Article

Multi-Wavelength Epi-Illumination in Fluorescence Microscopy

Fluorescence is a process where a substance after having absorbed light (photons) emitts a radiation the wavelength (colour) of which is longer than that of the absorbed light, and where this emission stops immediately after cessation of the excitation. This phenomenon is the basic element of fluorescence microscopy and its application.

Besides the "classical" excitation of fluorescence in a light microscope it is possible today to obtain the same emission effect via the modern technology of confocal laser scanning microscopy by an excitation with two or multiple photons having longer wavengths than those of the emitted ones.

Fluorescence occurs either as autofluorescenc of biological and/or inorganic structures or as so called secondary fluorescence after a treatment of the specimen with special dyes (fluorochromes, fluorescent markers). To perform fluorescence in a microscope the following requirements have to be met: powerful energy sources (highpressure mercury arc lamp, halogen lamp, etc.), adequate transmission filter systems (filterblocks) selecting the excitation light and the emitted radiation perfectly, and, last but not least, optical parts and outfits suitable for fluorescence, i.e. collector lenses, illuminators, beamsplitters, objectives, tube lenses and eyepieces.

Compared with the situation of today, fluorescence microscopy was for the first time applied with the use of transmitted light and darkfield microscopy. This was due to its limited rangers of application in those days. But with its increasing importance to histology, cytology, molecular biology and immuno-diagnosis the demand for a fundamental improvement of illumination and observation techniques came up more and more. This was the hour of birth of incident light fluorescence microscopy.

During its course of nearly 40 years of application and development this technique became one of the basic tools for routine and research work in biology, medicine, science and industry. The progress in incident light fluorescence microscopy was particularly determined by the research work of Ploem and his function as trend setter and also by the forward strategy of Leitz resp. Leica Wetzlar in developing the optical instruments required.

This course of development is described chronologically in the present contribution. A synoptical table of fluorescence microscopy techniques gives relevant information about microscopes, objectives, fluorochromes, light sources and filter systems concerning the fields of application and the methods referring to this.

Authors

- Johan Sebastiaan Ploem , Professor em.

- Friedrich Walter , Dr.

Topics & Tags

- Fluorescence Microscopy

- Fluorescence

- Fluorophore

- History

Fluorescence

Fluorescence is a molecular phenomenon in which a substance absorbs light (excitation) and radiates light of longer wavelength (emission) for a very short period of time. When a fluorochrome absorbs light, energy in the form of photons is taken up, leading to excitation of electrons to higher energy states. The process of absorption of photons is extremely rapid and is immediately followed by a return to lower energy states, which is then accompanied by emission of photons, which can be observed if light is emitted in the visual range.

This short duration (nanoseconds) distinguishes it from other forms of luminescence such as phosphorescence. The difference in wavelengths between the excitation and emission peaks is referred to as the Stokes shift. Fluorochromes with a large Stokes shift are easy to excite and to observe their emission in a fluorescence microscope. With transmitted light illumination, a small Stokes shift may make it impossible to illuminate a fluorochrome at its excitation peak and to observe the fluorescence colour at its emission peak.

Confocal laser scanning fluorescence microscopy enables two-photon excitation with photons of longer wave-length than the emitted light. The electron is then brought into its excited state with two photons of half the required energy, arriving simultaneously. In most cases the electron ends up in the same excited state as with normal single-photon excitation before it drops down to the ground state. Similarly, the fluorescence emitted is similar to that given off by normal single-photon excitation.

Fluorochromes

Many molecules, both non-organic and organic, show fluorescence emission, especially with excitation using high energy radiation. When plant or animal tissue is excited with -light very often a bluish fluorescence emission is observed which is called primary fluorescence, or autofluorescence [7, 8]. Naturally occurring substances often have very broad excitation and emission spectra. With the development of molecular biology, research has been focussed on special dyes with bright fluorescence to distinguish them from possible autofluorescence. The dyes used to selectively stain biologically important molecules are called fluorochromes. When conjugated to antibodies or nucleic acids, they are referred to as fluorescent markers or probes. Today fluorochromes are available with peak emissions in the violet, blue, green, orange, red and near-infrared regions of the spectrum. The possibility to excite fluorochromes with orange and red light and detect the red and infrared fluorescence with a photomultiplier or a CCD-camera has extended fluorescence microscopy beyond the visual range. The excitation and emission of a fluorochrome may shift with changes in cellular environment. Some dyes are especially chosen because their excitation or emission spectra are shifted, following changes in concentration of some ions in the intracellular medium such as calcium, natrium, and of the pH.

