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An Introduction to Live Cell Analysis

Cellular analysis has a long history. Anton van Leeuwenhoek (1632-1723) and other early Dutch optical pioneers created the first microscopes that allowed a unique, close up inspection of biology. What these early pioneers of microscopy discovered is that most cells are small, clear, and complicated. It can be difficult to observe their structure, behavior or composition without a visualization aid or stain. Because most stains kill cells, most analyzed cells then and today are fixed and static. Stains allow for good cellular resolution but the dynamic biology thought we know to exist is caught in a single moment of time, rather than “alive”. As with the introduction of microscopy, advances in live cell imaging technologies have created a new, unique way to study biology.

Live Cell Analysis: A Brief History

Live cell analysis also has a long history and even a Nobel Prize to its credit.

1907 Scientists began using crude cameras and film to capture time-lapse images of living cells. French researcher, Julius Ries made one of the earliest “microcinematographic” films of fertilization and development in sea urchins in 1907. Since the whole process of sea urchin larval development takes about 14 hours, these time lapse films allowed students and researchers to see the whole progression in about 2 minutes. Researchers viewing cells this way quickly realized they could see developmental details undetected by earlier static pictures.

A historical commentary on this work
1930 Dutch researcher Fritz Zernike discovers phenomenon of phase contrast microscopic imaging one evening in his completely black-painted optical laboratory. The significance and application of his discovery would wait until their potential use by Germany in World War II. Development of phase contrast microscopy allowed scientists to visualize living cells in great detail. (Cells are largely transparent and without color, so they appear very clear and unremarkable under simple, traditional light microscopy.)

The Nobel Prize in Physics was awarded to Frits Zernike "for his demonstration of the phase contrast method, especially for his invention of the phase contrast microscope".

Read the Noble Prize Press award ceremony speech.
1952 Merck:/Freestyle/BI-Bioscience/Cell-Culture/stem-cell-images/transparent-live-tissue.jpgFollowing Fritz Zernike’s phase contrast microscopy work, Georges Nomarski improved the technique by converting the optical gradients produced by the transparent live tissue into intensity differences.

The Nomarski interference contrast (DIC) method allowed greater depth of focus and surface detail of unstained, live cells and tissue.

Read Nomarski’s pioneering story.
1975 Robert Hoffman and Leo Gross publish paper describing a new microscope imaging system to visalize phase gradients called modulation contrast. Hoffman modulation (HMC) contrast microscopy incorporates optical amplitude spatial filters (modulators), slit plates and polarizers to restrict the phased light and allow for even more enhanced contrast of unstained biological specimens with better resolution.

Learn more about Hoffman modulation.
1999 Improved digital processing allows for quantitative holography of phase contrast images. This is accomplished by using the phase data to create a phase shift image which is independent of the intensity of the brightfield image. The phase shift images depend on the objects thickness and relative refractive index. The technique allows for exceptional live cell and tissue imaging.

Click here for links to some of the newest methods and pioneering groups offering quantitative phase contrast imaging.
2000 Today, improvements in cameras, fluorescent live dyes, glowing proteins, video compression, and time lapse microscopy have advanced our ability to visualize and analyze, living, unstained cells and their interactions with an astonishing level of detail and fidelity.

Transduced fluorescent protein
Transduced fluorescent proteins display diffuse distribution in growth media and a punctate distribution following starvation-induced autophagy
2014 In the late 1970’s theoretical research suggested that laser light focused from all sides, creating point-to-point illumination and detection could break the optical physics limitation known as the Abbe limit. By pushing beyond the Abbe limit, the realized microscopy could see down to nanometer resolution. In 2014 the Nobel Prize in Chemistry was awarded to Eric Betzig, W.E. Moerner and Stefan Hell for their work on pioneering “super-resolved fluorescence microscopy”, a technique that allows optical microscopy to peer into the molecular world.

Read the Noble Prize Press Release on Surpassing the limitations of the light microscope.

How Autophagy Is Studied Using Live Cell Lentiviral Biosensors

Live cell imaging coupled with video is a powerful tool for measuring cellular behavior.

M04G Neutrophil Migration Video

Click the video to see neutrophil migration response to a spatial gradient using CellASIC® Onix platform.