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Sittampalam GS, Gal-Edd N, Arkin M, et al., editors. Assay Guidance Manual [Internet]. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004-.

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Assay Guidance Manual [Internet].

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Basics of Assay Equipment and Instrumentation for High Throughput Screening

, , and .

Author Information

,1 ,2 and 3.

1 National Center for Advancing Translational Sciences
National Institutes of Health
2 National Center for Advancing Translational Sciences
National Institutes of Health
3 National Center for Advancing Translational Sciences
National Institutes of Health

Published ; Last Update: November 1, 2013.

Abstract

This chapter contains a synopsis of general and specialized instrumentation used in screening and lead optimization laboratories. The instrument type is described along with the general principles of operation to familiarize readers considering equipping drug discovery laboratories, principally directed to new investigators. The descriptions are introductory and detailed information on installation and applications should be obtained from instrument vendors and experienced drug discovery scientists and engineers.

Introduction

Instruments employed in quantitative biology laboratories can be broadly categorized into microplates, microplate readers, microplate handling equipment, liquid handling equipment and other miscellaneous instruments such as safety equipment, centrifuges, incubators, electronic balances, microscopes, pH meters, spectrophotometers and other bench-top equipment routinely used in high throughput screening (HTS) assay development laboratories. It is absolutely important to familiarize yourself with the equipment and its proper use.

Common Equipment in HTS Labs

1.

Microplates

2.

Microplate Readers

3.

Liquid Handling Equipment

4.

Microplate Handling Systems

5.

Misc. Benchtop Equipment

6.

Hoods, Incubators & Freezers

Microplates

The microplate is the standard format of miniaturization and automation for bioassays (Biochemical and cell-based assays) associated with drug discovery. Within each microplate is a 2D array of wells with a limited volume for the experimentation to take place. The most common well densities for screening come in 96, 384 and 1536 wells per plate. Numerous manufacturers make microplates in a wide range of materials specific to equipment and customer needs. The footprint of the microplate and well locations has been standardized by ANSI (American National Standards Institute) and SLAS (Society for Laboratory Automation and Screening).

In HTS laboratories, microplates are generally categorized into “Compound Plates” and “Assay Plates”. Compound plates are for storage of the molecular library to be screened against and durable such the same plate can be used for extended periods and across several screens. Assay plates are where the experimentation takes place, selected based on the assay conditions and only used for the extent of an individual screen.

Available Plate Configurations

Figure 1: . 24 Well Microplate, Source PerkinElmer http://www.

Figure 1:

24 Well Microplate, Source PerkinElmer http://www.perkinelmer.com

Figure 2: . 96 Well Microplate, Source PerkinElmer http://www.

Figure 2:

96 Well Microplate, Source PerkinElmer http://www.perkinelmer.com

Figure 3: . 384-Well Microplate, Source PerkinElmer http://www.

Figure 3:

384-Well Microplate, Source PerkinElmer http://www.perkinelmer.com

Figure 4: . 1536-Well Microplate, Source PerkinElmer http://www.

Figure 4:

1536-Well Microplate, Source PerkinElmer http://www.perkinelmer.com

Most commonly used microplates in HTS applications have 96-wells (12 columns × 8 rows) with 250-300 volume µL capacity. Recent introductions include 96-well half area, 384- and 1536-well plates using the same outside plate dimensions (or foot print). The 96-well half area and 384-well plates use less reagents (75-100 µL capacity), and a major advantage is the reduced reagent consumption. However, these plates require special care during reagent delivery. The 1536-well plates are used less commonly, and require special liquid delivery equipment.

Plate Color

Microplates are typically offered in opaque white, opaque black or translucent. Compound plates are typically translucent such that compound volumes and color can be seen. Opaque plates are used to enhance detection technologies. Generally white assay plates are for luminance assays and black assay plates are for florescence assays. The bottom material of an opaque plate may be clear to support bottom reads and colorimetric (absorbance) assays.

Microplate Materials

Microplates are available in numerous materials that have different characteristics and, as such, may be more appropriate for specific applications. The list below provides some examples of microplate materials, their characteristics, and applications for the different plate materials.

  • Polystyrene (PS)
    • Typical material for assay plates
    • Low production cost
    • Rigid and brittle
    • Some natural binding properties to biomolecules
  • Cyclic Olefin Copolymer (COC)
    • Material for assay and compound plates
    • Works well for acoustic dispensing
    • Less susceptible to breakage during handling
    • DMSO resistant
  • Polypropylene (PP)
    • Typical material for compound plates
    • DMSO resistant
    • Thermal Stability
    • More durable than PS

Well Bottom Shape

The shapes of the wells in microplates vary with the application. Flat bottoms are standard and also required for some detection technologies that use transmission of light through the bottom material. Round bottoms can aid in cell washing and V-bottoms reduce dead volumes when transferring volumes between plates.

Plate Bottom Material

The bottom material of a plate is often specific to the detection technology. Many assays require reading the plate from underneath therefore the plate bottom material must be optically clear. The thickness and quality of clarity varies with the materials used, often a plastic or glass. Microscopy biased detection systems with higher objectives would require more optical clarity then most microplate readers and therefor consideration in comparing materials to costs should be made.

Surface Treatments

To aid in assay performance many surface treatment options are available. Polystyrene inherently binds biomolecules with large hydrophobic regions through passive interactions without treatment. Surface treatment options can enhance or minimize this effect. Tissue culture treated surfaces are also common to aid in cell attachment. These either affect the binding of biomolecules to microplate surfaces or improve the attachment of cell lines.