Early developments in epi-illumination fluorescence microscopy

For a review of the history of fluorescence microscopy the reader is referred to Kasten [11]. Epi-illumination (vertically incident exciting light) was used already by Policard and Paillot [32]. Several instruments were made by Leitz (Leica), Bausch & Lomb, Reichert and Zeiss, which were partially based on suggestions by Ellinger and Hirt [4, 5], Singer [37] and Mehler and Pick [16, 19]. An early Leitz (Leica) fluorescence epi-illumination system was described by Haitinger [6]. For more details on the evolution of epi-illumination fluorescence microscopy the reader is referred to Rost [34] and for early applications of incident-light fluorescence microscopy to Hauser [7].

A major contribution in epi-illumination fluorescence microscopy was the introduction of a dichromatic mirror for incident illumination with  light by Brumberg and Krylova [2]. Epi-illumination has definite optical advantages because, unlike transmitted illumination where the condenser and the objective have independent optical axes which must be perfectly aligned. The objective functions both as a condenser and as a light-collecting objective, avoiding all alignment problems. The separation of fluorescence emission from excitation light, using a dichroic beamspitter, is much easier than with transmittted light fluorescence microscopy, These possibilities did, however, not lead to a general acceptance by industrial microscope manufacturers of epi-illumination for routine fluorescence microscopy. The main reason for this could have been that transmitted-light darkfield  excitation gave already excellent results in most applications of fluorescence microscopy [17]. Its replacement by  epi-illumination would not have had significant advantages. The use of transmitted light using a darkfield condenser remained the industry standard until the late sixties.

The rapidly growing interest in molecular biology, however, led to the development of many (monoclonal) antibodies for the detection of important macromolecules in the cell. To study the detailed morphological location of several macromolecules in the cellular organelles, fluorescent markers with different colours were increasingly used.  excitation – as used traditionally for fluorescence microscopy – was not optimally suited for detecting multiple fluorochromes simultaneously in a cell.

Around 1962 Ploem started work in collaboration with Schott on the development of dichroic beamspitters for reflection of blue and green light for fluorescence microscopy using epi-illumination. At the time of his first communication [1965] and publication on epi-illumination with narrow band blue and green light [21], he was not aware of the development of a dichroic beamsplitter for  excitation with incident light by Brumberg and Krylova [2]. Neither was the Leitz company, from which he obtained an "Opak" epi-illuminator with a neutral beamsplitter. This illuminator had to be modified to contain a slider in the incident light path containing four dichroic beamsplitters, for respectively , violet, blue and green excitation light. This device, developed at the University of Amsterdam, permitted the easy exchange of different dichroic beamsplitters in the incident light path (Figure 1a). The wavelength of the excitation light could thus be easily and rapidly changed.

, violet, blue and green excitation light. Constructed at the University of Amsterdam (Ploem, 1965).

Soon it became clear that excitation with narrow-band blue and green light opened optimal possibilities for the detection of the widely used immunofluorescence labels fluorescein-isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC). The use of blue and green excitation also minimized autofluorescence of tissue components, an undesired effect encountered with conventional transmitted illumination with  light. FITC could now be excited with narrow band blue light (using a band interference filter with a half width of 16 nm), close to the excitation maximum at 490 nm (long wavelength blue), with clear observation of the green fluorescence peak emission at 520 nm. Autofluorescence of tissue components was minimized (Figures 2a and b) resulting in a high image contrast. Excitation of FITC near its excitation maximum enabled such an efficient excitation that even a mercury high-pressure arc lamp, having no strong emission peak in the blue wavelength range, could be used. Furthermore epi-illumination with a green reflecting dichroic mirror enabled for the first time the excitation of Feulgen-pararosaniline with the strong mercury emission line at 546 nm (Figures 3a and b).

FITC) illuminated with wide-band  excitation. Note the tissue structure with blue autofluorescence.

