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2: Mastering the micropipette - Biology

2: Mastering the micropipette - Biology


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Learning Objectives

At the end of this laboratory, students will be able to:

  • select and adjust the most suitable micropipetteto transfer a given volume.
  • accurately transfer microliter volumes.
  • use a spectrophotometer to measure light absorbance.
  • explain how experimental errors affect measurements.

Welcome to the microworld! In this class, you will be working with microorganisms. These include yeast and bacteria, millions of which would fit into a period on this page. You will also be working with costly reagents, such as plasmids and enzymes. Therefore, in every experiment, you will be required to accurately measure volumes as small as a few microliters(μL). Micropipetteswill allow you to do this accurately and precisely.


Micropipettes

Arguably, the most important scientific equipment that you will use in this class are adjustable micropipettes, which you will use in nearly every experiment. Micropipettes are precision instruments that are designed to accurately and precisely transfer volumes in the microliter range. You may use microliters or milliliters as the units of volume in your lab notebooks and lab reports, but be careful to always state the volume unit that you are using. Recall the relationships between volume units:

1 microliter (abbreviated μL) = 10 -3 milliliter (mL) = 10 -6 liter (L)

Accuracy and precision

Accuracy depends on the micropipette delivering the correct volume. Precise results are reproducible. Let’s use a target analogy to demonstrate the difference between accurate and precise results. Imagine that four students try to hit the bulls-eye five times. Students A and B are precise, while students A and C are accurate.

Manufacturers determine the accuracy and precision of micropipettes by using them to transfer defined volumes of distilled water to a drop that is then weighed on an analytical balance. The density of water is 1.0 gram per mL at 25°C. The process is repeated several times during the calibration process, and the data is used to calculate the accuracy and precision of a micropipette.

Accuracy refers to the performance of the micropipette relative to a standard (the intended) value. Accuracy is computed from the difference between the actual volume dispensed by the micropipette and the intended volume. Note that this can be a negative or positive value. When micropipettes are calibrated, the accuracy is normally expressed as a percent of the selected value. Micropipettes are designed to operate with accuracies within a few percent (generally <3%) of the intended value. The accuracy of a micropipette decreases somewhat when micropipettes are set to deliver volumes close to the lowest values in their range.

Precision provides information about reproducibility, without any reference to a standard. Precision reflects random errors that can never be entirely eliminated from a procedure. Thus, a series of repeated measurements should generate a normal or binomial distribution. Precision is expressed as the standard deviation(s) of the set of measurements. In a normal distribution,

2/3 of measurements will fall within one standard deviation of the average or mean (x), and 95% of measurements will fall within two standard deviations of the mean. The standard deviation for a set of n measurements is calculated using the formula below.

Standard deviation describes the distribution of measurements relative to the mean value.

Choosing the micropipette

We use three different sizes of micropipettes in the laboratory, the P20, P200 and P1000. Our micropipettes have been purchased from several different manufacturers, but the principles of operation are the same. The numbers after the “P” refer to the maximum number of microliters that the micropipette is designed to transfer. Note that there is some overlap in the ranges of the different micropipettes. For example, both the P200 and P20 can be used to transfer 15 μl, but the P20 is more accurate within that range. As a rule of thumb, always select the smallest volume pipette that will transfer the desired volume.

Specifying the transfer volume

There are three numbers on the volume indicator. With each of the micropipettes, you will specify a volume to three digits by turning the volume adjustment knob. You will also be able to extrapolate between the lowest numbers with the vernier marks on the lower dial. Most of the measurements you will make with the micropipettes will be accurate to four significant figures!

Volume indicators on the P-1000, P-200 and P-20 micropipettes. The top digit on the P-1000 (red on some models) indicates milliliters and the bottom digit on the P-20 (red on some models) indicates tenths of microliters. Note the differences in accuracy between the micropipettes.

NEVER turn the indicator dial beyond the upper or lower volume limits of the micropipette!This could damage the piston.

Transferring volumes accurately

Micropipettes work by air displacement. The operator depresses a plunger that moves an internal piston to one of two different positions. The first stop is used to fill the micropipette tip, and the second stop is used to dispense the contents of the tip. As the operator depresses the plunger to the first stop, an internal piston displaces a volume of air equal to the volume shown on the volume indicator dial. The second stop is used only to dispense the contents of the tip.