Microplate Sealing

For the purpose of maintaining specific microplate well conditions there are several options for controlling environmental exposure and contamination including plate lids and plate seals.

Plate Lids

Plate lids rest on the top of a microplate and allow ease of access for biologists and are supported by many automated systems. The material may be machined metal or plastic with a gasket to seal along the parameter of the plate. Automated systems may use a vacuum holding system, specific features built into the lid shape or an empty plate nest to store the lid while the microplate wells are accessed for compound and reagent additions. Assay plate lids may also have a system such as an array of holes for gas exchange.

Plate Seals

The plate seal is an adhesive film that is pressed onto the top of the microplate. The material for the seal and the adhesive can be selected for the application and chemical compatibility required.

Automated Plate Sealing Systems

Automated plate sealing systems can be fed by hand or by robot (Figure 5). Most sealers use a thermal process where the microplate is pressed against a hot plate with the sealing material between. The seal adhesive, and to some extent the top of the microplate, melt making a seal. The seal can be permanent or removed by hand. Besides thermal sealing, there are automated plate sealing systems using press on adhesives. Press-on seals do not have the potential damaging effects of a thermal system such as plate deformation or having a significant heat source in close proximity to the microplate well contents. In addition to sealing plates, there is also instrumentation with the ability to robotically remove seals.

Figure 5: . PlateLocTM Thermal Microplate Sealer, Source Agilent Technologies http://www.

Figure 5:

PlateLocTM Thermal Microplate Sealer, Source Agilent Technologies http://www.chem.agilent.com

Microplate Readers

The microplate reader is designed to detect and quantify biological, chemical or physical events found within the well of a microplate. There is currently a wide range of detection technologies to suit specific assay requirements many of which can be combined into single multi-purpose instrumentation.

Single & Multi-Mode Readers

Single and multi-mode microplate readers are used for fluorescence, luminescence, absorbance and other light based detection technologies (Figure 6). These detection technologies share many of the same components differing in the light paths through the sample. Single mode microplate readers can be small and economical tailored to a specific technology. A bit larger and costlier then single mode readers, a multi-mode reader can be very advantageous for a lab combining multiple technologies and detection modes into a single more versatile unit. In many cases the use of a detection reagent is required to quantify a specific event.

Figure 6: . Synergy HT Multi-Mode Microplate Reader, Source Biotek http://www.

Figure 6:

Synergy HT Multi-Mode Microplate Reader, Source Biotek http://www.biotek.com

Common Detection Technologies

  • Fluorescence Intensity (FI) - A light source of a specific wavelength illuminates fluorescent molecules within a sample causing the simultaneous light emission from the sample (Figure 7). If the emission light is of a different wavelength it can be filtered from the excitation source light and can then be measured using a light detector such as a photomultiplier tube (PMT). Some variations of the technology.
    • Time-Resolved Fluorescence (TRF) - Uses specific fluorescent molecules called lanthanides that have long light emission times following the removal of an excitation source. The excitation light source is pulsed and the light detector measures the sample after other fluorescent molecule emissions have diminished resulting in lower backgrounds then FI however there is less assay compatibility and higher reagent costs.
    • Fluorescence Polarization - Polarization refers to the orientation waves, in this case waves of light. Similar to FI with the addition of polarizing light filters for the excitation light source and sample emission. Fluorescence polarization measures the mobility of florescent molecules. Fluorescent molecules attached to larger objects will rotate relatively slowly and will emit more polarized light when excited by a polarized light source. Smaller molecules will rotate more rapidly and the light emission will become depolarized. Useful in measuring molecular binding.
  • Luminescence - Measurement of light emission caused by a chemical or biochemical reaction (Figure 8). Luminescence does not require an excitation light source. Used in many luciferase-based assays such as gene expression, cytotoxicity and ATP detection.
  • Absorbance - Measuring the amount of light absorbed as it passes through the well. A light source of a selected wavelength illuminates the sample while the detector measures the amount of light from the opposing side of the well (Figure 9). The amount of light absorbed can be related to the biology of interest.
  • AlphaScreen - Assay technology developed using bead-based chemistry to study bimolecular interactions through homogeneous proximity. Binding of molecules of interest captured on anode and cathode beads leads to an energy transfer from one bead to the other producing a signal when subjected to a specific excitation. Requires the use of a 680nm laser excitation not available on all microplate readers.
Figure 7:

Figure 7:

Simplified Fluorescence Detection

Figure 8:

Figure 8:

Simplified Luminescence Detection

Figure 9:

Figure 9:

Simplified Absorbance Detection

Filters and Monochromators

Filters and monochromaters are two competing wavelength selection technologies integral to microplate reader design. Both have their advantages.

  • Monochromator - A defraction grating separates white light into a spectrum such that a slotted material can be positioned isolating the specific wavelength of light. Used for both excitation and emission filtering.
    • Convenient and flexible, does not require an inventory of compatible filters
    • Can perform a spectral scan to characterize unknown fluorophores or spectral shifts
    • Reduction of signal/sensitivity due to the significant loss of light in defraction gratings
  • Filter Based - Optical filters with specific wavelengths and bandwidths are incorporated into the excitation and detection light paths.
    • Less expensive components compared with monochromators
    • Minimal signal loss and effective separation of excitation and emission wavelenths
    • Several filters are typically maintained within the equipment and/or accessed for changing by the equipment operator.
    • Cannot perform spectral scan
    • An initial inventory of commonly used filters is required with the likelihood of purchasing more filters over time to accommodate changing needs

Many microplate readers are modular with numerous upgrade paths.