FITC label illuminated with epi-illumination using narrow-band blue (490 nm) light. Note the increased image contrast (Ploem, 1967).

In his second publication on the multi-wavelengths epi-illuminator, describing a Leitz prototype with four dichroic beam-spittters (Figure 1b), Ploem [22] could acknowledge the contribution of Brumberg and Krylova [2]. The inaccessibility of Russian research in that time period, and the absence of any major industrial development of epi-fluorescence microscopy in Russia or East Germany was the reason that Leitz had not been aware earlier of such a development. The possibility to introduce epi-illumination with  light, although useful for several applications, had not been a motive for a new technological development at Leitz, since they had already excellent transmitted dark field  excitation available. The increasing world-wide use of routine immunofluorescence microscopy in medical diagnosis and molecular biology research could, however, profit from the new possibility of epi-illumination using narrow band excitation with blue and green light. Since standard high-pressure mercury arc lamps could be used, this seemed a practical proposition.

Subsequently Leitz developed a novel multi-wavelength fluorescence epiilluminator (Leitz PLOEMOPAK) with four rotating dichroic beamsplitters for respectively , violet, blue and green light [13]. In successive generations of Leitz illuminators (containing four dichroic beamsplitters) barrier filters and a rotating turret for excitation filters were added. Finally an elegant epi-illuminator was constructed by Kraft [15] containing multiple sets of a combination of an excitation filter, a dichroic beamsplitter and a barrier or emission filter, mounted together in a filter cube, also called filter block (Figure 4). Since this illuminator permitted the filter cubes to be rapidly turned into the optical light path, multi-wavelength illumination of the same section of tissue became a practical proposition. Moreover, the four filter cubes in the illuminator could be exchanged by the user (Figure 1c). Different sets of four filter cubes could be assembled, chosen from many filter cubes, containing combinations of excitation, barrier filters and dichroic beamsplitters, developed for different applications. Following suggestions by Ploem, Leitz also produced an inverted microscope with epi-illumination (Figures 5a and b). For a review of the Leitz PLOEMOPAK illuminator for multiwavelength fluorescence microscopy, the reader is referred to a review by Pluta [31].

Left: Fig. 4: Complete Leitz (Leica) filter cube (block) for fluorescence microscopy containing: excitation filter, dichroic beam splitter and emission (barrier) filter.

The Leitz (Leica) filter cube system was so efficient that now, 39 years later, similar types of filter cubes are still used by most microscope manufacturers for multi-wavelength fluorescence microscopy. This development finally led within Leica to the development of automated multi-wavelength fluorescence epi-illuminators accommodating eight filter cubes for various wavelength ranges (Figure 5c). When switching between filter cubes, pixel shift on the computer monitor is avoided or stays below the resolution power of a 35 mm film due to a 0-pixel shift technology. This illuminator is now used for fluorescence in situ hybridisation methods (FISH) in the study of chromosomes.

Ploem [24, 25, 26, 27, 28], van der Ploeg and Ploem [20] and Nairn and Ploem [18] further explored the filter combinations that had to be developed for many biomedical applications. This was done in collaboration with Schott and Leitz. Rygaard and Olson [35] developed a novel shortwave pass high transmission interference filter with a very high transmission for blue light and a sharp cut-off towards wavelengths longer than 490 nm.

Ploem [23] combined this SP filter with a 1 mm GG 455 filter from Schott, which blocked  excitation, and suggested the development by Balzers of a similar filter (SP 560 = KP 560) for excitation with green light and a filter for excitation with violet light (LP 425 = KP 425). The latter filter was applied in the investigation of neurotransmitters [25]. In Figure 6a and b the resulting blue fluorescence can be observed.

SP (KP) 425 interference filter), a dichroic beam-splitter 495 nm, reflecting violet light and a barrier filter LP 460 nm. This filter-cube permittted for the first time the observation of blue fluorescent adrenergic nerve fibers, distinctly different from yellow fluorescent mast cells (Ploem, 1971).

From the optical industry side, early contributions and reviews on these developments were written by Kraft [14], Walter [39, 40], Trapp [38] and Herzog [8].

