Laser scanning cytometers have matured over the past decade to meet the needs of end-users for higher throughput cell analysis, to access to previously difficult applications and to keep pace with the diversity of new fluorophores, validated high content screening (HCS) assay kits and reagents now available.
Some of the latest instrument enhancements include multiple lasers, brightfield and light scatter for label-free detection of non-fluorescent objects, chromatic dye absorption, higher sensitivity low loss optics and simultaneous scanning with multiple lasers.
Although laser scanning cytometers are increasingly being used to transition image-based cell analysis into primary screening, their role in traditional high content analysis (HCA) can still be limited by their object resolution capabilities and both approaches (HCS and HCA) work best when deployed side by side.
Existing laser scanning cytometers already support a broad range of applications making it a key piece of kit in flow cytometry and HCS imaging facilities. In the future we can expect to see next-generation laser scanning cytometers encroaching further into the territory of flow cytometry.
Laser scanning cytometers capable of performing microplate-based high content screening (HCS) were first introduced more than a decade ago. Laser scanning cytometers combine the cell-recognition capabilities of microscope CCD-based imaging systems with the fast read speeds of bulk fluorescence microplate readers.
Typically they use scanning laser excitation with photomultiplier tube detection to resolve fluorescent objects. Signal thresholding algorithms identify fluorescence above the solution background from which fluorescent cells are identified for calculation of morphological and fluorescent parameters.
The principal enhancements to laser scanning cytometry over the past decade have mainly been related to:
1) increasing the number of laser lines offered (to enhance reagent compatibility and multiplexing capability)
2) the introduction of brightfield and laser scattering label-free modes of detection
3) increases in the throughput potential
Applications requiring quantitation of whole cell fluorescence initially was the application niche (‘the sweet spot’) where these instruments proved particularly valuable. However, it was suggested as early as 2005 (1) that HCS and high content analysis (HCA) require different tools and that the role of laser scanning cytometers was uniquely suited to HCS, where the emphasis was on higher throughput and the number of wells processed.
But it was not until the drive to bring HCS into the primary screening arena intensified when research groups began considering indirect or surrogate measurements did the use of laser scanning cytometers for HCS really take off. Perceptions regarding what is required for screening have been redefined over recent years. For example, in nuclear translocation assays although some purists want to see sub-cellular nuclear localisation and to determine cytoplasm/ nuclear ratios, there is an increasing acceptance that being able to quantify, using a laser scanning cytometer, how much fluorescence has moved into the nucleus may be a pragmatic alternative measure.
Such an approach facilitates the rapid selection of hits, so that follow-ups and secondary screens using more information-rich, lower throughput CCD imaging or flow cytometry assays can be limited to the most interesting compounds. Similarly, cell differentiation can be accurately assessed by laser scanning cytometers today and is increasingly being used as a screening surrogate for neurite outgrowth analysis.
However, this gradual transition of image-based HCA applications to HCS laser scanning cytometry has brought with it greater instrument demands for:
1) higher resolution (eg to be able to resolve what might be seen with a 40X microscope objective)
2) higher sensitivity (eg comparable to flow to detect what currently is poorly differentiated by laser scanning, eg weakly fluorescent reporter gene expressing cells)
3) label-free detection of cells or organisms that have not been fluorescently labelled (eg using laser scatter to visualise Zebrafish)
4) for lower cost instruments
This article uses as its starting point some recent market research undertaken in November 2009 by HTStec2 to investigate what impact improving laser scanning capabilities might have on applications currently run or what researchers would really like to run. These included cell-imaging assays, homogeneous mix-and-read binding assays, bead-based ELISA assays, oncology biomarkers assays, weakly fluorescent assays and analysis of multiplexed cellular fluorescence.
Some of the market research findings are considered relative to latest instrumentation and also the reagents, new HCS assay kits and labware that not only support, but are helping to maximise, the potential of laser scanners today.
The main weakness and benefits of HCS laser scanning cytometry
Survey respondents were asked to categorise a list of desirable cell imaging and analysis characteristics as either a weakness or a benefit of HCS laser scanning cytometry, of flow cytometry and of HCS CCD imaging. The resulting analysis revealed that the biggest perceived benefits of an HCS laser scanning cytometer was background rejection, area imaged and simultaneous multi-colour detection. Data storage and independent object recognition (laser scatter/ brightfield) were perceived as least beneficial, but overall were still seen as more of a benefit than a weakness (Figure 1).