Common Options to Consider

  • Top Read, Bottom Read or Both - Single mode microplate readers often have the detector light path set to read from only the top or bottom of the plate depending on the detection technology while multimode readers are often adapted to read plates from either the top or bottom of the microplate.
    • Bottom reads require a clear bottom microplate such that light can pass through often used for absorbance assays and florescence intensity often performs better for cells adherent to the bottom of the plate.
    • Top reads perform best with solid bottom plates and work well for detection of molecules that are suspended in solution.
  • Temperature Control & Incubation
  • Gas Purging
  • Reagent Injection- Some assays kits signals decay rapidly and require near simultaneous detection that can only be achieved by delivering reagents within the microplate reader.

Major manufacturers of microplate readers include:

  • PerkinElmer
  • Tecan
  • Thermo Scientific
  • BMG Labtech
  • Molecular Devices
  • Biotek

Ultra High Throughput Screening Microplate Reader

The ViewLuxTM uHTS Microplate Imager is a commonly used device in high throughput screening with some unique features of note (Figure 10). The ViewLuxTM operates on similar light based detection technologies as other microplate readers however, while most readers provide excitation light and detection on a well to well basis, the ViewLuxTM excites the entire microplate at once and using a highly sensitive CCD camera to capture an image of the emission signal. The image is rapidly processed to correct for any parallax error and to provide a numerical representation for each individual well. Though many microplate readers are more sensitive and considerably less costly the time to process an entire microplate on the ViewLux can be reduced from minutes to seconds. Due to its size and cost, the ViewLuxTM is typically reserved for assay development and automated screening of a high volume of compounds (manufactured by PerkinElmer).

Figure 10: . ViewLuxTM, Source PerkinElmer http://www.

Figure 10:

ViewLuxTM, Source PerkinElmer http://www.perkinelmer.com

High Content Imagers

High Content Screening (HCS) an extension of HTS utilizing much of the same processes and equipment for screening except that the microplate read portion of the assay is performed using an automated microscope or high content imager. Much more than a standard microscope, a high content imager incorporates an automated platform for plate handling and image processing software to quantify the data collected under specified parameters. Using one or more fluorescent dyes different excitation light sources can be applied and images saved of the emission. A separate image showing each fluorescent emission is taken in rapid succession and then overlaid onto each other resulting in a high resolution image of the results. High content imagers are able to collect data other microplate readers cannot such as cell morphology or spatially localized proteins.

Excitation light is provided using a lamp, lasers or light emitting diodes (LED). LEDs are currently the optimal choice due to their long life span and stable output. Emission light is collected from the bottom of the plate through microscope objectives that can be changed to different magnifications and the image is captured using a digital camera. Some high content imagers can be equipped with a confocal microscope system. Considerably more complex and expensive, a confocal system can provide further depth resolution and improved contrast by rejecting light from out of focus sources. A confocal system is particular useful in imaging small or 3D cellular systems/structures and samples with strong background fluorescence.

Manufactures include:

  • GE
  • Thermo Scientific
  • PerkinElmer
  • Molecular Devices
  • Yokogawa

Laser Scanning Cytometers

Laser scanning cytometers in comparison to other microplate readers can be classified as medium content imaging. Excitation is by laser across the surface of a microplate, as a molecule excites and fluoresces it is detected by photo multiplier tube. The technology is effective at detecting cells, colonies and model organisms but not subcellular features or processes. Has large depth of focus allowing differentiation between low and high concentration of signals.

Manufactures include:

  • TTP Labtech
  • Molecular Devices
  • Hamamatsu

Label Free Detection

Label free detection refers to the quantification of biological, chemical or physical events without the use of detection reagents. Detection reagents can be of considerable cost and though they are not used for label free detection the money saved is more than offset by the need of specialized plates. There are currently two methods of label-free detection on the market, monitoring the change in impedance and detecting shifts of the refractive index from the bottom of the plate.

Impedance-based label-free readers use microplates with integrated electrodes molded to bottom of the wells. A voltage is applied to each well and electrical currents flow around, between and through cells. The measurement of impedance or resistances to electrical flow is recorded during the duration of the event to be monitored. The microplate and electrical connections are often routed through and access port of an incubator to maintain environmental conditions while monitoring over long periods. Changes in cell adherence, shape, volume and interactions ultimately affect the recorded impedance logged in real time.

Using a specialized microplate changes near the bottom surface of the well are detected by monitoring the reflected wavelength. A refractive waveguide biosensor grating is imbedded into the bottom surface of the microplate. As a broadband light source illuminates the bottom of the well a reflected wavelength is detected indicating the refractive index near the well bottom. After a cell binding event or intracellular protein movement a shift in the refractive index occurs and is then detected by the change in the reflected wavelength (Figure 11).

Figure 11: . Epic Label Free Detection System, Source Corning http://www.

Figure 11:

Epic Label Free Detection System, Source Corning http://www.corning.com/lifesciences/epic/en/products/epic_system.aspx

Manufactures include:

  • Corning
  • PerkinElmer
  • Molecular Devices
  • SRU Biosystems

Spectrophotometry

The electromagnetic spectrum stretches from radio waves to gamma rays (Figure 12).

Figure 12: . Schematic depictions of the electromagnetic spectrum.

Figure 12:

Schematic depictions of the electromagnetic spectrum.

