Technology Feature<= /o:p>

Nature 437, 775-779 (29 September= 2005) | doi:10.1038/437775a

Imaging: The big pi= cture

Lisa Melton¹

Abstract

Over the pas= t ten years, microscopy has been transformed from slice, stain and fix, to the capacity to view living cells and even whole organisms in real time. Lisa Melton looks at what's on offer.

When it was = introduced in the late 1980s, confocal laser-scanning micr= oscopy opened up a new high-resolution world to biologists. But researchers are becoming more demanding of their microscopes. They expect not just to see a clearer image, but to monitor dynamic protein networks within cells, map the kinetics of intracellular organelles and track calcium signalling. Microscope developers have responded by producing conf= ocal microscope systems that have dramatically improved scanning speeds, greater resolution and the capacity to see detail in live cells labelled with multiple colours.

Researchers traditionally struggled to obtain sharp images from multi-stained specimens, the main obstacle being spectral emission overlap from different fluorescent probes. Nikon's recently launched Digital Eclipse C1 Spectral Imaging confocal system collects high-resolution data from the 400–750 nanometres range with a single pa= ss of the laser. A mathematical process unmixes close= ly overlapping spectral data to produce clean images with no cross-talk, even = for notoriously difficult reds. "We use chromatic aberration-free objectiv= es, so everything towards either end of the spectra focuses at the same point," says Chay Keogh, marketing manager= for Nikon, UK in Kingston upon Th= ames. "By eliminating the need for multiple scans, specimen damage is kept t= o a minimum."

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NIKON

Pancreatic cells imaged in the Dig= ital Eclipse.

A rival syst= em from Olympus, the Fluoview F= V1000, is a confocal laser-scanning microscope with two independent, synchronized laser scanners in a single instrument. While one laser provides high-resolution confocal images,= the second scanner, the SIM scanner, simultaneously stimulates the sample. This makes the FV1000 an ideal choice for live-cell applications such as fluorescence recovery after photobleaching (FRA= P), fluorescence loss in photobleaching (FLIP), uncaging, or photoactivation and photoconversion. "It offers, for the first time,= the opportunity to study the kinetics of rapid cellular reactions after laser stimulation, without a time lag, because you don't have to stop imaging when using laser light for stimulation," points out Martin Tewinkel, business manager at Olympus Life and Material Science = Europa in Hamburg, Germany. "Such studies of rapid cellular responses can pro= vide important insights into how various cellular mechanisms operate."=

Another high= -speed, high-resolution confocal imaging system is the = Nipkow-disk-based Ultraview ERS from PerkinElmer of Boston, Massachusetts, which offers a choice of cameras= for different applications: a standard interline CCD detector for the highest resolution with slower processes or bright samples that withstand higher la= ser intensities, or an electron-multiplying CCD detector for highly dynamic processes, very dim fluorescence, or light-sensitive samples.

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OL= YMPUS

3D rendering w= ith the Olympus Fluoview.

For research= ers who want speed but don't need the high resolution of a con= focal microscope, Olympus has designed live-cell imaging systems that can be fitt= ed to Olympus's upright BX or inverted IX series wide-field microscopes — the relatively inexpensive cell∧M imaging = station and the high-end station cell∧R. The cel= l∧R takes ten multicolour images per second a= t full resolution and can be used to track cell growth, metabolic transport and si= gnal transduction in real time. "It's always a trade-off: the quality of th= e data on the one hand and speed on the other," explains Christian Seel, head of information transfer management at Olym= pus BioSystems in Munich. The key to speed is finely synchronized illumination and camera controls. T= he high-intensity coloured light is switched off t= he specimen immediately after taking each photo, reducing photo-damage to a minimum, says Seel. Time-lapse series are store= d by the imaging software and presented as movies or charts.

Developers a= t Carl Zeiss believe there is no need to sacrifice resolutio= n for ultra-fast cellular dynamics with the confocal imaging system LSM 5 LIVE. "Our main motivation was to develop an instrument dedicated to fast live-cell imaging, with a much higher speed th= an any other system available today while maintaining a very good confocal resolution," says Richard Ankerhold, director of advanced development at Carl Zeiss = in Jena, Germany. Dynamic interactions can be viewed at different scales ̵= 2; a group of interacting molecules, a complete cell, a developing organ, or eve= n an entire zebrafish embryo, notes Ankerhold. LSM 5 LIVE operates even on weakly fluorescent specimens and collects up to= 120 confocal images per second at a resolution of 5= 12 =3Dtimes 512 pixels, scanning about 20 times fas= ter than a traditional confocal system.<= /span>

Developmental biologist Mary Dickinson and her group at the California Institute of Technology in Pasadena have capitalized on the instrument's fast-frame recording to image erythroblasts rushing through the heart of an 8-day-old mouse embryo, and to produce a time-lapse series of the beating heart of a = zebrafish embryo. This technology is expensive, however, and at present only affordab= le by large research institutes. Alternative optical approaches to imaging emb= ryos for developmental research are selective plane illumination microscopy and optical projection tomography microscopy (see 'An illuminating breakthrough' and 'Optical tomography for embryos').

