A Quantitative Measure of Field Illumination.

In this paper, we describe a statistically based algorithm to quantify the uniformity of illumination in an optical light microscopy imaging system that outputs a single quality factor (QF) score. The importance of homogeneous field illumination in quantitative light microscopy is well understood and often checked. However, there is currently no standard automatic quantitative measure of the uniformity of the field illumination. Images from 89 different laser-scanning confocal microscopes (LSCMs), which were collected as part of an international study on microscope quality assessment, were used as a "training" set to build the algorithm. To validate the algorithm and verify its robustness, images from 33 additional microscopes, including LSCM and wide-field (WF) microscopes, were used. The statistical paradigm used for developing the quality scoring scale was a regression approach to supervised learning. Three intensity profiles across each image-2 corner-to-corner diagonals and a center horizontal-were used to generate pixel-intensity data. All of the lines passed through the center of the image. The intensity profile data then were converted into a single-field illumination QF score in the range of 0-100, with 0 having extreme variation, and therefore, essentially unusable, and 100 having no deviation, i.e., straight lines with a constant uniform intensity. Empirically, a QF ≥ 83 was determined to be the minimum acceptable value based on manufacturer acceptance tests and reasonably achievable values. This new QF is an invaluable metric to ascertain objectively and easily the uniformity of illumination quality, provide a traceable reference for monitoring field uniformity over time, and make a direct comparison among different microscopes. The QF can also be used as an indicator of system failure and the need for alignment or service of the instrument.

Quantitative confocal microscopy: beyond a pretty picture.

Quantitative optical microscopy has become the norm, with the confocal laser-scanning microscope being the workhorse of many imaging laboratories. Generating quantitative data requires a greater emphasis on the accurate operation of the microscope itself, along with proper experimental design and adequate controls. The microscope, which is more accurately an imaging system, cannot be treated as a "black box" with the collected data viewed as infallible. There needs to be regularly scheduled performance testing that will ensure that quality data are being generated. This regular testing also allows for the tracking of metrics that can point to issues before they result in instrument malfunction and downtime. In turn, images must be collected in a manner that is quantitative with maximal signal to noise (which can be difficult depending on the application) without data clipping. Images must then be processed to correct for background intensities, fluorophore cross talk, and uneven field illumination. With advanced techniques such as spectral imaging, Förster resonance energy transfer, and fluorescence-lifetime imaging microscopy, experimental design needs to be carefully planned out and include all appropriate controls. Quantitative confocal imaging in all of these contexts and more will be explored within the chapter.

International test results for objective lens quality, resolution, spectral accuracy and spectral separation for confocal laser scanning microscopes.

As part of an ongoing effort to increase image reproducibility and fidelity in addition to improving cross-instrument consistency, we have proposed using four separate instrument quality tests to augment the ones we have previously reported. These four tests assessed the following areas: (1) objective lens quality, (2) resolution, (3) accuracy of the wavelength information from spectral detectors, and (4) the accuracy and quality of spectral separation algorithms. Data were received from 55 laboratories located in 18 countries. The largest source of errors across all tests was user error which could be subdivided between failure to follow provided protocols and improper use of the microscope. This truly emphasizes the importance of proper rigorous training and diligence in performing confocal microscopy experiments and equipment evaluations. It should be noted that there was no discernible difference in quality between confocal microscope manufactures. These tests, as well as others previously reported, will help assess the quality of confocal microscopy equipment and will provide a means to track equipment performance over time. From 62 to 97% of the data sets sent in passed the various tests demonstrating the usefulness and appropriateness of these tests as part of a larger performance testing regiment.

Measuring and interpreting point spread functions to determine confocal microscope resolution and ensure quality control.

This protocol outlines a procedure for collecting and analyzing point spread functions (PSFs). It describes how to prepare fluorescent microsphere samples, set up a confocal microscope to properly collect 3D confocal image data of the microspheres and perform PSF measurements. The analysis of the PSF is used to determine the resolution of the microscope and to identify any problems with the quality of the microscope's images. The PSF geometry is used as an indicator to identify problems with the objective lens, confocal laser scanning components and other relay optics. Identification of possible causes of PSF abnormalities and solutions to improve microscope performance are provided. The microsphere sample preparation requires 2-3 h plus an overnight drying period. The microscope setup requires 2 h (1 h for laser warm up), whereas collecting and analyzing the PSF images require an additional 2-3 h.

Quality assurance testing for modern optical imaging systems.

The days of being able to ascertain instrument performance by simply peering through the eye pieces at a specimen are gone. However, users and granting agencies need to be confident that data collected on these instruments is uniform and quantifiable both over time and between instruments. Ideally, a LASER should not fluctuate, illumination should be completely uniform, and colors should be perfectly aligned. To check the current performance of imaging equipment, we conducted a worldwide research study utilizing three image-based tests: long-/short-term illumination stability, co-registration of signals across various wavelengths, and field illumination uniformity. To differentiate between "acceptable" and "unacceptable" performance, the deviation in illumination power could not exceed 10% (long term) or 3% (short term), the difference in the center-of-mass of imaged multicolored beads could not exceed >1 pixel between different wavelengths, and field illumination values could not exceed 10% (horizontal) or 20% (diagonal) deviation. This study established the current state of microscope performance through simple, efficient, and robust tests, while defining relative standards to assist cores in maintaining their instruments in optimal operating conditions. We developed cross-platform performance standards that will improve the validity of quantitative measurements made using various light microscopes.

