants. The abdomen can be divided into four regions by two imaginary lines intersecting at the umbilicus: right upper quadrant (often designated as RUQ), left upper quadrant (LUQ), right lower quadrant (RLQ), and left lower quadrant (LLQ) are shown.
<img alt="Figure 2" src="/files/ftp_upload/10088/10088fig2.jpg" /> 
Figure 2. Nine abdominal regions. Midclavicular lines and subcostal and intertubercular planes separate the abdomen into nine regions: epigastric region, right hypochondriac region, left hypochondriac region, umbilical region, right lumbar region, left lumbar region, hypogastric region, right inguinal region, and left inguinal region. Terms for epigastric, umbilical, and hypogastric and suprapubic regions are the most commonly used in clinical practice.
<img alt="Figure 3" src="/files/ftp_upload/10088/10088fig3.jpg" /> 
Figure 3. Location of different organs in the four abdominal regions. Organs in the abdominal cavity and their location with respect to four abdominal quadrants.

Abdominal Exam I: Anatomy, Topography and Auscultation

Esame obiettivo dell'addome I: ispezione e auscultazione

Source: Alexander Goldfarb, MD, Assistant Professor of Medicine, Beth Israel Deaconess Medical Center, MA
Gastrointestinal disease accounts for millions ...

Abdominal Exam II: Percussion

Source: Alexander Goldfarb, MD, Assistant Professor of Medicine, Beth Israel Deaconess Medical Center, MA
Medical percussion is based on the difference in pitch between the sounds elicited by tapping on the body wall. The auditory response to tapping depends on the ease with which the body wall vibrates, and is influenced by underlying organs, strength of the stroke, and the state of the body wall. There are three main medical percussion sounds: resonance (heard over lungs), tympany (heard over the air-filled bowel loops), and dullness (heard over fluid or solid organs). The contrast between dullness vs. tympany or resonance allows for determination of the size and margins of organs and masses, as well as identification of fluid accumulation and areas of consolidation. Percussion remains an intricate part of the physical diagnosis since it was first introduced more than 200 years ago, and is especially useful in examination of the lungs and abdomen.
As a part of an abdominal examination, percussion follows visual inspection and auscultation. The examiner should first percuss over each of the nine abdominal regions (epigastric region, right hypochondriac region, left hypochondriac region, umbilical region, right lumbar region, left lumbar region, hypogastric region, right inguinal region, and left inguinal region). Tenderness elicited by percussion is abnormal, and peritoneal inflammation should be suspected. As the air-filled bowel loops are positioned in the closest proximity to the abdominal wall, the percussion over most parts of the abdominal cavity elicits a predominantly tympanic sound. The presence of large areas of dullness should prompt evaluation for organomegaly, intra-abdominal masses, or fluid.
In addition to assessing the amount and distribution of abdominal gas, an abdominal exam should include an estimation of liver and spleen size by percussion. As the liver and spleen are covered by the rib cage, the examiner should percuss over the lower anterior chest as well. Normally, one expects to hear dull sound on percussion of the right anterior chest over the liver, and tympanic sound on percussion of left anterior chest over the gastric air bubble and the splenic flexure of the colon (Figure 1).
<img alt="Figure 1" src="/files/ftp_upload/10090/10090fig1.jpg" />
Figure 1. Normal percussion notes over abdominal region. With the exception of an area of dullness over the liver in the right lower anterior chest, tympany is the predominant sound heard over the region.

Abdominal Exam II: Percussion Sounds, Dullness and Tympany

Esame obiettivo dell'addome II: percussione

Source: Alexander Goldfarb, MD, Assistant Professor of Medicine, Beth Israel Deaconess Medical Center, MA
Medical percussion is based on the difference in ...

Abdominal Exam III: Palpation

Source: Alexander Goldfarb, MD, Assistant Professor of Medicine, Beth Israel Deaconess Medical Center, MA
Abdominal palpation, if performed correctly, allows for examination of the large and relatively superficial organs; for a skilled examiner, it allows for assessment of the smaller and deeper structures as well. The amount of information that can be obtained by palpation of the abdominal area also depends on the anatomical characteristics of the patient. For example, obesity might make palpation of internal organs difficult and require that additional maneuvers be performed. Abdominal palpation provides valuable information regarding localization of the problem and its severity, as abdominal palpation identifies the areas of tenderness as well as presence of organomegaly and tumors. The specific focus of palpation is driven by the information collected during history taking and other elements of the abdominal exam. Palpation helps to integrate this information and develop the strategy for subsequent diagnostic steps.