Filling the micropipette
  • Remove the lid from the box containing the correct size micropipette tips. P-1000 tips may be blue or clear, while P-20 and P-200 tips are yellow or clear.
  • Attach the tip by inserting the shaft of the micropipette into the tip and pressing down firmly. This should produce an airtight seal between the tip and the shaft of the micropipette.
  • Replace the lid of the tip box to keep the remaining tips sterile. Avoid touching the tip (especially the thinner end), because the tips are sterile.
  • Depress the plunger of the micropipette to the FIRST stop.
  • Immerse the tip a few millimeters below the surface of the solution being drawn up into the pipette. Pipetting is most accurate when the pipette is held vertically. Keep the angle less than 20° from vertical for best results.
  • Release the plunger S L O W L Y, allowing the tip to fill smoothly. Pause briefly to ensure that the full volume of sample has entered the tip. Do NOT let the plunger snap up. This is particularly important when transferring larger volumes, because a splash could contaminate the shaft of the micropipette. If you inadvertently contaminate the shaft, clean it immediately with a damp Kimwipe. NEVER rest a micropipette with fluid in its tip on the bench!
Dispensing the contents of the micropipette
  • Place the micropipette tip against the side of the receiving test tube. Surface tension will help to dispense the contents of the micropipette. Do NOT attempt to eject the contents of the micropipette into “thin air.”
  • Smoothly depress the plunger to the first stop. Pause, then depress the plunger to the second stop. The contents of the pipette should have been largely released at the first stop. The second stop ensures that you’ve released the “last drop.”
  • Use the tip ejector to discard the tip. WARNING: The most common – and serious – operator error is depressing the plunger to the second stop before filling the micropipette tip. DO NOT DO THIS.

Mastering Biology Chapter 7 Answers

Cystic fibrosis is a genetic disease in humans in which chloride ion channels in cell membranes are missing or nonfunctional.

Chloride ion channels are membrane structures that include which of the following?

What name is given to the process by which water crosses a selectively permeable membrane?

A) phagocytosis
B) osmosis
C) passive transport
D) pinocytosis
E) diffusion

If there is a greater concentration of solute on the outside if the cell, it is considered to be what type of solution?

A) isotonic
B) hypertonic or isotonic
C) hypertonic
D) hypotonic and isotonic
E) hypotonic

The permeability of a biological membrane to a specific polar solute may depend on which of the following?

A) the amount of cholesterol in the membrane
B) the types of transport proteins in the membrane
C) the phospholipid composition of the membrane
D) the presence of unsaturated fatty acids in the membrane
E) the types of polysaccharides present in the membrane

According to the fluid mosaic model of membrane structure, proteins of the membrane are mostly

A) spread in a continuous layer over the inner and outer surfaces of the membrane.
B) free to depart from the fluid membrane and dissolve in the surrounding solution.
C) embedded in a lipid bilayer.
D) randomly oriented in the membrane, with no fixed inside-outside polarity.
E) confined to the hydrophobic interior of the membrane.

Which of the following factors would tend to increase membrane fluidity?

A) a lower temperature
B) a relatively high protein content in the membrane
C) a greater proportion of unsaturated phospholipids
D) a greater proportion of saturated phospholipids
E) a greater proportion of relatively large glycolipids compared with lipids having smaller molecular masses

Active and passive transport of solutes across a membrane typically differ in which of the following ways?

A) Active transport is usually down the concentration gradient of the solute, whereas passive transport is always against the concentration gradient of the solute.
B) Active transport always involves the utilization of cellular energy, whereas passive transport does not require cellular energy.
C) Active transport is always faster than passive transport.
D) Active transport uses protein carriers, whereas passive transport uses carbohydrate carriers.
E) Active transport is used for ions, passive transport is used for uncharged solutes

The movement of glucose into a cell against a concentration gradient is most likely to be accomplished by which of the following?

A) receptor-mediated endocytosis
B) passive diffusion of the glucose through the lipid bilayer
C) movement of glucose into the cell through a glucose channel
D) facilitated diffusion of the glucose using a carrier protein
E) cotransport of the glucose with a proton or sodium ion that was pumped across the membrane using the energy of ATP hydrolysis

A white blood cell engulfing a bacterium is an example of _____.