The main classes of filters used in epi-illumination fluorescence microscopy were defined in (a) the primary excitation filters LP (long pass) and SP (short pass) – in the German literature known as KP filter – and (b) the secondary filters such as barrier filters and emission filters [23]. The latter were also described as fluorescence selection filters; these are for instance used to limit the observation to the peak fluorescence at 520 nm of FITC. A recent extensive review on filters for fluorescence microscopy has been given by Reichman [33].

Cormane [3] was the first to demonstrate that narrow band blue light epi-illumination of the fluorescent label FITC gave an optimal contrast in immunofluorescence studies of human skin disease. Transmitted-light excitation with  light used to cause such a strong autofluorescence of elastic fibres in the skin, so that visualization of the fluorescent antibody was severely hindered.

The pioneering work of Leitz in epi-illumination fluorescence microscopy coincided in the seventies with a worldwide increase in the application of immunofluorescence and other molecular biology methods like FISH in medical diagnosis and research. Hijmans et al. [9, 10] were the first to demonstrate the usefulness of the new Leitz multi-wavelength excitation epi-illuminator for the selective detection of certain classes of immunoglobulins in cells, using antibodies conjugated with green fluorescent FITC and red fluorescent TRITC. They applied the two-wavelengths excitation method using blue and green light and the selection of the peak fluorescence of FITC by an emission filter at 520 nm (Figure 7). Brandtzaeg [1] and Klein et al. [12] made similar discoveries in identifying immunologically important cell types, using two-wavelength excitation with the Leitz epiilluminator. In a staining of blood with "rosette" formation, the two-wavelengths excitation method using  and green light can demonstrate erythrocytes around a mononuclear cell (Figure 8).

FITC conjugate. Epi-illmination with narrow band green and blue light, resulting in red fluorescence of cells containing TRITC and green fluorescence of cells containing FITC. Some cells contain both FITC and TRITC.

light. Lymphocytes stained with the orange-red stain eosine after excitation with green light (1965).

Epi-illumination

In epi-illumination, a dichroic beamsplitter is used to deflect the incident light towards the specimen. The spectral characteristics of the dichroic mirrors have been designed in such a way that only the desired excitation wavelengths are deflected downwards through the objective onto the specimen, while the unwanted wavelengths are transmitted by the dichroic mirror and collected in a light trap [15] behind the dichroic mirror. The elimination of this unwanted excitation light results in a significant decrease of stray light and thus improves the image contrast. The dichromatic mirror deflects the desired (short wavelength) excitation light through the objective onto the specimen, but is transparent to the longer fluorescence wavelengths. The suppression filter (barrier filter) absorbs (or reflects) the excitation light reflected from the specimen and the lens surfaces of the objective, but is highly transparent to the fluorescence, which can thus reach the eyepieces. The efficiency of epi-illumination is related to the fourth power of the numerical aperture (NA) of an objective, serving in epi-illumination first as a condenser and then for observation as a light-collecting lens. At the time of marketing the first multi-wavelengths epi-illuminators only high-power objectives (x70, x100) were available with a high NA (0.95, 1.30). Following suggestions by Ploem [21, 22], Leitz was the first manufacturer to produce moderate power objectives like the oil-immersion x40 objective with a NA of 1.30 (Figure 9). This new type of objective, especially designed for epi-illumination fluorescence microscopy, resulted in very bright images permitting short exposure times in routine fluorescence microphotography.

Right: Fig. 9: Leitz (Leica) early (1967) prototype ("Versuchs") oil immersion objective x40 with a NA 1.30 developed for trial experiments in fluorescence epi-illumination technology.

Optimal filling of the entrance pupil (aperture) of an objective

A problem in epi-illumination fluorescence microscopy has been the optimal filling of the entrance pupil of the objective with an image of the light source. A medium magnification of the light source of approximately 8 times was typical. This is related to the different arc sizes of the various high-pressure mercury and xenon lamps. In addition, entrance pupils of objectives vary considerably, e.g. 3.6 mm for an x100, NA 0.90 objective and 12 mm for an x10, NA 0.30 objective. Furthermore if only part of the arc can enter the entrance pupil, the fluorescence intensity obtained will be diminished. Recently Schönenborn [36] has reported on a completely innovated incident light path in the Leica DM R (HCS) microscopes. The path for epi-illumination has now been designed to allow individual adaptation of the illumination light path to the specific light sources used for different applications. Assuming a Koehler illumination in the incident light path, the combination of the illumination optics and the collector is to image the light source in the entrance pupil of the objective. For a given objective, the choice of the magnification of the light source depends directly on its geometric dimensions. Halogen lamps should be very well adjusted, since the coils of the lamp filament cause an extreme sensitivity to filament maladjustment. Relatively low magnifications are then advisable.