Spectrometric Techniques

  • Absorption
  • Emission
  • Scattering
  • Ultraviolet and Visible Absorption Spectroscopy
  • Dual-beam uv-vis spectrophotometer
  • Fluorescence Spectroscopy

Electromagnetic Spectrum

Spectroscopy is the use of the absorption, emission, or scattering of electromagnetic radiation by atoms or molecules (or atomic or molecular ions) to qualitatively or quantitatively study the atoms or molecules, or to study physical processes. The interaction of radiation with matter can cause redirection of the radiation and/or transitions between the energy levels of the atoms or molecules. A transition from a lower level to a higher level with transfer of energy from the radiation field to the atom or molecule is called absorption. A transition from a higher level to a lower level is called emission if energy is transferred to the radiation field or non-radiative decay if no radiation is emitted. Redirection of light due to its interaction with matter is called scattering, and may or may not occur with transfer of energy, i.e., the scattered radiation has a slightly different or the same wavelength.

Absorption

When atoms or molecules absorb light, the incoming energy excites a quantized structure to a higher energy level. The type of excitation depends on the wavelength of the light. Electrons are promoted to higher orbitals by ultraviolet or visible light, vibrations are excited by infrared light, and microwaves excite rotations. An absorption spectrum is the absorption of light as a function of wavelength. The spectrum of an atom or molecule depends on its energy level structure, and absorption spectra are useful for identification of compounds. Measuring the concentration of an absorbing species in a sample is accomplished by applying the Beer-Lambert Law.

The Beer-Lambert law (or Beer's law) is the linear relationship between absorbance and concentration of an absorbing species. The general Beer-Lambert law is usually written as:

A=a(λ)*b*c

where A is the measured absorbance, a(λ) is a wavelength-dependent absorptivity coefficient, b is the path length, and c is the analyte concentration. When working in concentration units of molarity, the Beer-Lambert law is written as:

A=ε*b*c

where ε is the wavelength-dependent molar absorptivity coefficient with units of M-1 cm-1. Experimental measurements are usually made in terms of transmittance (T), which is defined as T = I / Io, where I is the light intensity after it passes through the sample and Io is the initial light intensity (Figure 13). The relation between A and T is:

Figure 13:

Figure 13:

Schematic of Beer-Lambert law

A=-logT=-log(I/Io).

The linearity of the Beer-Lambert law is limited by chemical and instrumental factors. Causes of nonlinearity include:

  • Deviations in absorptivity coefficients at high concentrations (>0.01 M) due to electrostatic interactions between molecules in close proximity.
  • Scattering of light due to particulates in the sample.
  • Fluoresecence or phosphorescence of the sample.
  • Changes in refractive index at high analyte concentration.
  • Shifts in chemical equilibria as a function of concentration.
  • Non-monochromatic radiation. (Deviations can be minimized by using a relatively flat part of the absorption spectrum such as the maximum of an absorption band).
  • Stray light leaking into the sample compartment.

Emission

Atoms or molecules that are excited to high energy levels can decay to lower levels by emitting radiation (emission or luminescence). For atoms excited by a high-temperature energy source this light emission is commonly called atomic or optical emission (see atomic-emission spectroscopy), and for atoms excited with light it is called atomic fluorescence (see atomic-fluorescence spectroscopy). For molecules it is called molecular fluorescence if the transition is between states of the same spin and phosphorescence if the transition occurs between states of different spin. The emission intensity of an emitting substance is linearly proportional to analyte concentration at low concentrations, and is useful for quantifying emitting species (Figure 14).

Figure 14:

Figure 14:

Jablonski Diagram

Scattering

When electromagnetic radiation passes through matter, most of the radiation continues in its original direction but a small fraction is scattered in other directions. Light scattered at the same wavelength as the incoming light is called Rayleigh scattering. Light that is scattered in transparent solids due to vibrations (phonons) is called Brillouin scattering. Brillouin scattering is typically shifted by 0.1 to 1 cm-1 from the incident light. Light that is scattered due to vibrations in molecules or optical phonons in solids is called Raman scattering. Raman scattered light is shifted by as much as 4000 cm-1 from the incident light.

Ultraviolet and Visible Absorption Spectroscopy

UV-vis spectroscopy is the measurement of the wavelength and intensity of absorption of near-ultraviolet and visible light by a sample. Ultraviolet and visible light are energetic enough to promote outer electrons to higher energy levels. UV-vis spectroscopy is usually applied to molecules and inorganic ions or complexes in solution. The UV-vis spectra have broad features that are of limited use for sample identification but are very useful for quantitative measurements. Measuring the absorbance at some wavelength and applying the Beer-Lambert Law can determine the concentration of an analyte in solution. The light source is usually a hydrogen or deuterium lamp for UV measurements and a tungsten lamp for visible measurements. The wavelengths of these continuous light sources are selected with a wavelength separator such as a prism or grating monochromator. Spectra are obtained by scanning the wavelength separator and quantitative measurements can be made from a spectrum or at a single wavelength (Figure 15).

Figure 15:

Figure 15:

Schematic of a single beam UV-vis spectrophotometer

Dual-beam UV-VIS spectrophotometer

In single-beam UV-vis absorption spectroscopy, obtaining a spectrum requires manually measuring the transmittance (see the Beer-Lambert Law) of the sample and solvent at each wavelength. The double-beam design greatly simplifies this process by measuring the transmittance of the sample and solvent simultaneously (Figure 16). The detection electronics can then manipulate the measurements to give the absorbance. Table 1 provides specifications for a typical spectrophotometer.