In comparison the biggest perceived benefit of a flow cytometer was detection sensitivity. This was followed by simultaneous multicolour detection, then background rejection. The biggest weakness of a flow cytometer was inability to assay adherent cells (Figure 2).
In direct contrast, the biggest perceived benefit of HCS CCD imaging was the ability to assay adherent cells, this was followed by ability to resolve cellular morphology and image output. The biggest weaknesses of HCS CCD imaging were throughput and data storage, and then background rejection (Figure 3).
Overall, this analysis defines current end-user thinking of the optimal role for these three approaches to cellular imaging analysis, highlights the gulf between them and opportunity that exists if an instrument with broader applicability were possible.
Most important or key applications
Cytotoxicity/viability was rated as the most important or key application survey respondents ran on an HCS laser scanning cytometer today. This was very closely followed by cell proliferation. The next most important applications were apoptosis, followed by cell cycle analysis and then reporter gene activation. Of least importance was animal imaging (eg Zebrafish or C.elegans) and beadbased anisotropy (Figure 4).
Biggest obstacles facing laser scanning today
Survey respondents ranked optical resolution as the biggest obstacle facing HCS laser scanning today, and limiting its wider application. This was closely followed by the need to make multiple scans (one per laser). The next biggest obstacles were the range of laser excitation options, then detection sensitivity. Microplate type (variation) was ranked the smallest obstacle facing HCS laser scanning today (Figure 5).
Most difficult fluorescent applications
When respondents were asked about the fluorescent applications they had most difficultly assaying with a laser scanning cytometer they selected weakly fluorescent assays (eg stem cells, cell surface markers, multiple biomarkers) (49% with difficulty). This was followed by analysis of multiplexed cellular fluorescence (eg in blood cell phenotyping) (44% with difficulty). In contrast, only 16% of respondents indicated they had difficulty with multiplexed cellular fluorescence (Figure 6).
40% of survey respondents were aware of applications that would benefit from simultaneous multi-colour laser scanning, ie simultaneous excitation with up to three lasers and collection of up to six channels of fluorescence emissions avoiding the need for multiple scans and cross-laser data correlation. 38% of survey respondents were aware of applications that would benefit from increased detection sensitivity, ie are weakly fluorescent and barely doable by existing plate-based imaging technologies. 26% of survey respondents were aware of applications that would benefit from scanning an SBS plate-sized area in one go.
Impact of new laser scanning capabilities on current cell imaging assays
True multi-colour simultaneous laser scanning (excitation and emission) was rated as the new HCS laser scanning capability which, if enabled, would most impact most on respondents current (existing) cell-imaging assays. This was closely followed by higher sensitivity laser scanning (comparable to flow cytometry) and then large scan area (including whole plate). Rated of least impact current (existing) cell-imaging assays was GXP certification (Figure 7).
Impact of new laser scanning capabilities on ability to perform bead-based assays
Higher sensitivity (comparable to flow cytometry) was rated as the laser scanning capability which, if enabled, would most impact respondent’s ability to perform bead-based assays. This was followed by true multi-colour simultaneous laser scanning (excitation and emission). Rated of least impact on respondent’s ability to perform bead-based assays was GXP certification (Figure 8).
Latest instrument developments in laser scanning cytometry
Table 1 compares available HCS laser scanning cytometers and related instrumentation over a range of parameters.
The following vendor snapshots provide additional details and describe some of the latest developments in laser scanning cytometry instrumentation:
Compucyte (www.compucyte.com) has recently introduced several new fundamental hardware and software developments to its iCys® Imaging Cytometer. Following the market need to combine HCS and HCA capabilities, Compucyte introduced the concept of variable resolution scanning, allowing the throughput/resolution ratio to be tailored to the requirements of a given application. In laser scanning cytometry (LSC), analysis image resolution is determined by both objective lens magnification and stage resolution.