A) phagocytosis
B) exocytosis
C) pinocytosis
D) receptor-mediated endocytosis
E) facilitated diffusion

In facilitated diffusion, what is the role of the transport protein?

A) Transport proteins organize the phospholipids to allow the solute to cross the membrane.
B) Transport proteins provide a low-resistance channel for water molecules to cross the membrane.
C) Transport proteins provide a hydrophilic route for the solute to cross the membrane.
D) Transport proteins provide a protein site for ATP hydrolysis, which facilitates the movement of a solute across a membrane.
E) Transport proteins provide the energy for diffusion of the solute

If the concentration of phosphate in the cytosol is 2.0 mM and the concentration of phosphate in the surrounding fluid is 0.1 mM, how could the cell increase the concentration of phosphate in the cytosol?

A) passive transport
B) diffusion
C) active transport
D) osmosis
E) facilitated diffusion


Welcome to the Goldberg Lab

Since 1973, Goldberg laboratory has been investigating the molecular processes controlling the development of specialized cells in higher plants. Our long-term goal is to understand the genes and regulatory networks required to make a seed. Our research projects are supported by the National Science Foundation (NSF) Plant Genome Program.

The major questions our research addresses are (1) how are genes organized in the genome, (2) what are the mechanisms that control the regulation of plant gene expression, (3) what are the sequences that program plant gene expression during development, (4) what are the genes that control the differentiation of specific plant cell types, and (5) what events cause an undifferentiated cell to take on a specialized state. We use a variety of genomic approaches and model plants to answer these questions &mdash with a particular focus on identifying and using the best suited approach for answering each specific question.

Professor Goldberg is wholeheartedly committed to teaching and public education. He created and currently teaches a novel course sponsored by the NSF that utilizes long-distance learning to teach students simultaneously at UCLA, UC Davis, and Tuskegee University.


Professor
Member of the National Academy of Sciences
Howard Hughes Medical Institute Professor
Founder of Plant Cell Journal


Mastering Biology Chapter 7 Pretest Answers & Notes

d. The two sides of the plasma membrane have different lipid and protein composition.

Because the membrane serves different functions on the cytoplasmic and exterior surfaces, the structure and composition of the surfaces must be different.

Which of the following best describes the structure of a biological membrane?

a. two layers of phospholipids with proteins either crossing the layers or on the surface of the layers
b. two layers of phospholipids (with opposite orientations of the phospholipids in each layer) with each layer covered on the outside with proteins
c. two layers of phospholipids with proteins embedded between the two layers
d. a fluid structure in which phospholipids and proteins move freely between sides of the membrane
e. a mixture of covalently linked phospholipids and proteins that determines which solutes can cross the membrane and which cannot

a. two layers of phospholipids with proteins either crossing the layers or on the surface of the layers

*The membrane proteins can be found either embedded in or attached to the surface of the phospholipid bilayer.

The presence of aquaporins (proteins that form water channels in the membrane) should speed up the process of osmosis.

*Aquaporins facilitate water movement across membranes and thus speed up the process of osmosis.


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Microlit RBO Single Channel Variable Volume Micropipette is a High Precision Micropipette that is designed with ergonomics in mind. It facilitates remarkable user experience and impeccable accuracy in practical laboratory environments. The product is highly recommended for Molecular biology, Microbiology, Immunology, cell culture, Analytical Chemistry, Biochemistry, Genetics etc. 1ml pipette is our most popular micropipette model followed by the 200ul pipette.

What’s in the box?

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The Plunger has been carefully designed with a high quality spring mechanism to ensure snag free and soft movement.

Use Various Tips with a Universal Tipcone

A Universal Tipcone enhances the compatibility of the instrument and enables it to easily work with most of the internationally accepted standard tips. However, Microlit tips are recommended.

Set the Volume with Perfection

A soft click sound at every increment ensures perfect volume setting and prevents any accidental changes.

Store Safely with a Holder

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Eject Tips Easily with a Tip Ejector

Tip Ejector enables easy tip ejection and comfortable access to bottles and tubes with narrow necks.