Figure 16: . Schematic of a dual-beam UV-VIS spectrophotometer.

Figure 16:

Schematic of a dual-beam UV-VIS spectrophotometer.

Table 1:

Table 1:

Specifications of a typical spectrophotometer:

Fluorescence Spectroscopy

Light emission from atoms or molecules can be used to quantify the amount of the emitting substance in a sample. The relationship between fluorescence intensity and analyte concentration is:

F=k*QE*Po*(1-10[-*b*c])

where F is the measured fluorescence intensity, k is a geometric instrumental factor, QE is the quantum efficiency (photonsemitted/photons absorbed), Po is the radiant power of the excitation source, is the wavelength-dependent molar absorptivity coefficient, b is the path length, and c is the analyte concentration (, b, and c are the same as used in the Beer-Lambert law). Expanding the above equation in a series and dropping higher terms gives:

F=k*QE*Po*(2.303*b*c)

This relationship is valid at low concentrations (<10-5 M) and shows that fluorescence intensity is linearly proportional to analyte concentration. Determining unknown concentrations from the amount of fluorescence emitted from a sample requires calibration of a fluorimeter with a standard (to determine k and QE) or by using a working curve.

Many of the limitations of the Beer-Lambert law also affect quantitative fluorimetry. Fluorescence measurements are also susceptible to inner-filter effects. These effects include excessive absorption of the excitation radiation (pre-filter effect) and self-absorption of atomic resonance fluorescence (post-filter effect).

pH Meters

The pH meter measures the pH of a solution using an ion-selective electrode (ISE) that responds to the H+ concentration of the solution. The pH electrode produces a voltage that is proportional to the concentration of the H+ concentration, and making measurements with a pH meter is therefore a form of potentiometry. The pH electrode is attached to control electronics that convert the voltage to a pH reading and display it on a meter. A pH meter consists of a H+-selective membrane (ISE), an internal reference electrode, an external reference electrode, and a meter with control electronics and display (Figure 17). Commercial pH electrodes usually combine all electrodes into one unit that are then attached to the pH meter.

Figure 17: . Schemaic of an ISE measurement.

Figure 17:

Schemaic of an ISE measurement.

An ion-selective electrode (ISE) produces a potential that is proportional to the concentration of an analyte. Making measurements with an ISE is therefore a form of potentiometry. The most common ISE is the pH electrode, which contains a thin glass membrane that responds to the H+ concentration in a solution. The potential difference across an ion-sensitive membrane is

E=K-(2.303RT/nF)log(a)

where K is a constant to account for all other potentials, R is the gas constant, T is temperature, n is the number of electrons transferred, F is Faraday's constant, and a is the activity of the analyte ion. A plot of measured potential versus log(a) will therefore give a straight line. ISEs are susceptible to several interferences. Samples and standards are therefore diluted 1:1 with total ionic strength adjuster and buffer (TISAB). The TISAB consists of 1 M NaCl to adjust the ionic strength, acetic acid/acetate buffer to control pH, and a metal complexing agent. ISEs consist of the ion-selective membrane, an internal reference electrode, an external reference electrode, and a voltmeter. A typical meter is shown in the document on the pH meter. Commercial ISEs often combine the two electrodes into one unit that are then attached to a pH meter.

NOTE: All pH meters should be calibrated daily, and preferably, before each use. It is important to know that the ISE potential can drift due to drying of the LaF3 membrane and the evaporation of internal reference solutions.

Electronic Balances

Analytical balances are accurate and precise instruments to measure weights (Figure 18). They require a draft-free location on a solid bench that is free of vibrations. Modern balances have built-in calibration weights to maintain accuracy. Older balances should be calibrated periodically with a standard weight. A few weighing tips follow:

Figure 18: . Representative image of a balance.

Figure 18:

Representative image of a balance.

  • Do not bump or place objects on the bench after zeroing the balance.
  • Weigh powders on weighing paper or in weighing dishes. Handle objects with tongs, gloves, or weighing paper to prevent fingerprints.
  • Let hot objects cool before weighing.
  • Weigh hygroscopic materials rapidly since they will absorb water during weighing.
  • When making repetitive weighings always use the same procedure.
  • Ensure that the balances are calibrated routinely.
  • It is absolutely important to keep the balance pans and the area around precision balances clean. Clean up after each use for safety and convenience of others.
  • In dry cold weather, weighing fine powders can be a problem due to static charges that develop on particles. Devices are now available for neutralizing electric charges of the material while it is still in the container. Use of these devices is highly recommended for accurate weighings.

Microscopes

  • Microscopes
  • Microscope Parts
  • Objectives
  • Condensers
  • Iris Diaphram
  • Eyepiece
  • 2.5 Field Limiting Aperture

A microscope is not designed to magnify small objects. For example, you can find in any hobby or toy store a $49.95 instrument capable of magnifying objects to 1200 times. And that includes a zoom lens and light source. Most student and research microscopes magnify no more than 1000 times with costs starting at around $1500.00, with research microscopes going into the tens of thousands of dollars. Is the academic community being taken for a ride? No. The $49.95 microscope only gives you an image that is a soft blur at 1000x magnification, whereas the research microscope’s image is crystal sharp. This is called resolution, the ability to see fine details. Once you can resolve fine details then you can magnify them. Every optical system has a finite resolution; if you magnify objects beyond the resolution the result will be empty magnification. So, the actual purpose of a microscope is to see small things clearly.