Light-emitting diodes are better illumination sources for biological microscopy than conventional sources.

Light-emitting diodes (LEDs) can be easily and inexpensively integrated into modern light microscopes. There are numerous advantages of LEDs as illumination sources; most notably, they provide brightness and spectral control. We demonstrate that for transmitted light imaging, an LED can replace the traditional tungsten filament bulb while offering longer life; no color temperature change with intensity change; reduced emission in the infrared region, which is important for live cell imaging; and reduced cost of ownership. We show a direct substitution of the typical tungsten bulb with a commercially available LED and demonstrated the color stability by imaging a histology section over a wide range of light intensities. For fluorescent imaging, where the typical illumination sources are mercury or xenon lamps, we demonstrate that LEDs offer advantages of providing a longer lifespan, having a more constant intensity output over time, more homogeneous illumination, and significantly lower photon dose. Our LED equipped system was used to image and deconvolve dual fluorescently labeled cells, as well as image cells undergoing mitosis expressing green fluorescent protein-histone 2B complex. The timing of the stages of mitosis is well established as an indicator of cell viability.

Inhibition of MCF-7 breast cancer cell proliferation by MCF-10A breast epithelial cells in coculture.

A coculture system was developed to investigate the interactions between MCF-10A breast epithelial cells and MCF-7 breast cancer cells stably expressing the green fluorescent protein (MCF-7-GFP). Studies with this MCF-10A/MCF-7-GFP coculture system on microtiter plates and on reconstituted basement membrane (Matrigel), revealed paracrine inhibition of MCF-7-GFP cell proliferation. Epidermal growth factor, which in monocultures modestly enhanced MCF-7-GFP and markedly increased MCF-10A cell proliferation, greatly inhibited MCF-7-GFP cell proliferation in MCF-10A/MCF-7-GFP cocultures. 17beta-Estradiol, which stimulated MCF-7-GFP but not MCF-10A cell proliferation in monoculture, inhibited MCF-7-GFP cell proliferation in MCF-10A/MCF-7-GFP cocultures, an effect that was blocked by the antiestrogen, ICI 182,780. On Matrigel, complex MCF-10A/MCF-7-GFP cellular interactions were observed in real time that resulted in the formation of acinus-like structures. These results indicate a role of normal epithelial cells in inhibiting tumor-cell proliferation and demonstrate the utility of this coculture system as a model of early paracrine control of breast cancer.

The small molecule Hesperadin reveals a role for Aurora B in correcting kinetochore-microtubule attachment and in maintaining the spindle assembly checkpoint.

The proper segregation of sister chromatids in mitosis depends on bipolar attachment of all chromosomes to the mitotic spindle. We have identified the small molecule Hesperadin as an inhibitor of chromosome alignment and segregation. Our data imply that Hesperadin causes this phenotype by inhibiting the function of the mitotic kinase Aurora B. Mammalian cells treated with Hesperadin enter anaphase in the presence of numerous monooriented chromosomes, many of which may have both sister kinetochores attached to one spindle pole (syntelic attachment). Hesperadin also causes cells arrested by taxol or monastrol to enter anaphase within <1 h, whereas cells in nocodazole stay arrested for 3-5 h. Together, our data suggest that Aurora B is required to generate unattached kinetochores on monooriented chromosomes, which in turn could promote bipolar attachment as well as maintain checkpoint signaling.

Cold-shock and the Mammalian cell cycle.

Progression through the cell cycle is temperature sensitive, but the relationship is not straightforward. In culture, many types of mammalian cells fail to undergo the G(2)/M transition after cooling from 37 degrees C to 16-20 degrees C (moderate hypothermia). However, progression through G(1) and S is not blocked at these temperatures, nor is progression through mitosis in cells cooled after they have become committed to the division process. Thus, at least one pathway is present during G(2)-but not during G(1), S or mitosis-that is selectively disrupted at or below a critical temperature. As a result, a prolonged (24-48 hr) exposure to moderate hypothermia can be used to enrich cultures for G(2) cells. A brief (1 hr) exposure to severe hypothermia (4-10 degrees C) is also reported to induce a high degree of mitotic synchrony (up to 80%) in some mammalian cultures. Although the mechanism behind this synchronization remains vague, it may involve a cell cycle checkpoint, triggered in response to the cold shock, that transiently inhibits the G(1)/S transition.

DNA damage during mitosis in human cells delays the metaphase/anaphase transition via the spindle-assembly checkpoint.