This new function permits the operator selection of microscope stagestepping resolution, making it adjustable from 20 microns (low resolution, high speed screening) down to 0.05 microns (very high resolution, higher content analysis). In late 2009 Compucyte announced the release of four-laser iCyte®, iCys® and iColor® imaging cytometers with excitation wavelength options that can be chosen from a palette of 405, 488, 532, 561, 594 and 633 nanometers.
These newly added wavelengths (561 and 594nm) allow researchers to take full advantage of the most efficient members of the new ‘fruit’ series of fluorescent proteins, while at the same time providing full coverage of the chromatic dye absorption spectrum, taking the definition of high-content cellular and tissue analysis and screening (HCA and HCS) to a new level.
New software developments include automatic exposure determination for all three modes of analysis (fluorescence, absorption and scatter) to allow for faster scan set-up of new samples, cross-boundary segmentation that will identify events that cross scan field borders thus increasing the number of analysable cells per area, a ‘seeded watershed’ segmentation algorithm that adds an important new tool for difficult to contour samples to the already robust LSC software toolkit (iNovator).
LSC’s current outstanding ability to relate analytical features and images has been recently augmented with the ability to relate individual cells to their location in scattergrams and histograms as well as the ability to annotate individual cells (Figure 9).
The Molecular Devices (https://www.moleculardevices.com/) IsoCyte Laser Scanning Cytometer is a fast, automation-friendly system that enables real-time imaging for cell- and bead-based assays, array scanning, colony assays, small organisms and tissue slices. The system’s telecentric lens captures emissions from 400μm thick optical slices, providing high sample signals with efficient background rejection. These thick optical slices also eliminate the need for focusing and are ideal for large cell populations, 3-D cultures, small organisms as well as homogenous assays.
In addition, object-byobject identification and enumeration allows cell sub-populations analysis such as live versus dead, cells in mitosis. The IsoCyte System’s high assay sensitivity is enabled by PMTs in up to four channels to support multiplexed assays and maximum experiment efficiency. A label-free light scatter imaging mode also allows interrogation of non-fluorescent objects (eg cells and cell colonies) where exogenous dyes are unacceptable. Scanning is ‘format- independent’ and an entire microplate (6- to 1536-well format) or slide can be scanned in 2-10 minutes depending on the selected image resolution.
Object-based ‘on-the-fly’ image analysis provides immediate full plate results in the time it takes to scan. Images or data can be further analysed with MetaXpress® and AcuityXpress™ Software or exported in FCS format for plotting in flow cytometry software. The versatile IsoCyte Cytometer bridges the gap between microscopybased high content screening and plate readers, simplifying and accelerating high-content discovery (Figure 10).
TTP LabTech (www.ttplabtech.com) is one of the forerunners in laser scanning microplate cytometry and has been developing this technology for more than a decade. The company’s proprietary cytometric approach to analysis is well established for primary high content screening. The product line has recently been updated and extended through the introduction of a triple laser Acumen, the Acumen® eX3 and the launch of Mirrorball™, a high sensitivity microplate cytometer.
The Acumen eX3 has enhanced multiplexing capabilities which make this instrument more compatible with the growing number of live cell stains and fluorescent proteins that enable the monitoring of cell health and signalling events. This is especially powerful given that high content assays are increasingly being used to report phenotypic changes associated with disease rather than target specific endpoints.
One of the most exciting new application areas for Acumen eX3 is genome-wide RNAi screening and a number of studies have employed this instrument for these screens. For example, a recent screen used the Acumen platform in the identification of human host factors crucial for influenza virus replication (3). Other application areas include cell colony formation, quantification of cell migration and invasion through the use of the Oris™ cell migration assay (Platypus Technologies), and the study of chemotaxis using the iuvo™ chamber (Bellbrook Labs).
The final update to Acumen has been the introduction of the new microscope slide holder which facilitates scanning tissue sections. Acumen is an established high content, high throughput screening platform that is amenable to automation. This instrument lends itself to primary and secondary screens predominantly, but can be utilised throughout the drug discovery process. One of the unmet needs of current laser scanning instrumentation is the high sensitivity required to detect low abundance proteins.