A desirable attribute of a microscope is depth of field, which is the range of depth that a specimen is in acceptable focus. A microscope that has a thin depth of field will have to be continuously focused up and down to view a thick specimen. A third feature that a microscope has is its mechanism for contrast formation. Contrast is the ratio between the dark and the light. Typically, most microscopes use absorption contrast; that is, the specimen is subjected to stains in order to be seen. This is called bright field microscopy. There are other types of microscope that use more exotic means to generate contrast, such as phase contrast, dark field, and differential interference contrast. The fourth desirable feature is a strong illumination source. The higher a microscope magnifies the more light will be required. Also, there will be more optical trade off leeway when more light is present. The illumination source should also be at a wavelength (color) that will facilitate the interaction with the specimen. All microscopes fall into either of two categories based on how the specimen is illuminated. In the typical compound microscope the light passes through the specimen and is collected by the image forming optics. This is called diascopic illumination. Dissecting (stereo) microscopes generally use episcopic illumination for use with opaque specimen. The light is reflected onto the specimen and then into the objective lens. The four attributes of an optical system may have trade offs with each other. For example, resolution and brightness is antagonistic towards contrast and depth of field. One cannot have maximum resolution and maximum contrast simultaneously. Theoretically speaking, if you had an infinite resolving system there would be no contrast to discern the image. It is up to the microscopist to decide which attribute is needed to view a particular specimen. All of which are controlled by the iris diaphragm.

Microscope Parts

Objectives

The objective lens is the lens that is closest to the object or specimen (Figure 19). It is essentially the information-gathering lens of an optical system. Therefore, it is regarded as the most important lens of the microscope. There are many different types of objective lenses. The most common and inexpensive is the achromat. This lens is usually found on student microscopes. It is corrected for spherical aberration for only green light. Chromatic aberration is corrected in only two colors. The apochromat objective is far superior and generally very expensive. Chromatic aberration is corrected for all three colors and it is spherically corrected for two colors. These objectives quite often will require a special compensating eyepiece. Semiapochromat objectives have correction in between the apochromat and achromat. Flat field or plano objectives compensate for curvature of field and are excellent for histology work. The flat field objectives can be optically constructed to be also an achromat, semiapochromat or apochromat. In the latter case the lens would be called a plano apochromat which is generally regarded as the finest lens available. The price of a single plano apochromat will run into the many thousands of dollars. Figure 20 provides a comparison of the resolution using different types of objectives.

Figure 19:

Figure 19:

Image of objective lens

Figure 20: . Comparison of the resolution using different types of objectives.

Figure 20:

Comparison of the resolution using different types of objectives.

Each objective has information critical for the maximum resolution possible written on the side of the barrel. Generally the magnification is printed in the largest text with the manufacturer type designation. The second value is the numerical aperture. Beneath that, in a smaller font the tube length and the cover glass thickness is given. Any special information will also be added such as if it is an oil lens, infinity etc. The tube length usually 160 refers to the distance between the objective and the eyepiece in millimeters. It must be maintained if the aberrations are to be corrected. You can recognize a superior microscope if when adjusting the interpupillary distance you can see the eyepiece extend which happens to maintain the proper tube length. The cover slip thickness usually around 0.17mm is also critical. This corresponds to a cover glass of No. 1.5. The more sophisticated objectives even have a cover glass compensation control that you dial in the thickness of the cover glass.

Condensers

The sub-stage condenser of a microscope is designed to focus the light onto the specimen. In addition it must also fill the numerical aperture of the objective (Figure 21). Like objective lenses there are several different types. The most common being the Abbe condenser. This type is not corrected for optical aberrations. The achromatic condenser is corrected for both spherical and chromatic aberrations. Both types of condenser have their numerical aperture printed on the side. This needs to be of equal or greater value than that of the objective N.A., otherwise, the full resolution of the objective will not be utilized. Most substage condensers can use immersion oil like that of the objectives to achieve their full N. A. This is not recommended unless you are doing very demanding photomicroscopy work.

Figure 21: . Example of a substage microscope condenser.

Figure 21:

Example of a substage microscope condenser.

Iris Diaphram

The iris diaphragm is the most important single control on the microscope (Figure 22). There is a misconception that it is used to regulate the amount of light. The light intensity control is the sole means to adjust the brightness. The iris diaphragm is the resolution verses contrast control. It does this by varying the size of the numerical aperture of the objective lens. Usually, lenses such as those found on cameras have the iris diaphragm built in the objective lens. In a microscope objective the iris diaphragm would have to be very small, which would be difficult to manufacture. So the optical engineers put the iris diaphragm at the optical equivalent of being in the objective lens, in the condenser assembly. This is one of the reasons why the condenser lens has to be set at the correct distance to the objective. In addition the iris diaphragm controls the depth of field.

Figure 22: . Schematic of iris diaphgram control.

Figure 22:

Schematic of iris diaphgram control.

Eyepiece

The eyepiece is basically a projection lens system (Figure 23). There are three types generally used in light microscopy. The most common is the Huygenian type. This eyepiece is used with low and medium magnification and is designed to project the image into a human eye. Some of these eyepieces will have a long eyepoint, the spot where your eye should be, so you can focus with your glasses on. If you suffer from astigmatism you should ware your glasses while using the microscope. If you are near or far sighted then you can adjust the eyepiece for your personal correction using the diopter corrector and leave your glasses off. The second type of eyepiece is the compensating eyepiece and is generally used with apochromate or flat field objectives. These provide superior image quality. The third type is the photo eyepiece. These are designed to project a corrected image onto film plane in a camera. These are generally considered the finest of eyepieces. All eyepieces will have a relative magnification written on the side of the barrel. They range in magnification from 2.5X to 15X with the lower magnifications used with the photo eyepiece.