A specially designed large Grippy provides a good grip and great ease of use while operation

Model No. Vol. Range(uL) Increment(uL) Accuaracy CV
+% +uL +% +uL
RBO-2 0.2-2.0ul 0.002 2 0.04 1.2 0.024
RBO-10 0.5-10ul 0.02 1 0.1 0.5 0.05
RBO-20 2-20ul 0.02 0.8 0.16 0.4 0.08
RBO-50 5-50ul 0.1 0.8 0.4 0.4 0.2
RBO-100 10-100ul 0.2 0.6 0.6 0.2 0.2
RBO-200 20-200ul 0.2 0.6 1.2 0.2 0.4
RBO-1000 100-1000ul 1.0 0.6 6 0.2 2
RBO-5000 0.5-5ml 10.0 0.6 30 0.2 10
RBO-10000 1-10ml 20.0 0.6 60 0.2 20

The error limits (Accuracy and Coefficient of Variation) mentioned above are in accordance with the nominal capacity (or maximum volume) indicated on the instrument. These are obtained by using the instrument with distilled water at equilibrium, the ambient temperature of 20 °C while operating it smoothly and steadily. The error limits are in accordance with DIN EN ISO 8655-2.

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Contents

Classical electrophysiological techniques Edit

Principle and mechanisms Edit

Electrophysiology is the branch of physiology that pertains broadly to the flow of ions (ion current) in biological tissues and, in particular, to the electrical recording techniques that enable the measurement of this flow. Classical electrophysiology techniques involve placing electrodes into various preparations of biological tissue. The principal types of electrodes are:

  1. simple solid conductors, such as discs and needles (singles or arrays, often insulated except for the tip),
  2. tracings on printed circuit boards or flexible polymers, also insulated except for the tip, and
  3. hollow tubes filled with an electrolyte, such as glass pipettes filled with potassium chloride solution or another electrolyte solution.

The principal preparations include:

  1. living organisms,
  2. excised tissue (acute or cultured),
  3. dissociated cells from excised tissue (acute or cultured),
  4. artificially grown cells or tissues, or
  5. hybrids of the above.

Neuronal electrophysiology is the study of electrical properties of biological cells and tissues within the nervous system. With neuronal electrophysiology doctors and specialists can determine how neuronal disorders happen, by looking at the individual's brain activity. Activity such as which portions of the brain light up during any situations encountered. If an electrode is small enough (micrometers) in diameter, then the electrophysiologist may choose to insert the tip into a single cell. Such a configuration allows direct observation and recording of the intracellular electrical activity of a single cell. However, this invasive setup reduces the life of the cell and causes a leak of substances across the cell membrane. Intracellular activity may also be observed using a specially formed (hollow) glass pipette containing an electrolyte. In this technique, the microscopic pipette tip is pressed against the cell membrane, to which it tightly adheres by an interaction between glass and lipids of the cell membrane. The electrolyte within the pipette may be brought into fluid continuity with the cytoplasm by delivering a pulse of negative pressure to the pipette in order to rupture the small patch of membrane encircled by the pipette rim (whole-cell recording). Alternatively, ionic continuity may be established by "perforating" the patch by allowing exogenous pore-forming agent within the electrolyte to insert themselves into the membrane patch (perforated patch recording). Finally, the patch may be left intact (patch recording).

The electrophysiologist may choose not to insert the tip into a single cell. Instead, the electrode tip may be left in continuity with the extracellular space. If the tip is small enough, such a configuration may allow indirect observation and recording of action potentials from a single cell, termed single-unit recording. Depending on the preparation and precise placement, an extracellular configuration may pick up the activity of several nearby cells simultaneously, termed multi-unit recording.

As electrode size increases, the resolving power decreases. Larger electrodes are sensitive only to the net activity of many cells, termed local field potentials. Still larger electrodes, such as uninsulated needles and surface electrodes used by clinical and surgical neurophysiologists, are sensitive only to certain types of synchronous activity within populations of cells numbering in the millions.

Other classical electrophysiological techniques include single channel recording and amperometry.