The high performance, low-loss optics of the new Mirrorball addresses this issue and enables the system to perform high sensitivity mix-and-read assays for applications such as hybridoma screening. These include screening against soluble antigens and proteins that are only expressed at low levels, for example cell surface receptors. Cell-based assays may be conducted on live or fixed cells from either adherent or suspension cell cultures.
Alternatively, multiplexed bead-based assays can be undertaken using different fluorescence encoding in conjunction with label-free detection of different bead sizes. The highest specification Mirrorball offers dual laser excitation (488nm and 640nm), four fluorescence data channels and a single laser scatter channel. Mirrorball is the first laser scanning cytometer capable of simultaneous scanning with multiple lasers, a quality which enhances its multiplexing and analytical capabilities.
Such functionality is integral to flow cytometry, where it permits direct correlation of fluorescent emissions across lasers for highly multiplexed assays; in microplate format, simultaneous laser scanning increases throughput by eliminating a need for sequential analysis. The capabilities of Mirrorball are ideally suited to antibody discovery, in particular hybridoma screening and cell surface receptor expression (Figures 11 and 12).
The LEAP Cell Processing Workstation from Cyntellect (www.cyntellect.com) is an automated, high-throughput cell imaging and laser-based cell purification/processing instrument that rapidly images entire wells of multiwell plates, and then targets up to 1,000 points per second to achieve laser-based cell purification and processing.
Highthroughput is obtained by using a large field-ofregard (FOR) F-theta lens with high-speed galvanometers to both scan a large area (to obtain images) and steer the laser to hit target cells, all without moving the cells or culture plate. The engineering design relies on proven optics, microelectronics and software technologies. Whereas many cell imaging systems are based on the ‘microscope-in-a-box’ approach, LEAP is based instead on an approach resembling robust semiconductor manufacturing equipment (eg, for inspecting and etching/cutting/welding of materials).
Since the Ftheta lens is flat-field corrected over the entire FOR, this approach significantly reduces the number of stage movements and refocus steps required as compared with microscope-based imaging, resulting in a >10-fold throughput improvement. Adding the lasers to this optical platform enables rapid cell processing across the large FOR. LEAP contains two compact solid-state diode-pumped Nd:YAG lasers, at 355nm and 532nm, which deliver sub-nanosecond pulses at repetition rates up to 1kHz.
LEAP not only provides high-speed cell image scanning cytometry, but also the ability to perform closed system purification of cells (including potentially biohazardous cells) by laserbased cell elimination. Adherent (and non-adherent) cells can be analysed and purified directly in well plates right where they are grown, down to the clonal level, based on numerous image-based morphological and fluorescent parameters.
LEAP provides the ability to purify small samples with far greater yield and purity than other technologies, because the cells are never removed from the culture surface. Further, LEAP can directly process complex ES or iPS cell colonies in a well without having to disaggregate the cells into single cell suspensions, a great advantage given the sensitivity of these cells to excessive manipulations (Figure 13).
Latest reagents or HCS assay kit developments that support laser scanning cytometry
The following vendor snapshots provide details of some of the latest reagents and HCS assay kit developments that support laser scanning cytometry:
Active Motif’s (www.activmotif.com) Chromeo™ Py-Dyes are a new class of amine-reactive fluorescent pyrylium dyes that change their colour upon reacting with primary amines. They undergo a large shortwave spectral shift, as well as a large increase in quantum yield resulting in a bright fluorescent conjugate. This effectively eliminates background from any unbound dye. These dyes have been used by Active Motif to develop a collection of Cell and Organelle Staining kits that provide specific and efficient staining of subcellular structures.
The kits can be used to highlight changes in cellular morphology, to monitor changes in the localisation and shape of organelles, or to monitor cell-cell contacts within a population of cells. The LavaCell™ Live Cell Membrane Staining Kit, which has been shown to work in a 3D colony assay to monitor proliferation on the MDS Isocyte™ platform, and the Chromeo™ Live Cell Mitochondrial Staining Kit stain their specific cellular targets in live and fixed cells. The excellent retention of the dyes within cells makes them useful for long-term labelling experiments.
The dye of the Chromeo™ Red Fluorescent Fixed Cell Staining Kit stains the nuclear membrane, mitochondria, fibres and nucleoli in fixed cells. It is ideal for use as a counterstain in immunofluorescence experiments in combination with FITC or other 488nm excitable fluorescent dyes.