Figure 23: . Example of a microscope eyepiece.

Figure 23:

Example of a microscope eyepiece.

Field Limiting Aperture

The field-limiting aperture is used to determine the correct position and center of the condenser lens. It is used in conjunction with the condenser centering knobs to place the illumination in the center. It also helps in reducing the amount of optical flare.

Liquid Handling Devices

The high throughput screening lab has several liquid handling applications and device technologies are chosen to best suit the specific task.

Hand-held Pipette

The pipette transfers precise volumes of liquids through movement of a piston and the displacement of air (Figure 24). Pipettes have specified volume ranges and the user selects the increment. The usable range of volume is 100nl to 1ml. Pipette tips are disposable and available in numerous shapes, sizes and treatments. Accurate manual pipetting is a lab skill acquired with practice. Common variations of pipettes are as follows.

Figure 24:

Figure 24:

Example pipettes

  • Single-channel
  • Multi-channel – Typically 8 or 12 simultaneous channels
  • Electronic
  • Repeaters – Allows for multiple dispenses following a single aspiration

Automated Pipetting Devices

Automated pipetting platforms are scalable to the application and level of automation desired (Figure 25). Microplates are positioned in specific locations on the instrument deck or into a plate stacker. The number of available positions can range from 2 - >16 positions and further plate capacity can be added when using stackers. The head, consisting of the liquid handling apparatus, may be outfitted with a single tip, single column of tips or 96/384 2D array of tips for accommodating whole plate transfers referred to as plate stamping. Tips used may be disposable or reusable with washing applications.

Figure 25: . 96 Channel Automated Pipette, Source Tecan http://www.

Figure 25:

96 Channel Automated Pipette, Source Tecan http://www.tecan.com

Creating methods on these devices can be very involved and uses elements of programming logic such as variable parameters, database access and conditional looping. Once a method is optimized it is then very reproducible. These devices are often used as the main integrating platform for compact systems with options to include plate transferring capabilities and auxiliary equipment.

Major manufacturers of automated pipetting systems include:

  • Beckman Coulter
  • Hamiliton
  • Perkin Elmer
  • Cybio
  • Agilant
  • Tecan

Solenoid Valve Based Dispensers

Using a pressurized bottle of fluid, a fast acting valve and robotic positioning, these systems can deliver precise volumes (>0.1 μl) into the microplate well at very high rates of speed. Assuming a constant air pressure in the bottle, the volume to be dispensed is controlled by adjusting valve timing.

Most systems offer the ability to dispense multiple fluids simultaneously using separate valves and fluid paths. Dispensing is controlled by a spreadsheet correlating the specific valve and volume to the microplate well. Common fluids for this equipment include cell media, buffers and detection reagents. Dispensing of viscous fluids or cells prone to clumping can be problematic and interfere with valve operation. Dispensing DMSO is possible though may cause rapid valve degeneration.

Most components of the equipment fluid path are reused and therefore cleaning operations are a must. In most cases a combination of 70% ethanol, cleaning detergents and high purity water are sufficient.

These systems are used heavily in high throughput screening due to their dispense accuracy and speed of operation. Dispensing a nominal volume across an entire microplate can take 1 to 3 minutes.

Manufacturers of Solenoid Valve Based Dispensers:

  • Beckman Coulter
  • Thermo Fisher Scientific

Peristaltic Pump Based Dispensers

Peristaltic pumps are found in numerous applications in the laboratory and medical fields (Figure 25). Peristaltic pumps move fluids using positive displacement by pinching flexible tubing with rotating sets of rollers. The pumping liquid is maintained within the tubing and no external contact is made between pump components and fluid. This lack of contact allows for a large range of chemical capabilities based solely on the tubing material. Tubing is intended to be remove or replaced. Direction and speed are easily varied.

For microplate dispensing the peristaltic pump has some specific features. The fluid path is part of a cassette assembly consisting of tubing, tips, tension adjustment screws and plastic housing. The cassettes are consumable with lifespans varied by manufacturer and operating volume ranges. The equipment has a plate positioning system, motor controlled rotating rollers and the user interface.

The main disadvantage of the peristaltic pump microplate dispenser is the cost of consumable cassettes. Each cassette costs $500-$1000 and is considered accurate for dispensing a few hundred plates. The lifespan may be increased by proper cleaning, storage and recalibration techniques. The advantage is that cassettes are accurate out of the box.

Manufacturers of Peristaltic Pump Based Dispensers:

  • Thermo Fisher Scientific
  • Biotek

Pintools

The pintool is used for fixed low volume liquid transfers between microplates. For HTS this equipment is used for the transfer of compounds from the library into the assay plate. The pins are stainless steel with precision machined features to set the volume of liquid transfer (Figure 26). As the pin enters the source, pin surfaces make liquid contact. When the pin is withdrawn, small amounts of liquid adhere to the surfaces until the pin is submerged again and surface tension of the adherent liquid can be broken. Following any liquid transfer, pintool pins must be washed and dried prior to their next use. Washing steps typically include solvent baths, blotting and air drying.

Figure 26: . Example Slotted Pin Selection, Source V&P Scientific http://www.