Abdominal Exam III: Light and Deep Palpation

Esame obiettivo dell'addome III: palpazione

Source: Alexander Goldfarb, MD, Assistant Professor of Medicine, Beth Israel Deaconess Medical Center, MA
Abdominal palpation, if performed correctly, ...

Abdominal Exam IV: Acute Abdominal Pain Assessment

Source: Joseph Donroe, MD, Internal Medicine and Pediatrics, Yale School of Medicine, New Haven, CT
Abdominal pain is a frequent presenting concern in both the emergency department and the office setting. Acute abdominal pain is defined as pain lasting less than seven days, while an acute abdomen refers to the abrupt onset of severe abdominal pain with features suggesting a surgically intervenable process. The differential diagnosis of acute abdominal pain is broad; thus, clinicians must have a systematic method of examination guided by a careful history, remembering that pathology outside of the abdomen can also cause abdominal pain, including pulmonary, cardiac, rectal, and genital disorders.
Terminology for describing the location of abdominal tenderness includes the right and left upper and lower quadrants, and the epigastric, umbilical, and hypogastric regions (Figures 1, 2). Thorough examination requires an organized approach involving inspection, auscultation, percussion, and palpation, with each maneuver performed purposefully and with a clear mental representation of the anatomy. Rather than palpating randomly across the abdomen, begin palpating remotely from the site of tenderness, moving systematically toward the tender region, and thinking about what lies below the fingers at each position. A helpful technique is to imagine a clock face with the xiphoid process at 12:00 and the pubic symphysis at 6:00 (Figure 3). When palpating at 8:00, there is skin, muscle, cecum, appendix, and ureters. Performing the exam in this fashion assists in clinical reasoning and minimizes the chance of missing pathology.
<img alt="Figure 1" src="/files/ftp_upload/10120/10120fig1.jpg" /> 
Figure 1. Four abdominal quadrants. Abdomen can be divided into four regions by two imaginary lines intersecting at umbilicus. Right upper quadrant (often designated as RUQ), left upper quadrant (LUQ), right lower quadrant (RLQ), and left lower quadrant (LLQ) are shown.
<img alt="Figure 2" src="/files/ftp_upload/10120/10120fig2.jpg" /> 
Figure 2. Nine abdominal regions. Midclavicular lines and subcostal and intertubercular planes separate abdomen into nine regions: epigastric region, right hypochondriac region, left hypochondriac region, umbilical region, right lumbar region, left lumbar region, hypogastric region, right inguinal region, and left inguinal region.
<img alt="Figure 3" src="/files/ftp_upload/10120/10120fig3.jpg" /> 
Figure 3. Visualizing a clock face over the abdomen for thinking about the underlying anatomy while performing the exam.

Esame obiettivo dell'addome IV: valutazione del dolore addominale acuto

Source: Joseph Donroe, MD, Internal Medicine and Pediatrics, Yale School of Medicine, New Haven, CT
Abdominal pain is a frequent presenting concern in ...

Male Rectal Exam

Source: Joseph Donroe, MD, Internal Medicine and Pediatrics, Yale School of Medicine, New Haven, CT
While its usefulness in cancer screening is debated, the male rectal examination remains an important part of the physical exam. The exam is indicated in selected patients with lower urinary tract symptoms, urinary and/or fecal incontinence or retention, back pain, anorectal symptoms, abdominal complaints, trauma patients, unexplained anemia, weight loss, or bone pain. There are no absolute contraindications to the rectal exam; however, relative contraindications include patient unwillingness to undergo the exam, severe rectal pain, recent anorectal surgery or trauma, and neutropenia.
When performing the rectal exam, the examiner should conceptualize the relevant anatomy. The external anal sphincter is the most distal part of the anal canal, which extends three to four centimeters before transitioning into the rectum. The prostate gland lies anterior to the rectum, just beyond the anal canal. The posterior surface of the prostate, including its apex, base, lateral lobes, and median sulcus, can be palpated through the rectal wall (Figure 1). The normal consistency of the prostate is similar to the thenar eminence when the hand is in a tight fist. The thumb knuckle is representative of what a hard nodule may feel like. The normal size of the prostate increases with age and is approximately 4.0 cm by 3.5 cm, or 20-30 g, in a young man.
The steps of the male rectal exam, including preparation, positioning, neurologic assessment, external inspection and palpation, examination of the prostate and rectum, and evaluation of the stool, are detailed in this video.
<img alt="Figure 1" src="/files/ftp_upload/10102/10102fig1.jpg" /> 
Figure 1. Male rectal anatomy (left) and regions of prostate gland posterior surface (right).