Electrographic modalities by body part Edit

Electrophysiological recording in general is sometimes called electrography (from electro- + -graphy, "electrical recording"), with the record thus produced being an electrogram. However, the word electrography has other senses (including electrophotography), and the specific types of electrophysiological recording are usually called by specific names, constructed on the pattern of electro- + [body part combining form] + -graphy (abbreviation ExG). Relatedly, the word electrogram (not being needed for those other senses) often carries the specific meaning of intracardiac electrogram, which is like an electrocardiogram but with some invasive leads (inside the heart) rather than only noninvasive leads (on the skin). Electrophysiological recording for clinical diagnostic purposes is included within the category of electrodiagnostic testing. The various "ExG" modes are as follows:

Modality Abbreviation Body part Prevalence in clinical use
electrocardiography ECG or EKG heart (specifically, the cardiac muscle), with cutaneous electrodes (noninvasive) 1—very common
electroatriography EAG atrial cardiac muscle 3—uncommon
electroventriculography EVG ventricular cardiac muscle 3—uncommon
intracardiac electrogram EGM heart (specifically, the cardiac muscle), with intracardiac electrodes (invasive) 2—somewhat common
electroencephalography EEG brain (usually the cerebral cortex), with extracranial electrodes 2—somewhat common
electrocorticography ECoG or iEEG brain (specifically the cerebral cortex), with intracranial electrodes 2—somewhat common
electromyography EMG muscles throughout the body (usually skeletal, occasionally smooth) 1—very common
electrooculography EOG eye—entire globe 2—somewhat common
electroretinography ERG eye—retina specifically 2—somewhat common
electronystagmography ENG eye—via the corneoretinal potential 2—somewhat common
electroolfactography EOG olfactory epithelium in mammals 3—uncommon
electroantennography EAG olfactory receptors in arthropod antennae 4—not applicable clinically
electrocochleography ECOG or ECochG cochlea 2—somewhat common
electrogastrography EGG stomach smooth muscle 2—somewhat common
electrogastroenterography EGEG stomach and bowel smooth muscle 2—somewhat common
electroglottography EGG glottis 3—uncommon
electropalatography EPG palatal contact of tongue 3—uncommon
electroarteriography EAG arterial flow via streaming potential detected through skin [2] 3—uncommon
electroblepharography EBG eyelid muscle 3—uncommon
electrodermography EDG skin 3—uncommon
electrohysterography EHG uterus 3—uncommon
electroneuronography ENeG or ENoG nerves 3—uncommon
electropneumography EPG lungs (chest movements) 3—uncommon
electrospinography ESG spinal cord 3—uncommon
electrovomerography EVG vomeronasal organ 3—uncommon

Optical electrophysiological techniques Edit

Optical electrophysiological techniques were created by scientists and engineers to overcome one of the main limitations of classical techniques. Classical techniques allow observation of electrical activity at approximately a single point within a volume of tissue. Classical techniques singularize a distributed phenomenon. Interest in the spatial distribution of bioelectric activity prompted development of molecules capable of emitting light in response to their electrical or chemical environment. Examples are voltage sensitive dyes and fluorescing proteins.

After introducing one or more such compounds into tissue via perfusion, injection or gene expression, the 1 or 2-dimensional distribution of electrical activity may be observed and recorded.

Intracellular recording involves measuring voltage and/or current across the membrane of a cell. To make an intracellular recording, the tip of a fine (sharp) microelectrode must be inserted inside the cell, so that the membrane potential can be measured. Typically, the resting membrane potential of a healthy cell will be -60 to -80 mV, and during an action potential the membrane potential might reach +40 mV. In 1963, Alan Lloyd Hodgkin and Andrew Fielding Huxley won the Nobel Prize in Physiology or Medicine for their contribution to understanding the mechanisms underlying the generation of action potentials in neurons. Their experiments involved intracellular recordings from the giant axon of Atlantic squid (Loligo pealei), and were among the first applications of the "voltage clamp" technique. [3] Today, most microelectrodes used for intracellular recording are glass micropipettes, with a tip diameter of < 1 micrometre, and a resistance of several megohms. The micropipettes are filled with a solution that has a similar ionic composition to the intracellular fluid of the cell. A chlorided silver wire inserted into the pipet connects the electrolyte electrically to the amplifier and signal processing circuit. The voltage measured by the electrode is compared to the voltage of a reference electrode, usually a silver chloride-coated silver wire in contact with the extracellular fluid around the cell. In general, the smaller the electrode tip, the higher its electrical resistance, so an electrode is a compromise between size (small enough to penetrate a single cell with minimum damage to the cell) and resistance (low enough so that small neuronal signals can be discerned from thermal noise in the electrode tip).

Voltage clamp Edit

The voltage clamp technique allows an experimenter to "clamp" the cell potential at a chosen value. This makes it possible to measure how much ionic current crosses a cell's membrane at any given voltage. This is important because many of the ion channels in the membrane of a neuron are voltage-gated ion channels, which open only when the membrane voltage is within a certain range. Voltage clamp measurements of current are made possible by the near-simultaneous digital subtraction of transient capacitive currents that pass as the recording electrode and cell membrane are charged to alter the cell's potential.

