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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.