In some applications, the fluorescence intensity could be increased by factor 1.8 and for industrial applications in the  range even by factor 3.5 in the DM R (HCS) microscopes. The increase in the  range has been supported by a new chromatically corrected 6-lens collector of special high transmission glass types. For the Leica DM R (HCS) microscope system two modules are now available: Illumination optics with the HC F module have a magnification factor of 11.5 and for the HC RF module the magnification factor is 4.8. In addition the Leica DM R (HCS) epi-illumination stand has an extra illuminating lens in combination with the HC F module that produces very high fluorescence intensity and good homogeneity in a field of view of 25 mm. Users who need particularly good homogeneity even have the possibility to disengage an illumination lens of the HC F module, which only slightly decreases the fluorescence intensity. The image scale of the light source in the objective pupil is reduced from 11.5 to a value of about 9.5, resulting in an increase of about 20 % of the useful field of view. To further increase the homogeneity, optical diffusion disks can be inserted in the module. Interestingly, it has been observed that the most pleasant image impression for viewing fluorescence is obtained when image intensity is not completely constant but slightly falls to the edge of the image. This effect is the result of the physiology of the eye (so-called "lateral retardation"). Conversely, video techniques with image analysis demand extremely constant illumination. This can be achieved by switching the optical diffusion disk in the HC RF module to the video mode, resulting in more homogeneity in a central field of about 16 mm (corresponding to the area covered by a 1/2" camera with a TV adaptor x0.5).

Leica has also further improved objectives for fluorescence microscopy. The selection of optical glasses with low autofluorescence properties resulted in an enhancement of the image contrast.

A problem in fluorescence microscopy was that no sharp images could be obtained from relatively thick specimens. This is due to the unwanted contribution of pre-focal and post-focal planes – using objectives with a high numerical aperture – to the final microscope image. This results in less well-defined images. The Leica spectral confocal microscope can however obtain sharp images taken at high resolution from several focal planes within the specimen at high lateral and vertical resolution.

The colour separation of multiple fluorochromes in a specimen has been further improved by spectral analysis. Computer assisted spectral confocal scanning microscopy has thus become the ultimate tool for fluorescence microscopy in biomedical and materials research.

Filter cubes for various applications in molecular biology

The explosion in molecular-biology applications in the last decade has led to a development of many filter combinations for the various applications. These sets of filters are now mounted in a large variation of filter cubes (blocks). Fluorescence epi-illuminators enable the exchange of 2–8 filter cubes by hand-operation or motorized exchange, as for example needed in fluorescence hybridisation studies. This paper is not intended to give a comprehensive and detailed discussion of the multitude of applications enabled by the various filter cubes. Instead only a schematic overview of filter cubes and keywords is given, indicating possible applications (Table 1).

Table 1: Examples of fluorescence microscopy applications

Only the optical parts and fluorochromes are listed here when they were mentioned in the cited papers. The language and terminology in this table are restricted to the terminology used in the text of the cited papers. Filter name terminology in German is listed in Italics. The half-width of band interference filters (BP) may be referred to by authors as either e.g. +00 or /00 nm, indicating the center wavelength and the half power bandwidth (e.g. BP 525/20) or the short and long-wave half power points (e.g. BP 450–490). Short and long pass filters are identified by the letters SP and LP and the edge wavelength in nm (e.g. SP 490, LP 520 or >520 nm). For references of the authors to manufacturers of optical parts the reader is referred to the cited literature.