Current clamp Edit

The current clamp technique records the membrane potential by injecting current into a cell through the recording electrode. Unlike in the voltage clamp mode, where the membrane potential is held at a level determined by the experimenter, in "current clamp" mode the membrane potential is free to vary, and the amplifier records whatever voltage the cell generates on its own or as a result of stimulation. This technique is used to study how a cell responds when electric current enters a cell this is important for instance for understanding how neurons respond to neurotransmitters that act by opening membrane ion channels.

Most current-clamp amplifiers provide little or no amplification of the voltage changes recorded from the cell. The "amplifier" is actually an electrometer, sometimes referred to as a "unity gain amplifier" its main purpose is to reduce the electrical load on the small signals (in the mV range) produced by cells so that they can be accurately recorded by low-impedance electronics. The amplifier increases the current behind the signal while decreasing the resistance over which that current passes. Consider this example based on Ohm's law: A voltage of 10 mV is generated by passing 10 nanoamperes of current across 1 MΩ of resistance. The electrometer changes this "high impedance signal" to a "low impedance signal" by using a voltage follower circuit. A voltage follower reads the voltage on the input (caused by a small current through a big resistor). It then instructs a parallel circuit that has a large current source behind it (the electrical mains) and adjusts the resistance of that parallel circuit to give the same output voltage, but across a lower resistance.

Patch-clamp recording Edit

This technique was developed by Erwin Neher and Bert Sakmann who received the Nobel Prize in 1991. [4] Conventional intracellular recording involves impaling a cell with a fine electrode patch-clamp recording takes a different approach. A patch-clamp microelectrode is a micropipette with a relatively large tip diameter. The microelectrode is placed next to a cell, and gentle suction is applied through the microelectrode to draw a piece of the cell membrane (the 'patch') into the microelectrode tip the glass tip forms a high resistance 'seal' with the cell membrane. This configuration is the "cell-attached" mode, and it can be used for studying the activity of the ion channels that are present in the patch of membrane. If more suction is now applied, the small patch of membrane in the electrode tip can be displaced, leaving the electrode sealed to the rest of the cell. This "whole-cell" mode allows very stable intracellular recording. A disadvantage (compared to conventional intracellular recording with sharp electrodes) is that the intracellular fluid of the cell mixes with the solution inside the recording electrode, and so some important components of the intracellular fluid can be diluted. A variant of this technique, the "perforated patch" technique, tries to minimise these problems. Instead of applying suction to displace the membrane patch from the electrode tip, it is also possible to make small holes on the patch with pore-forming agents so that large molecules such as proteins can stay inside the cell and ions can pass through the holes freely. Also the patch of membrane can be pulled away from the rest of the cell. This approach enables the membrane properties of the patch to be analysed pharmacologically.

Sharp electrode recording Edit

In situations where one wants to record the potential inside the cell membrane with minimal effect on the ionic constitution of the intracellular fluid a sharp electrode can be used. These micropipettes (electrodes) are again like those for patch clamp pulled from glass capillaries, but the pore is much smaller so that there is very little ion exchange between the intracellular fluid and the electrolyte in the pipette. The electrical resistance of the micropipette electrode is reduced by filling with 2-4M KCl, rather than a salt concentration which mimics the intracellular ionic concentrations as used in patch clamping. [5] Often the tip of the electrode is filled with various kinds of dyes like Lucifer yellow to fill the cells recorded from, for later confirmation of their morphology under a microscope. The dyes are injected by applying a positive or negative, DC or pulsed voltage to the electrodes depending on the polarity of the dye.