With calcium-sensitive dyes such as Fura-2 you can visualize spikes, waves and oscillations of calcium in living cells. An instrument dedicated to imaging intracellular ion kinetics — and with= a price tag within the reach of most research labs — is the InCyt Imaging system from Intracellular Imaging of Cincinnati, Ohio, co-founded by Eric Gruenstein, director of the Center for Image Analysis at the Uni= versity of Cincinnati.=

Starting at US$30,000, it includes a fluorescence microscope, low-light-level CCD camera, filter changer and image-processing computer. "It's a very cost-effective and feature-rich solution for ion imaging and cell kinetics," says Tim Fletcher, product specialist at I= mage Solutions of Preston, the UK distributors for Intracellular Imaging. InCyt h= as been tailored to image and quantify calcium, but can measure other ions, and works with all commonly used dyes. Designed by cell biologists, the software follows the logical flow of an experiment. Images can be displayed in real = time or saved and played back as an animated sequence. "The InCyt system needs very little training, and researchers pick it up quickly," says Fletcher.

The whole animal

For some ima= ging applications, getting a picture from inside the living body is the goal (se= e 'Optical biopsies'). And for others, a shift from imaging in vitro to the whole living animal is desirable. "Many pathways interact on a systems level, that is, the immune system and the endocrine system," says Pam = Contag, co-founder of imaging company Xenogen in Alameda, California. Xenog= en puts together non-invasive optical imaging systems with transgenic mice and rats containing the required luciferase-tagged = genes. The Xenogen IVIS 200 will image either bioluminescence or fluorescence, and its adjustable field of view makes it versatile enough to image single cells at high resolution or up to five anaesthetized mice at a time. Biophotonic imagi= ng has proved a success with the pharmaceutical industry to test drug candidates a= nd make more educated predictions. "In drug discovery, the cell is used as the gold standard, but you don't treat single cells, you treat the whole person," Contag insists. "Our goal is= to make animal models to be predictive for what happens in humans."<= /o:p>

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INTRACELLULAR IMAGING

Calcium signals: the InCyt imaging system.

Lightools of Encinitas, California, concentrates on whole-body imaging of animals carrying fluorescent-tagged genes. "We can zoom in = and out from a couple of centimetres to 10–15= centimetres in the field of view," says John Fox= , Lightool's president. The company has recently introd= uced two novelties that are proving popular. One is a device for simultaneously viewing green and red fluorescent proteins (GFP and RFP) in transgenic anim= als, with independent controls for each fluorophore. "It allows you to turn up the RFP excitation and turn down the GFP = 212; which is usually brighter — to obtain a more balanced image," sa= ys Fox.

The second i= s the Pan-A-See-Ya panoramic imaging system, which gi= ves a 270° view of the animal in one evenly illuminated image. "It's like being able to see round the corner," says Fox. A target such as a fluorescent tumour can be viewed from two diffe= rent angles at the same time with a single camera. The process is even fast enou= gh to allow animals to be imaged without anaesthesia.

Breaking the resolution = barrier

Abbe's law, postulating that optical res= olution is impossible below 200 nanometres, went unchal= lenged for 120 years. Until recently, that is, when physicist Stefan Hell, a direc= tor of the Max Planck Institute for Biophysical Chemistry in Göttingen, Germany, established a new la= w that promises greater resolution in fluorescence microscopy. The first commercial application of his new ideas is the 4Pi fluorescence c= onfocal imaging system, created in cooperation with Leica Microsystems in Mannheim, Germany.

The Leica TCS 4Pi improves resolution of fluorescent imag= es to 110 nanometres along the z axis. Martin = Hoppe, marketing manager for Leica Microsystems, says = that the system addresses a resolution gap between optical and electron microsco= pes. "That's what I see when I offer this system to researchers," he s= ays. Structures down to 110 nanometres can now be re= solved in living cells — a malaria parasite can now be localized precisely inside a red blood cell, for example.