Microscope, objective, macromolecule

Literature references

Fluoro-

chrome

Light source

Excitation filter

Dichroic

beam splitter

Filter cube

Emission filter

Laser scan microscopy (LSC) x20

Surface markers

DNA staining

Körnemann et al.: Cytometry 41: 172–177 (2000)

FITC

PI

Argon laser

blue 488 nm*

530 + 30 nm&

BP green

625 + 28 nm&

BP red

Inverted fluorescence microscope x40

Fluorescence lifetime imaging

Murata et al.: Cytometry 41: 178–185 (2000)

Ho

7-AAD

DCM laser

335 nm*

FT 395%

(DM, DBS)

450 + 33 nm&

BP violet

Digital fluorescence microscopy x100/0.60–1.32, SP (KP) 630 in front of CCD camera to block far red and infrared light

PNA FISH

de Pauw et al.: Cytometry 32: 163–167 (1998)

CY3

DAPI

green 515–560&

340–380& nm BP

LP 580 red#

LP 430 blue#

Confocal fluorescence microscopy x60/1.4

Photobleaching studies

van Oostveldt et al.: Cytometry 32: 137–146 (1998)

FITC

Ar+-ion line

blue 488 nm*

BHS&

LP OG 530#

yellow

Digital fluorescence microscopy x63/1.4

Excitation filter wheel

Chromosome studies

Poon et al.: Cytometry 36: 267–278 (1999)

DAPI

Cy3

Hybrid/xenon mercury lamp

405* nm violet

546* nm green

DAPI/Cy3 filter set

Fluorescence microscopy

Intratumor heterogeneity of chromosomes

da Vinci et al.: Cytometry 37: 369–375 (1999)

TRITC

FITC

DAPI

Triple-bandpass filter* (MBP)

FM%

(Emission)

Fluorescence microscope x100/1.35

Daunorubicin sequestration

Bour-Dill et al.: Cytometry 39: 16–25 (2000)

LB

Daunorubicin

NBD

DiOC

Mercury 100 W

BP 330–385&

BP 460–449&

BP 460–490&

DM 410%

DM 570%

DM 510%

(Emission)

BP 430–460$

LP 590#

BP 510–550$

Fluorescence microscope

Automated comet assay

Böcker et al.: Cytometry 35: 134–144 (1999)

PI

Mercury 100 W

SP 580^

E2@

(Emission)

LP 590#

Motorized epi-illuminator x100/1.3

Chromosome studies with multicolor

FISH

Kuzobek et al.: Cytometry 36: 279–293 (1999)

DAPI

FITC

Texas-red

Mercury 100 W

blue

green

Four filter cubes@ (FC)

(Emission)

BP 425@ blue

BP 525@ green

BP 615@ red

Inverted fluorescence microscope, CCD

Three-color imaging of chromosomes

Coco-Martin et al.: Cytometry 32: 327–336 (1998)

DAPI

FITC

TRITC

BP 365/12&

BP 450–490&

BP 546/12&

Emission

>379# nm

>520# nm

>593# nm

Digital fluorescence microscope and (CCD) x40/0.75

Image analysis of granulocytes

Szecs et al.: Cytometry 33: 19–31 (1998)

R 123

EB

Multi-band pass filter (MBP)

490~ nm blue

570~ nm green

Triple-band (polychroic) beamsplitter (PBS)%~

Triple-band emission filter

520~ nm

580~ nm

Fluorescence microscope with motorized epi-illuminator, delayed luminescence imaging and CCD, x63/1.32

FISH Karyotyping

Tanke et al.: Cytometry 33: 453–459 (1998)

cascade bl

F

LR

Cy5

Cy7

Platinum coproporphyrin

Mercury 100 W

BP& 500–560 nm

A@

HQ-FITC@

HQ-TRITC@

HQ-Cy5@

HQ-Cy7@

DBS 580@ nm

BP$ 600–700 nm

Fluorescence microscope with motorized filter changer and CCD camera

Two colour ratio method in FISH

Morrison and Legator: Cytometry 27: 314–326 (1997)

DNA probes:

1 green

2 orange

(1) WCP

(2) CEP

BP& 510/20 nm

BP& 572/18 nm

Triple-band (polychroic) beamsplitter (PBS)%~

Triple band-pass emission filter:

MBP~ 535 nm

MBP~ 606 nm

Fluorescence microscope with CCD camera

Comparative genomic hybridization

Bornfleth et al.: Cytometry 24: 1–13 (1996)