Male Rectal Anatomy and Exam Procedure

Esame rettale maschile

Source: Joseph Donroe, MD, Internal Medicine and Pediatrics, Yale School of Medicine, New Haven, CT
While its usefulness in cancer screening is debated, ...

Comprehensive Breast Exam

Source: 
Alexandra Duncan, GTA, Praxis Clinical, New Haven, CT 
Tiffany Cook, GTA, Praxis Clinical, New Haven, CT 
Jaideep S. Talwalkar, MD, Internal Medicine and Pediatrics, Yale School of Medicine, New Haven, CT
Breast exams are a key part of an annual gynecological exam and are important for all patients, no matter their sex or gender expression. One out of every 8 women will be diagnosed with breast cancer; male breast cancer, though less common, has a lifetime incidence of 1 in 1000.
Breast exams can feel invasive to patients, so it is important to do everything possible to make the patients feel comfortable and empowered, rather than vulnerable. Examiners should be aware of what they are communicating, both verbally and non-verbally, and give their patients control wherever possible (for instance, always allowing them to remove their own gowns). Examiners may choose to utilize chaperones for the patients&#39; (as well as their own) comfort. Some institutions require the use of chaperones.
While it is always important to avoid overly clinical language, certain colloquial words can cross the line from caring to overly intimate in this exam. It is helpful to avoid the words &#34;touch&#34; and &#34;feel&#34; in this exam, as this language can feel sexualized. Instead, use words like &#34;assess,&#34; &#34;check,&#34; or &#34;examine.&#34;
Additionally, the best practice dictates avoiding assumptions about patients&#39; gender, as patients with female anatomy may identify as another gender (e.g., transgender, genderqueer, etc.). This video depicts the approach to patients whose history has revealed no specific complaints or risk factors related to breast health.
In order to avoid missing potential findings, the breast exam should be performed in a systematic approach and consist of three main components: visual inspection of the breast tissue, palpation of the lymph nodes, and palpation of breast tissue.
The breast tissue extends from directly under the clavicle to around the fifth rib (or bra line). Laterally, it extends from the midaxillary line to the sternal border. The breast is viewed in four quadrants; the upper outer quadrant has the most tissue and is the location of many lymph nodes, and the tail of Spence (or axillary tail) extends to the edge of the axilla, where it attaches to the chest wall (Figure 1).
<img alt="Figure 1" src="/files/ftp_upload/10118/10118fig1.jpg" /> 
Figure 1. Breast anatomical landmarks.

Breast Tissue Anatomy; Tissue and Lymph Node Examination

Esame completo del seno

Source:
Alexandra Duncan, GTA, Praxis Clinical, New Haven, CT
Tiffany Cook, GTA, Praxis Clinical, New Haven, CT
Jaideep S. Talwalkar, MD, Internal ...