For life-sci= ences researchers, the advantage of the TCS 4Pi over electron microscopy is that specimens can be kept alive and fluorescent stains can be used. "People don't have to redo their staining techniques," says Hoppe.<= /span>

The remarkab= le gain in sharpness is due to the use of two opposing lenses with high numerical aper= ture to illuminate a single focal spot. The two wavefronts<= /span> of the opposing beams interfere constructively at the focal point, which gi= ves much higher resolution.

But at US$1,= 000,000, the TCS 4Pi doesn't come cheap. "What drives up the cost is the combination of precision mechanics, interferometer optics and state-of-the-= art electronics. Also, the 4Pi objectives have to be paired, this makes the manufacturing yield very low," Hoppe points out.

A world of colour

Quantum dots= may be the newest kid on the block (see 'Quantum dots keep on glowing'), but since its cloning a decade ago, the green fluorescent protein has become one of the most powerful molecular tools in = the cell biology tool-box. Either by itself or as part of a fusion protein, GFP= is used to visualize proteins inside living cells in a vast number of applications, from detecting gene expression to tracking cell fate in developing embryos.

The range of fluorescent proteins from jellyfish now includes red (RFP), cyan (CFP) and yellow (YFP) relatives of GFP. CFP and YFP, in particular, make a suitable contrasting pair for multicolour imaging for differential gene expression and protein localization.

Species othe= r than jellyfish are now being mined to expand the colour palette. The DsRed–Monomer fluorescent protein, an engineered variety of a protein from the sea anemone <= i>Discosoma, was recently launched by BD Biosciences Clontech of Mountain View, California, now part of Shiga-based Japanese life-sciences supply company Takara Bio.

"What i= s driving users' interest towards red is a better signal-to-noise ratio, because the background fluorescence from the culture medium is in the green range," explains Andrew Farmer, director of cellular and molecular biology for Clontech Business Research. The = monomeric form of the new protein is an added advantage, particularly for subcellular labelling. &q= uot;The DsRed–Monomer is less likely to misbehave than = the tetrameric reds, which can sometimes disrupt the func= tion of the fusion protein," says Farmer.

Stony corals= have yielded a remarkable collection of new fluorescent proteins. The CoralHue range was originally isolated by Atsushi Miyawaki at the RIKEN Brain Science Institute in Saitama, Japan. Kaede (Japanese for maple leaf), the first of t= he family, is a brilliant green fluorescent protein that changes colour to a stable red when exposed to a short pulse = of ultraviolet or violet light. Miyawaki's team have used Kaede to st= udy hippocampal neuronal connections. Cultured neurons ar= e labelled with green Kaede= by gene transfection then, with a focused violet light = pulse, a single cell body is illuminated. As the red spots spread rapidly througho= ut the cell's cytosol, all the nerve-cell processe= s, including an axon and illuminated dendrites, stand out from the green background, delineating the neuron and its multiple contact sites.

A recent add= ition to the CoralHue family is Kus= abira orange, the first monomeric true-orange fluores= cent protein. "Its greatest value is in combination with Midoriishi–Cyan for fluorescence resonance excitation transfer analysis," says Suzan <= span class=3DSpellE>Oberle, product manager for MBL International of Wobu= rn, Massachusetts, which distributes the CoralHue r= ange. The CoralHue pair is brighter and shows better spectral separation of donor and acceptor signals than the widely used CFP = and YFP pair.

For FRAP and= FLIP applications, CoralHue Dro= npa green bleaches following excitation at 500 nanometres<= /span> but completely regains its bright green fluorescence after minimal irradiat= ion at 400 nanometres, without losing signal intens= ity. The switching can be repeated without losing brightness. Miyawaki's group has used this reversible protein highlighting technique to track prot= eins shuttling across the nuclear membrane after cell stimulation.

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R. ANDO & H. MIZUNO

Violet light makes HeLa cells expressing Kaede fluorescent protein chan= ge from green to red.

Another phot= o-switchable fluorescent protein is produced by Evrogen, a biotechnology company based in Moscow, Russia. Their cyan-to-green photo-converting protein PS-CFP2, gives a 2,000-fold increase in the green-to-cyan fluorescence ratio, making it the highest-contrast monomeric photoactivatable fluorescent protein so far.

The microsco= pe may be 400 years old, but microscopy is refusing to show its age. High-resolution = live imaging is giving researchers a whole new look at the biological world.

Lisa Melton is science writer in reside= nce at the Novartis Foundation in London.