DAPI

FITC

Texas red

Mercury 50 W

Triple band-pass excitation filter

Triple band-pass emission filter

Fluorescence microscope and CCD camera, x20/0.75

Segmentation of nuclear images

Price et al.: Cytometry 25: 303–316 (1996)

DAPI

Mercury 100 W

BP& 365/10

DM% 400 nm

none

Microscope with CCD camera x30/0.5

Multiparameter fluorescence imaging of lymphocytes using labelled monoclonal antibodies

Galbraith et al: New Technologies in Cytometry, in: Proc. of the SPIE 1063 (1989)

FITC

PE

Cy5

Cy3

Manual, six position slider for fluorescence filters

Special optical system for epi-illumination microscopy with wavelength switching using a polychroic beam-splitter

Heiden and Tribukait: Cytometry 20: 95–101 (1995)

DAPI

TRITC

Mercury 100 W

BP& 365/33 nm

BP& 546/23 nm

Polychroic PBS%

365 reflection

546 reflection

BP$ 435–500 nm

BP$ 500–750 nm

*Emission or Laser line.

^Short Pass SP (KP) excitation filter.

&Narrow band-pass (BP) excitation filter.

~Multi band-pass filter (MBP).

%Dichroic beam splitter (DBS) or dichroic mirror (DM) and

%~Polychroic beam spitter (PBS).

#Barrier filter (LP).

$Emission band-pass (BP) interference filter, and emission multiple band-pass filter (MBP).

@Filter cube, block (FC).

Reflection contrast microscopy (RCM)

The development in epi-illumination microscopy by Leica has led to a further development: reflection contrast microscopy [29, 30]. Reflection contrast microscopy provides strong signals from specimens stained with routine absorbing immunomarkers such as peroxidase-DAB, immunogold-silver and immuno-phosphatase (Figures 10 and 11). Because of the strong signals

that can be obtained – the high image contrast and no deterioration of the image by pre- and post focal images –, RCM of thin sections fulfils all the theoretical optical criteria for optimal light microscope resolution. The optical results can be defined as high definition images.

The strong reflection of most immunostains provides such a high contrast that this staining can be combined with many classical (absorbing) histochemical stains for important macromolecules that show a moderately strong reflection. The latter stains can often be used to provide fine morphological orientation and in many cases a more precise location of the immunomarker is achieved than with fluorescent markers alone. In addition, such light microscope observations can be confirmed by electron microscopy through examination of a next ultra-thin section (having the same immunostaining) with EM.

In confocal laserscanning microscopy, RCM can be performed without a special objective or other stray light reducing optical measures, due to the elimination of stray light by illumination through a pinhole. Most absorbing immunostains give a very strong reflection of laser light and show very limited fading. They often permit the use of a small pinhole size (e.g. 10 micron), which is about five to ten times smaller than can be used with most fluorochromes in confocal fluorescence laserscanning microscopy. The use of reflecting immunomarkers can therefore result in a very significant increase in optical resolution. Specimens double-stained with both a fluorochrome and the Leica Spectral Confocal Laserscanning Microscope providing new possibilities for multiple markers can easily examine a reflecting immunomarker.

RCM uses a fluorescence microscope stand, epi-illuminator and high pressure lamps. In the fluorescence epi-illuminator an extra polarizer filter cube, containing a polarizer, reflector, and an analyser must be inserted (Figure 12). Also a reflection contrast (RC) diaphragm module (containing the central stop system) must be brought in the incident light path. This RC diaphragm module adapts sliding sets with central stops and/or aperture diaphragms. Furthermore a special objective developed for reflection contrast microscopy, the Leica RC M-plan oil-immersion x100/1.25 RCM objective equipped with a λ/4 plate at the front lens, should be added to the set of objectives (for fluorescence microscopy) on the turret of the objective revolver.