Single-unit recording Edit

An electrode introduced into the brain of a living animal will detect electrical activity that is generated by the neurons adjacent to the electrode tip. If the electrode is a microelectrode, with a tip size of about 1 micrometre, the electrode will usually detect the activity of at most one neuron. Recording in this way is in general called "single-unit" recording. The action potentials recorded are very much like the action potentials that are recorded intracellularly, but the signals are very much smaller (typically about 1 mV). Most recordings of the activity of single neurons in anesthetized and conscious animals are made in this way. Recordings of single neurons in living animals have provided important insights into how the brain processes information. For example, David Hubel and Torsten Wiesel recorded the activity of single neurons in the primary visual cortex of the anesthetized cat, and showed how single neurons in this area respond to very specific features of a visual stimulus. [6] Hubel and Wiesel were awarded the Nobel Prize in Physiology or Medicine in 1981. [7]

Multi-unit recording Edit

If the electrode tip is slightly larger, then the electrode might record the activity generated by several neurons. This type of recording is often called "multi-unit recording", and is often used in conscious animals to record changes in the activity in a discrete brain area during normal activity. Recordings from one or more such electrodes that are closely spaced can be used to identify the number of cells around it as well as which of the spikes come from which cell. This process is called spike sorting and is suitable in areas where there are identified types of cells with well defined spike characteristics. If the electrode tip is bigger still, in general the activity of individual neurons cannot be distinguished but the electrode will still be able to record a field potential generated by the activity of many cells.

Field potentials Edit

Extracellular field potentials are local current sinks or sources that are generated by the collective activity of many cells. Usually, a field potential is generated by the simultaneous activation of many neurons by synaptic transmission. The diagram to the right shows hippocampal synaptic field potentials. At the right, the lower trace shows a negative wave that corresponds to a current sink caused by positive charges entering cells through postsynaptic glutamate receptors, while the upper trace shows a positive wave that is generated by the current that leaves the cell (at the cell body) to complete the circuit. For more information, see local field potential.

Amperometry Edit

Amperometry uses a carbon electrode to record changes in the chemical composition of the oxidized components of a biological solution. Oxidation and reduction is accomplished by changing the voltage at the active surface of the recording electrode in a process known as "scanning". Because certain brain chemicals lose or gain electrons at characteristic voltages, individual species can be identified. Amperometry has been used for studying exocytosis in the nervous and endocrine systems. Many monoamine neurotransmitters e.g., norepinephrine (noradrenalin), dopamine, and serotonin (5-HT) are oxidizable. The method can also be used with cells that do not secrete oxidizable neurotransmitters by "loading" them with 5-HT or dopamine.

Planar patch clamp is a novel method developed for high throughput electrophysiology. [8] Instead of positioning a pipette on an adherent cell, cell suspension is pipetted on a chip containing a microstructured aperture. A single cell is then positioned on the hole by suction and a tight connection (Gigaseal) is formed. The planar geometry offers a variety of advantages compared to the classical experiment:

  • It allows for integration of microfluidics, which enables automatic compound application for ion channel screening.
  • The system is accessible for optical or scanning probe techniques. of the intracellular side can be performed.

Schematic drawing of the classical patch clamp configuration. The patch pipette is moved to the cell using a micromanipulator under optical control. Relative movements between the pipette and the cell have to be avoided in order to keep the cell-pipette connection intact.

Scanning electron microscope image of a patch pipette.

In planar patch configuration, the cell is positioned by suction. Relative movements between cell and aperture can then be excluded after sealing. An antivibration table is not necessary.

Scanning electron microscope image of a planar patch clamp chip. Both the pipette and the chip are made from borosilicate glass.

Solid-supported membrane (SSM)-based Edit

With this electrophysiological approach, proteoliposomes, membrane vesicles, or membrane fragments containing the channel or transporter of interest are adsorbed to a lipid monolayer painted over a functionalized electrode. This electrode consists of a glass support, a chromium layer, a gold layer, and an octadecyl mercaptane monolayer. Because the painted membrane is supported by the electrode, it is called a solid-supported membrane. It is important to note that mechanical perturbations, which usually destroy a biological lipid membrane, do not influence the life-time of an SSM. The capacitive electrode (composed of the SSM and the absorbed vesicles) is so mechanically stable that solutions may be rapidly exchanged at its surface. This property allows the application of rapid substrate/ligand concentration jumps to investigate the electrogenic activity of the protein of interest, measured via capacitive coupling between the vesicles and the electrode. [9]