The fluorescent Mitotic Index Assay Kit provides a quick and accurate assay of the proportion of cells undergoing mitosis in a given cell population, whereas the ToxCount™ Cell Viability Assay is a two-colour fluorescent cell viability assay that detects live and dead cells simultaneously. Both assays have been validated on MDS Isocyte™ and identified as fast and simple methods for homogenous laser scanning cytometry (Figure 14).
Cell Signaling Technology (CST) (www.cellsignal.com) specialises in the development of activation state-specific antibodies that detect post-translational modifications such as phosphorylation, methylation, acetylation, ubiquitinisation and methylation. CST has also developed novel mutation- specific (eg, EGFR L858R, Bcr/Abl) and motif-specific antibodies (eg, phospho-tyrosine, acetyl-lysine, Akt substrate, MAPK substrate).
Signalling antibodies can be used in cell-based assays to examine the cellular signalling underlying complex biological processes, identify aberrant disease- related signalling, or examine treatment- or compound-induced differences in protein activity, expression level or subcellular localisation. They can be used individually in HCS assays to monitor activity of specific kinases, or arranged in arrays to profile signalling across many different pathways or to examine biological processes such as proliferation, apoptosis, cell cycle, DNA damage, inflammation, metabolism or stress.
The robust on/off responses commonly seen with activation statespecific antibodies make them well-suited for laser scanning cytometers. The photomultiplier tubes in these platforms have a large dynamic range that can accurately quantify dim and bright signals. This is particularly useful in assays that involve the use of multiple antibodies with very different signal intensities on the same plate. Such assays can be difficult on CCD imagers as they may require the use of multiple exposures to avoid signal saturation or loss of dim signals that are below the threshold of detection.
CST scientists have rigorously validated more than 700 signalling antibodies for use in fluorescent cell-based assays. This provides HCS users with a large collection of reliable and robust reagents that can be used to monitor endogenously expressed signalling proteins in the cell models that best represent the disease of interest (Figure 15).
Molecular Probes®’ (www.invitrogen.com/HCS) products, for example its Alexa Fluor® labelled secondary antibodies and many of its probes for cellular function and structure, have been proven on various high content imaging, fluorescence microscopy or flow cytometry platforms, including those based on laser scanning cytometry. Molecular Probes has also applied its long experience of developing fluorescent dyes to create solutions for the specific requirements of HCS.
For instance, the CellMask™ and NuclearMask™ segmentation tools have minimal bleed-through into adjacent channels and are available in a range of colours for easy combination with other assay components. Recent developments have focused on applying the extremely powerful Click chemistry to generate assays for biological problems that previously required radioactive components or techniques that were time-consuming and insufficiently robust for the rigours of multiplexed HCS analyses.
Click technology is based upon copper-catalysed reaction between an azide and an alkyne. Either the azide or the alkyne moieties can be used to tag the molecule of interest, while the other is used for subsequent detection. The specificity of the reaction between the azide-alkyne tag pair affords detection with unprecedented sensitivity and low background even in complex biological matrices. With the Click-iT® EdU cell proliferation assay, the harsh treatments required by antibody-based BrdU methods are avoided. Therefore antigen recognition sites and DNA integrity are preserved, enabling truly indepth, multiplexed analyses.
The Click-iT® RNA assays allow detection of global RNA transcription Imaging Assays for DNA fragmentation are available with a choice of several bright and photostable Alexa Fluor® dyes, ranging from green to far-red fluorescence, providing unprecedented flexibility when working with other apoptosis detection reagents.
For researchers interested in monitoring off-target effects on translation during drug discovery, it offers the Click-iT® AHA Alexa Fluor® Protein Synthesis Assay, which uses a methionine analogue that specifically and efficiently is incorporated into nascent proteins. The expanding menu of powerful and easy-to-use Click-iT® HCS kits from Life Technologies provide robust solutions for complex biological problems (Figure 16).
Platypus Technologies (www.platypustech.com) has developed an innovative line of 96-well, cell exclusion zone assays for performing cell migration and cell invasion experiments. The original Oris™ assays utilise cell seeding stoppers to create a cell-free, central Detection Zone around which an annular monolayer of cells can be seeded. The new Oris™ Pro Cell Migration Assay uses a non-toxic biocompatible gel (BCG) to create the cell-free, central Detection Zone.