BACKGROUND: DNA damage during mitosis triggers an ATM kinase-mediated cell cycle checkpoint pathway in yeast and fly embryos that delays progression through division. Recent data suggest that this is also true for mammals. Here we used laser microsurgery and inhibitors of topoisomerase IIalpha to break DNA in various mammalian cells after they became committed to mitosis. We then followed the fate of these cells and emphasized the timing of mitotic progression, spindle structure, and chromosome behavior. RESULTS: We find that DNA breaks generated during late prophase do not impede entry into prometaphase. If the damage is minor, cells complete mitosis on time. However, more significant damage substantially delays exit from mitosis in many cell types. In human (HeLa, CFPAC-1, and hTERT-RPE) cells, this delay occurs during metaphase, after the formation of a bipolar spindle and the destruction of cyclin A, and it is not dependent on a functional p53 pathway. Pretreating cells with ATM kinase inhibitors does not abrogate the metaphase delay due to chromosome damage. Immunofluorescence studies reveal that cells blocked in metaphase by chromosome damage contain one or more Mad2-positive kinetochores, and the block is rapidly overridden when the cells are microinjected with a dominant-negative construct of Mad2 (Mad2deltaC). CONCLUSIONS: We conclude that the delay in mitosis induced by DNA damage is not due to an ATM-mediated DNA damage checkpoint pathway. Rather, the damage leads to defects in kinetochore attachment and function that, in turn, maintain the intrinsic Mad-2-based spindle assembly checkpoint.

Separating centrosomes interact in the absence of associated chromosomes during mitosis in cultured vertebrate cells.

We detail here how "free" centrosomes, lacking associated chromosomes, behave during mitosis in PtK(2) homokaryons stably expressing GFP-alpha-tubulin. As free centrosomes separate during prometaphase, their associated astral microtubules (Mts) interact to form a spindle-shaped array that is enriched for cytoplasmic dynein and Eg5. Over the next 30 min, these arrays become progressively depleted of Mts until the two centrosomes are linked by a single bundle, containing 10-20 Mts, that persists for > 60 min. The overlapping astral Mts within this bundle are loosely organized, and their plus ends terminate near its midzone, which is enriched for an ill-defined matrix material. At this time, the distance between the centrosomes is not defined by external forces because these organelles remain stationary when the bundle connecting them is severed by laser microsurgery. However, since the centrosomes move towards one another in response to monastrol treatment, the kinesin-like motor protein Eg5 is involved. From these results, we conclude that separating asters interact during prometaphase of mitosis to form a spindle-shaped Mt array, but that in the absence of chromosomes this array is unstable. An analysis of the existing data suggests that the stabilization of spindle Mts during mitosis in vertebrates does not involve the chromatin (i.e., the RCC1/RanGTP pathway), but instead some other chromosomal component, e.g., kinetochores.

Partner telomeres during anaphase in crane-fly spermatocytes are connected by an elastic tether that exerts a backward force and resists poleward motion.

As chromosomes move polewards during anaphase in crane-fly spermatocytes, trailing arms commonly stretch backwards for a brief time, as if tethered to their partners. To test that notion, a laser microbeam was used to sever trailing arms and thereby release telomere-containing arm segments (called acentric fragments because they lack kinetochores) from segregating chromosomes. Analysis of the movement of acentric fragments after their release provided clear evidence that previously conjoined partners were indeed tethered at their telomeres and that tethers exerted backward forces that were sufficient to move the fragment across the equator and into the opposite half-spindle. To address concerns that tethers might be artifacts of in vitro cell culture, spermatocytes were fixed in situ, and stretched arms within fixed cells provided strong evidence for tethers in vivo. The substantial resistance that tethers impose on the poleward movement of chromosomes must normally be over-ridden by the poleward 'pulling' forces exerted at kinetochores. In spermatocytes, poleward forces are supplied primarily by the 'traction fibers' that are firmly attached to kinetochores through end-on attachments to the plus ends of kinetochore microtubules.

Polar ejection forces are operative in crane-fly spermatocytes, but their action is limited to the spindle periphery.

Laser microsurgery was employed to reveal kinetochore-independent forces acting on chromosome arms in crane-fly spermatocytes. When a portion of an arm situated along the interpolar axis between the equator and a pole was cut off, the resultant acentric fragment was transported poleward and outward into the peripheral domain of the spindle. If the fragment was generated well in advance of the onset of anaphase, then at the spindle periphery, it changed direction and moved away from the pole and back toward the equator. That domain-specific movement-poleward in the central spindle and away from the pole at the spindle periphery-not only provides the first evidence for polar ejection forces acting on acentric fragments in a meiotic system, but it is the first example of kinetochore-independent forces in both directions at the same stage of division. Sniglets generated by laser pulses directed at specific sites in the spindle revealed that the mechanism underlying ejection forces was specific to chromosomes. At anaphase onset, polar ejection forces ceased, and pole-directed forces took over. At that time, chromosome fragments that had been ejected to the equator moved poleward again, providing clear evidence for kinetochore-independent forces on chromosome arms during anaphase.