Pelvic Exam I: Assessment of the External Genitalia

Source: 
Alexandra Duncan, GTA, Praxis Clinical, New Haven, CT 
Tiffany Cook, GTA, Praxis Clinical, New Haven, CT 
<a href="https://www.jove.com/author/Jaideep+S._Talwalkar">Jaideep S. Talwalkar</a>, MD, Internal Medicine and Pediatrics, Yale School of Medicine, New Haven, CT
The pelvic exam can feel invasive to patients, so it is important to do everything possible to make patients feel comfortable and empowered, rather than vulnerable. Clinicians should be aware of how they are communicating, both verbally and nonverbally, and should give their patients control whenever possible. There are many ways to do this, from how the exam table is positioned to how the patient is engaged throughout the exam. As many as 1 in 5 patients may have experienced sexual trauma; therefore, it is important to avoid triggering those patients, but it&#39;s not always possible to know who they are. The exam in this video demonstrates neutral language and techniques that can be employed with all patients to create the best experience possible.
It&#39;s important to keep the patient covered wherever possible and to minimize extraneous contact. A clinician should be careful to tuck fingers that aren&#39;t being used to examine the patient to avoid accidental contact with the clitoris or anus.
Before performing the pelvic examination, examiners should find out how knowledgeable the patients are about the exam and their own bodies, and establish the expectation that the patients can communicate questions or concerns throughout the exam. While it is always important to avoid extremely clinical language, certain colloquial words can cross the line from caring to overly intimate during this exam. It is helpful to avoid the words &#34;touch&#34; and &#34;feel,&#34; which can feel sexualized in this context; instead, the words &#34;assess,&#34; &#34;check,&#34; &#34;inspect,&#34; or &#34;examine&#34; should be used. The words &#34;bed&#34; and &#34;sheet&#34; should be avoided, and &#34;table&#34; and &#34;drape&#34; should be used instead. Also, a clinician should use the word &#34;footrests&#34; rather than &#34;stirrups&#34; to avoid connoting horses. It&#39;s a good idea to avoid telling patients to &#34;relax,&#34; because it&#39;s a hard order for a patient to follow when anxious. Asking patients to &#34;soften&#34; or &#34;release&#34; specific muscles can be more useful, and having a patient do a Kegel exercise or Valsalva maneuver can serve as a specific relaxation technique.
Best practice dictates avoiding assumptions about patients&#39; gender, as patients with female anatomy may identify as another gender (e.g., transgender or genderqueer). This video depicts the approach to a patient whose history has revealed no specific complaints or risk factors related to gynecological health.
The pelvic exam consists of three parts: the visual and manual exam of the external genitalia, a speculum exam, and a bimanual exam. This video covers the introduction to the pelvic exam and the external genitalia exam. To avoid missing potential findings, the external pelvic exam should be performed in a systematic approach consisting of two main components: a visual inspection of the vulva (Figure 1), and internal palpation and assessment of glands and tone.
<img alt="Figure 1" src="/files/ftp_upload/10144/10144fig1.jpg" /> 
Figure 1. Diagram of the vulva. A diagram showing external genitals with the structures labelled.