Bioelectric recognition assay (BERA) Edit

The bioelectric recognition assay (BERA) is a novel method for determination of various chemical and biological molecules by measuring changes in the membrane potential of cells immobilized in a gel matrix. Apart from the increased stability of the electrode-cell interface, immobilization preserves the viability and physiological functions of the cells. BERA is used primarily in biosensor applications in order to assay analytes that can interact with the immobilized cells by changing the cell membrane potential. In this way, when a positive sample is added to the sensor, a characteristic, "signature-like" change in electrical potential occurs. BERA is the core technology behind the recently launched pan-European FOODSCAN project, about pesticide and food risk assessment in Europe. [10] BERA has been used for the detection of human viruses (hepatitis B and C viruses and herpes viruses), [11] veterinary disease agents (foot and mouth disease virus, prions, and blue tongue virus), and plant viruses (tobacco and cucumber viruses) [12] in a specific, rapid (1–2 minutes), reproducible, and cost-efficient fashion. The method has also been used for the detection of environmental toxins, such as pesticides [13] [14] [15] and mycotoxins [16] in food, and 2,4,6-trichloroanisole in cork and wine, [17] [18] as well as the determination of very low concentrations of the superoxide anion in clinical samples. [19] [20]

A BERA sensor has two parts:

A recent advance is the development of a technique called molecular identification through membrane engineering (MIME). This technique allows for building cells with defined specificity for virtually any molecule of interest, by embedding thousands of artificial receptors into the cell membrane. [22]

Computational electrophysiology Edit

While not strictly constituting an experimental measurement, methods have been developed to examine the conductive properties of proteins and biomembranes in silico. These are mainly molecular dynamics simulations in which a model system like a lipid bilayer is subjected to an externally applied voltage. Studies using these setups have been able to study dynamical phenomena like electroporation of membranes [23] and ion translocation by channels. [24]

The benefit of such methods is the high level of detail of the active conduction mechanism, given by the inherently high resolution and data density that atomistic simulation affords. There are significant drawbacks, given by the uncertainty of the legitimacy of the model and the computational cost of modeling systems that are large enough and over sufficient timescales to be considered reproducing the macroscopic properties of the systems themselves. While atomistic simulations may access timescales close to, or into the microsecond domain, this is still several orders of magnitude lower than even the resolution of experimental methods such as patch-clamping. [ citation needed ]

Clinical electrophysiology is the study of how electrophysiological principles and technologies can be applied to human health. For example, clinical cardiac electrophysiology is the study of the electrical properties which govern heart rhythm and activity. Cardiac electrophysiology can be used to observe and treat disorders such as arrhythmia (irregular heartbeat). For example, a doctor may insert a catheter containing an electrode into the heart to record the heart muscle's electrical activity.

Another example of clinical electrophysiology is clinical neurophysiology. In this medical specialty, doctors measure the electrical properties of the brain, spinal cord, and nerves. Scientists such as Duchenne de Boulogne (1806–1875) and Nathaniel A. Buchwald (1924–2006) are considered to have greatly advanced the field of neurophysiology, enabling its clinical applications.

Clinical reporting guidelines Edit

Minimum Information (MI) standards or reporting guidelines specify the minimum amount of meta data (information) and data required to meet a specific aim or aims in a clinical study. The "Minimum Information about a Neuroscience investigation" (MINI) family of reporting guideline documents aims to provide a consistent set of guidelines in order to report an electrophysiology experiment. In practice a MINI module comprises a checklist of information that should be provided (for example about the protocols employed) when a data set is described for publication. [25]


Is there a demand for biotechnology?

BLS projects a 7 percent growth for biological laboratory technicians from 2018-2028, which is faster than the average for all occupations. Other biotechnology jobs are expected to grow at least as fast as average as more industries look for people who understand science and are adept at data analysis.

“People looking for jobs that deal with large amounts of data using bioinformatics approaches are especially well-positioned due to a growing need,” Bean says. “Overall, there will be no shortage of opportunities in biotechnology. It is a really fun field where you can combine your interest in science with so many other areas, so that makes it enticing for a lot of people.”


Course Description

During Fall 2020, all MIT students and the general public were welcomed to join Professors Richard Young and Facundo Batista as they discussed the science of the pandemic during this new class. The livestream of the lectures was available to the public, but only registered students were able to ask questions during the Q&A.

Special guest speakers included: Drs. Anthony Fauci, David Baltimore, James Bradner, Victoria Clark, Kizzmekia Corbett, Britt Glaunsinger, Akiko Iwasaki, Eric Lander, Michael Mina, Michel Nussenzweig, Shiv Pillai, Arlene Sharpe, Skip Virgin, and Bruce Walker.