After cells are seeded and allowed to adhere, the BCG dissolves and cells can then migrate into the centre of each well. The Oris™ Pro Cell Migration Assay offers unlimited access to test wells by automated liquid handling equipment to facilitate efficient delivery of cells, media and test compounds, thereby decreasing laboratory personnel hands-on-time. Data can be generated using multiple stains and analysed using fluorescence microplate readers, inverted microscopes, HCS laser scanning cytometers or HCS CCD imaging instruments.
One such HCS instrument, the Molecular Devices IsoCyte™ Dual Laser scanner, utilises software to accurately calculate the area occupied by fluorescently stained cells that have migrated into the Detection Zone in the centre of the Oris™ assay wells. The IsoCyte™ scanner establishes a region of interest, provides images, total area values and intensity data to analyse an Oris™ assay plate within 2-4 minutes.
To establish a reference for the area of the cell-free Detection Zone that is revealed upon dissolution of the BCG, cells were treated with the actin polymerisation inhibitor, Cytochalasin D (3μM), which completely arrests cell migration.
Figure 17 demonstrates MDA-MB-231 cell migration using an Oris™ Pro Cell Migration Assay – Collagen I Coated. In this study, a Z’ factor of 0.75 was achieved, thereby demonstrating the robustness and suitability of this assay for high throughput screening.
Thermo Scientific (www.thermo.com/hcs) HCS assays use multiplexed imaging to quantitatively monitor the cellular changes of fluorescent-labelled components in cells after drug treatments. Thermo Scientific offers optimised and validated tools to enable simple access to a wide range of imaging assays amiable to the laser excited fluorescence of scanning cytometry instrumentation.
Using appropriate combinations of fluorescent probes, antibodies and reagents is essential in developing these assays, and Cellomics® HCS Kits provide this combination of components and optimised procedures for easy assay implementation. Thermo Scientific BioImage Redistribution® Assays uses GFP-tagged proteins stably expressed in cells to track intracellular protein translocation.
Thermo Scientific Redistribution Cell Lines and kits are designed for a variety of research fields including oncology, inflammation, apoptosis and toxicology. These different assays can also be coupled together to maximise the amount of information extracted from a cell imaging experiment. When used with any laser scanning cytometry instrument containing multiple lasers, these technologies enable easily implemented fully automated HCS assays in a high throughput manner. For example, cell proliferation and apoptosis are two important areas of interest in cell biology and drug-discovery research.
The new Thermo Scientific Cellomics Multiplex Mitosis-Apoptosis Kit is for simultaneous quantification of nuclear DNA content, BrdU incorporation and active caspase 3 and p53 proteins. This kit allows simultaneous measurements of cell proliferation (cell number, DNA replication) and apoptosis in cells grown on standard high-density microplates. DAPI is a DNA-binding dye used to determine the nuclear size and nuclear morphology as well as cell cycle phases by DNA content.
Cell proliferation and apoptosis, which denote life and death of the cell, are critical areas of cell biology and drug-discovery research. This kit identifies cell proliferation (BrdU), caspase-dependent cell death pathway and p53 pathway in the cell, which enables systematic investigation of apoptosis and mitosis events (Figure 18).
Discussion
In this review we have seen how HCS laser scanning cytometers from Compucyte, Molecular Devices and TTP LabTech have matured over the past decade to meet the changing needs of end-users (ie to cater for higher throughput HCS); in enabling HCS of previously intractable applications (eg cell migration assays); and to keep pace with the ever broadening range of fluorophores (eg by adding extra laser lines to maximise multiplexing) and antibodies that monitor endogenously expressed signalling proteins in the cell models.
HCS reagent manufacturers (eg Active Motif, Cell Signalling Technology, Molecular Probes, Platypus Technologies and Thermo Scientific) have also made a significant contribution to this maturation by offering a greater diversity of validated reagents and kits for popular applications, which can be used on both laser scanning cytometers and HCS CCD imagers. These kits not only reduce assay development times by their ease of implementation, but they improve assay robustness and when different assays are coupled together (multiplexed) they maximise the amount of information extracted from a single cell experiment.