Figure 26:

Example Slotted Pin Selection, Source V&P Scientific http://www.vp-scientific.com

The pintool head is the fixture to hold the pins the proper positions (Figure 27). The head is made to match the diameter of the pins used and plate density, typically 96, 384 or 1536 pins to be used with 96-, 384-, or 1536-well plates, respectively. Though fixtures can be made for hand held operations, higher density plates require higher levels of precision for accurate transfers thus necessitating the need for robotic control. Most liquid handling robotic systems can be adapted to use pintools, while dedicated systems are also available.

Figure 27: . Example Pintool Head, Source V&P Scientific http://www.

Figure 27:

Example Pintool Head, Source V&P Scientific http://www.vp-scientific.com

The pintool is advantageous for HTS due to its high speed and direct correlation of compound plate well position to assay plate well position. The principal provider of pintool pins and fixtures is V&P Scientific.

Acoustic Dispensers

Acoustic dispensers use focused bursts of sound energy to propel 1-10 nanoliter (nL) sized droplets between source and destination microplates without direct liquid contact (Figure 28). Droplet dispenses are very rapid and the final volume transfer is achieved using increments of droplet size. Contamination is minimized since there is no contact carryover between operations. Contrasting the pintool, the acoustic dispensers is exceedingly flexible using a user-created dispense map that specifically defines source well, destination well and volume for each transfer.

Figure 28: . Simplified Acoustic Dispense, Source http://en.

Figure 28:

Simplified Acoustic Dispense, Source http://en.wikipedia.org/wiki/File:Acoustic_transfer1.jpg

The acoustic dispenser brings flexibility into the screening workflow. In addition to 1-1 plate stamping complex liquid handling operations can easily be achieved at low volumes such as dispensing into dry wells, serial dilutions, matrix/poly-pharmaceutical screening and cherry picking. Dispensing compound into dry plates then storing for later use is known as making “Assay Ready” plates. An “Assay Ready” plate can be created in advance of a screen as resources are available and on a separate system. If screening multiple compound concentrations, the number of concentrations multiplies the number of compound plates needed. Using an acoustic dispenser, different concentrations can be achieved from a single stock greatly reducing the number of compound plates needed. Follow up assays often require the creation compound plates with compounds of specific interest at different concentrations. The creation of a single follow up plate can take a considerable amount of resources. Using the acoustic dispenser screening of only the specific follow up compounds is possible without the need of new plates and potentially done on the fly.

The advantages of acoustic dispensing have some contrasting limitations. The cost for equipment varies with model and options but is typically >$300k per unit. The speed of operation is significantly slower when compared to the pintool requiring multiple units to maintain throughput. A 384-well transfer can take about 4min while 1536 wells can take > 10min. The compound plate must have 384 or 1536 wells and be acoustically compatible, meaning that the well shape and material must effectively transfer sound energy. Many microplate venders are now offering microplates for this specific purpose. The droplet destination can be any labware that fits into an SLAS/SBS microplate footprint though it must inverted for operation. For 384 and 1536 plates, liquid tension maintains fluids in the well during inversion though other labware may have to be initially dry.

Manufacturers of Acoustic Dispensers:

  • Labcyte
  • EDC Biosystems

The quality of assay data depends critically on the ability of individual scientists to use the liquid delivery devices appropriately. It is highly recommended that you make yourself familiar with all pipeting equipment in your lab. Also note that these liquid dispensers will have to be calibrated on a regular basis. For automated liquid handlers, special procedures recommended by the manufacturers are employed. These procedures usually involve serial dilutions of dyes from stock solutions and the determination of the accuracy and precision of the dilutions.

References

1.

Analytical Instrumentation Review: http://www​.uam.es/docencia​/quimcursos/Scimedia​/chem-ed/analytic/ac-meths.htm

2.

Assay Development Guidelines for Image-Based High Content Screening, High Content Analysis and High Content Imaging: http://www​.ncbi.nlm.nih​.gov/books/NBK100913/

3.

Impedance-Based Technologies: http://www​.ncbi.nlm.nih​.gov/books/NBK91999/

4.

Corning Microplate Selection Guide: http://catalog2​.corning​.com/lifesciences​/media/pdf/productselectionguide​_microplates11​_02_cls_mp_014.pdf

5.

The Use of AlphaScreen Technology in HTS: Current Status: http://www​.ncbi.nlm.nih​.gov/pmc/articles/PMC2775125/

6.

Luminescence lifetimes in biological systems. [Analyst. 1994] - PubMed – NCBI: http://www​.ncbi.nlm.nih​.gov/pubmed/8024118

7.

Time-resolved fluorescence spectroscopy. [Curr Opin Struct Biol. 1996] - PubMed – NCBI: http://www​.ncbi.nlm.nih​.gov/pubmed/8913686

8.

Corning Life Sciences | Corning® Epic® Technology | Corning Epic System: http://www​.corning.com​/lifesciences/epic​/en/products/epic_system.aspx

9.

Agilent PlateLoc Thermal Microplate Sealer Consumables Selection Guide: http://www​.chem.agilent​.com/Library/selectionguide​/Public/5990-3659en_lo%20CMS​.pdf

10.

Everything You Need To Know About Pin Tools But Were Afraid To Ask: http://www​.vp-scientific​.com/pin_tools.htm

Copyright Notice

All Assay Guidance Manual content, except where otherwise noted, is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported license (CC BY-NC-SA 3.0), which permits copying, distribution, transmission, and adaptation of the work, provided the original work is properly cited and not used for commercial purposes. Any altered, transformed, or adapted form of the work may only be distributed under the same or similar license to this one.

Bookshelf ID: NBK92014PMID: 22553880
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