Pelvic Exam I: External Genitalia Anatomy and Exam Procedure

Esame obiettivo ginecologico I: valutazione dei genitali esterni

Pelvic Exam II: Speculum Exam

Source: 
Alexandra Duncan, GTA, Praxis Clinical, New Haven, CT
Tiffany Cook, GTA, Praxis Clinical, New Haven, CT
Jaideep S. Talwalkar, MD, Internal Medicine and Pediatrics, Yale School of Medicine, New Haven, CT
Providing comfortable speculum placement is an important skill for providers to develop, since the speculum is a necessary tool in many gynecological procedures. Patients and providers are often anxious about the speculum exam, but it is entirely possible to place a speculum without patient discomfort. It&#39;s important for the clinician to be aware of the role language plays in creating a comfortable environment; for instance, a provider should refer to the speculum &#34;bills&#34; rather than &#34;blades&#34; to avoid upsetting the patient.
There are two types of speculums: metal and plastic (Figure 1). This demonstration utilizes plastic, as plastic speculums are most commonly used in clinics for routine testing. When using a metal speculum, it&#39;s recommended to use a Graves speculum if the patient has given birth vaginally, and a Pederson speculum if the patient has not. Pederson and Graves speculums are different shapes, and both come in many different sizes (medium is used most often). Prior to placing a metal speculum, it is helpful to perform a digital cervical exam to assess for the appropriate speculum size. The depth and direction of the cervix is estimated by placing one finger into the vagina. If the patient&#39;s cervix can be located while the patient is seated, it is likely that the patient has a shallow vagina, and therefore should be most comfortable with a short metal speculum.
<img alt="Figure 1" src="/files/ftp_upload/10141/10141fig1.jpg" /> 
Figure 1. A photograph of commercially available speculums in different sizes. 
Plastic speculums are all shaped like Pederson metal speculums and come in different sizes. To assess the appropriate size for a plastic speculum, the examiner places two fingers in the patient&#39;s vagina, palm down, and tries to separate the fingers: if there is no space between the fingers, a small plastic speculum should be used; if there is space between the fingers, a medium one should be used. The exam should never be performed with a large speculum (as it is significantly longer) without first determining the length of the vaginal canal.
The speculum is used to perform the Papanicolaou test as part of cervical cancer screening examinations. Cervical cancer was once the leading cause of cancer deaths for women in the United States, but in recent decades the number of cases and deaths has declined significantly1. This change is credited to the discovery made by Georgios Papanicolaou in 1928 that cervical cancer could be diagnosed by vaginal and cervical smears. The Pap test, as it is now called, detects abnormal cells in the cervix, both cancerous and pre-cancerous. Current guidelines for recommended screening intervals can be found through the U.S. Preventive Services Task Force (USPSTF) website2.
The test can be performed using either 1) a conventional glass slide and fixative with a spatula and endocervical brush (the traditional &#34;Pap smear&#34;) or 2) the more commonly utilized liquid-based cytology with a cervical broom or a spatula and endocervical brush (Figure 2). No matter what tools are used, the samples are collected from just inside the external os and the squamocolumnar junction, or transition zone around the os (Figure 3). This video demonstrates the spatula and endocervical brush with liquid-based cytology, as the liquid preparation is a more effective technique for the detection of cervical lesions, and the spatula and endocervical brush improve specimen collection.
<img alt="Figure 2" src="/files/ftp_upload/10141/10141fig2.jpg" /> 
Figure 2. Pap smear tools. Shown in sequence are: a liquid cytology canister, cervical broom, spatula, and endocervical brush.
<img alt="Figure 3" src="/files/ftp_upload/10141/10141fig3.jpg" /> 
Figure 3. Diagram of the cervix withrelevant structures labeled.

Pelvic Exam II: Speculum Exam and Pap Smear Test

Esame obiettivo ginecologico II: esame con lo speculum

Source: 
Alexandra Duncan, GTA, Praxis Clinical, New Haven, CT
Tiffany Cook, GTA, Praxis Clinical, New Haven, CT
Jaideep S. Talwalkar, MD, Internal ...

Pelvic Exam III:  Bimanual and Rectovaginal Exam

Source:
Alexandra Duncan, GTA, Praxis Clinical, New Haven, CT
Tiffany Cook, GTA, Praxis Clinical, New Haven, CT
<a href="https://www.jove.com/author/Jaideep+S._Talwalkar">Jaideep S. Talwalkar</a>, MD, Internal Medicine and Pediatrics, Yale School of Medicine, New Haven, CT
A bimanual exam is a thorough check of a patient&#39;s cervix, uterus, and ovaries. It can tell an experienced provider a great deal, as it may lead to the discovery of abnormalities, such as cysts, fibroids, or malignancies. However, it&#39;s useful even in the absence of such findings, as it allows the practitioner to establish an understanding of the patient&#39;s anatomy for future reference.
Performing the bimanual exam before the speculum exam can help relax patients, mentally and physically, before what is often perceived as the &#34;most invasive&#34; part of the exam. A practitioner already familiar with the patient&#39;s anatomy can insert a speculum more smoothly and comfortably. However, lubrication used during the bimanual exam may interfere with processing certain samples obtained during the speculum exam. Providers must be familiar with local laboratory processing requirements before committing to a specific order of examination.
This demonstration begins immediately after the end of the speculum exam; therefore, it assumes the patient has provided a history and is in the modified lithotomy position.
A rectovaginal exam is not always necessary, but it may be performed to fully assess a retroverted uterus and ovaries (this may be the only way to accomplish full assessment depending on uterine position) or to assess the rectum.

Education Physical Examinations Ii

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Education Physical Examinations Ii

@media screen and (min-width: 320px){.css-u03n47{font-size:var(--chakra-fontSizes-xs);}}Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)

Science Education (Competenze cliniche)