Typical cell-based assays performed using laser scanning cytometry now include cell cycle/mitosis, DNA damage/ apoptosis, mitochondrial function, reporter assays (GFP, ‘fruit’ fluorescent proteins, etc), cell-cell interactions, cell migration, cell signalling, immunophenotyping, circulating tumour cells and many more. The tissue-based assays capabilities of laser scanning cytometry should not be overlooked and now cover in situ protein expression on a per cell or per area basis, nuclear, cytoplasmic and membrane biomarkers, immunophenotyping, counts of cellular populations of interest and tissue microarray screening, eg in biomarker discovery programmes.
Instrument manufacturers have continued to evolve their hardware and in recent years have added new features, eg brightfield imaging and label-free light scattering are now widely available permitting interrogation of non-fluorescent objects (eg cells, colonies or organisms like Zebrafish) or the visualisation of sample morphology either used alone or applied as a composite with fluorescent imaging.
In addition, Compucyte’s iGeneration laser scanning cytometers have gone one step further and offer chromatic dye absorption. This technique utilises photodiodes to measure the difference between unobstructed laser light and laser light transmitted directly through chromatically stained samples, typically tissue sections or tissue microarrays. Software functionality has also evolved. For example, some systems use cytometric analysis to identify cells in real-time ‘on-the-fly’ without the generation of intermediate images.
Following recent updates, such software now supports simultaneous export of FCS format for plotting in flow cytometry software or export of open source TIFF images alongside cytometric processing. TIFF allows capture of images covering large fields of view (eg whole well in 24-well plate) and subsequent image analysis in fully-featured third-party software. Such an approach is particularly suited to the study of angiogenesis and tissue sections.
Overall, laser scanning cytometers can be considered today to have effectively bridged the gap between microscopy-based high content screening and the bulk fluorescence of plate readers. However, although the space between HCS and HCA has been narrowed the object resolution capabilities of most laser scanning cytometers are still not comparable to that of confocal microscopy and the two approaches largely remain complementary, working best when deployed side by side.
The next generation of laser scanning cytometers, as exemplified by TTP LabTech’s new Mirrorball, looks set to change thinking and deployment strategies. Mirrorball’s higher sensitivity low loss optics facilitate the detection of soluble antigens and proteins that are only expressed at low levels (eg on cell surface receptors in hybridoma screening) and its ability to simultaneously scan with multiple lasers permits direct correlation of fluorescent emissions across lasers and significantly increases throughput by eliminating a need for sequential analysis.
These new capabilities more and more blur the differentiators that previously separated laser scanning cytometry from flow cytometry, and we can expect further encroachment of microplate-based laser scanning cytometry into the flow arena in the future, particularly if the sample prep limitations of flow are factored in. Platforms such as Mirrorball may also impact bead-based ELISA or homogeneous mix-and-read binding assays, eg previously done using the discontinued FMAT (ABI 8200) or could provide a 384 or 1536 plate-based alternative for XMAP Luminex assays.
In conclusion, existing laser scanning cytometers already support a broad range of applications making it a ‘must have’ instrument in every core flow cytometry and HCS imaging facility. The availability of next generation laser scanning cytometers, such as Mirrorball, will further enhance the versatility and application repertoire of this unique cytometry platform. DDW
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This article originally featured in the DDW Spring 2010 Issue
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Dr John Comley is Managing Director of HTStec Limited, an independent market research consultancy whose focus is on assisting clients delivering novel enabling platform technologies (liquid handling, laboratory automation, detection instrumentation and assay reagent technologies) to drug discovery and the life sciences. Since its formation seven years ago, HTStec has published more than 50 market reports on drug discovery technologies and Dr Comley has authored more than 30 review articles in Drug Discovery World. Please contact info@htstec.com for more information.
References
1 Comley, J (2005). High Content Screening: emerging importance of novel reagents/probes and pathway analysis. Drug Discovery World 6 (3): 31-53.
2 Unpublished results. HTStec Limited, Cambridge UK, November 2009.
3 Karlas et al. Genome-wide RNAi screen identifies human host factors crucial for influenza virus replication. Nature 2010 463:818-822.