In reflected-light microscopy, contrast is based on differences in reflection intensities (= reflectance). In this type of microscopy it should be reminded that reflection of light occurs at every optical boundary, that is when the refractive index and/or the absorption index change. For biological specimens showing reflectance of less than 1 % and mostly less than 0.2 %, it is almost impossible to obtain a reflected light image with a conventional microscope, due to the unwanted reflections inside the microscope tube. In reflection contrast microscopy, a combination of two methods is used to reduce glare due to scattered light. The incident light is made into a ringshaped cone of light by the insertion of a central stop (an aperture diaphragm provided with a central stop, creating an annular aperture) in the incident light path at a plane conjugated with the back focal (aperture) plane of the objective. As a second method, unwanted scattered reflected light is reduced by the use of the "antiflex" method. Light reflected inside the microscope is suppressed by using crossed polarizers, with the result that only the light reflected from the specimen is passed on to the eyes. To that purpose an objective is provided with a quarter-wave plate in the front lens of the objective. The passing of the light through the λ/4 plate (downwards to the specimen and upwards after reflection from the specimen) change the polarization direction of the light with 2 x 45° = 90°.

References

1. Brandtzaeg P: Two types of IgA Immunocytes in Man. Nature (New Biology) 243: 142–143 (1973).

2. Brumberg EM, Krylova TN: O fluoreschentnykh mikroskopopak. Zh. obshch. biol. 14: 461 (1953).

3. Cormane RH, Szabo E, Hague LS: Immunofluorescence of the skin: the interpretation of the staining of blood vessels and connective tissue aided by new techniques. Br. J. Derm. 82 (Supplement 5): 26–43 (1970).

4. Ellinger P, Hirt A: Mikroskopische Untersuchungen an lebenden Organen. I. Mitteillung: Methodik: Intravitalmikroskopie. Zeitschrift für Anatomie und Entwicklungs-Geschichte 90: 791–802 (1929).

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Scientific and Technical Information CDR 5: 1–16 (2001)

Article

The Fundamentals and History of Fluorescence and Quantum Dots

At some point in your research and science career, you will no doubt come across fluorescence microscopy. This ubiquitous technique has transformed the way in which microscopists can image, tag and trace anything from whole organisms to single proteins and beyond.

In this article, we will examine what is meant by "fluorescence", the history and basic physics behind its definition, the discovery and application of Green Fluorescent Protein (GFP) and a look at the rapidly expanding field of fluorescent probes including Quantum Dots.

Authors

- Martin Wilson , PhD

Topics & Tags

- Fluorescence Microscopy

- Basics in Microscopy

- Fluorescence

- Fluorescent Protein

- Fluorophore

- GFP

How do we define "fluorescence"?

Typing the phrase "definition of fluorescence" into any search engine and you will get the following phrase, or something very similar;

"The emission of visible or invisible radiation from certain substances as a result of incident radiation of a shorter wavelength such as X-rays or ultraviolet light."

How does this definition relate to fluorescence microscopy (Figure 1)? The "incident radiation of a shorter wavelength" is simply the light source used to "excite" the fluorophores in a sample. This light can include the visible spectrum, ultraviolet () and infra-red () and the source in a microscope can range from a mercury or xenon arc lamp to lasers. The fluorophores ("certain substances" in the above definition) are chemical compounds which have special properties whereby they can reemit higher wavelength light/photons upon excitation by light which has a lower wavelength.

Figure 1: Fluorescently labelled cells visualized by fluorescence microscopy.

The basic unit of wavelength is the meter and "wavelength" is defined as the distance between two successive peaks or troughs of a light wave. The symbol for wavelength is the Greek letter lambda (l). The wavelengths which are typically used in microscopy are in the nanometre (nm) range within the visible spectrum between 400 and 700 nm, the  spectrum below 400 nm and the  spectrum which starts at 700 nm (Figure 2).

Fluorophores are categorised by their excitation and emission wavelengths which is usually in the form of a graph displaying the maximum peaks. Fluorophores are naturally stable in what is termed the "ground state". When they absorb a photon of light (from the "shorter wavelength" light), the energy of the photon raises the electrons of the fluorophore to a higher energy "excited" state. The excited electrons do not remain in this state, but lose the vibrational energy and emit a photon of a longer wavelength on their return to the ground state. For the fluorophores used in microscopy, one complete cycle from excitation to the return to ground takes in the region of 0.5 to 20 nanoseconds.

Fluorophore excitation and emission wavelengths are commonly abbreviated as the Greek letter lambda with a subscript "ex" (excitation) or "em" (emission; lex or lem).

Each fluorophore has a distinct maximum excitation and emission spectra and this property is used to distinguish different targets in the same specimen of interest. For example, using the fluorescent stain DAPI (4',6-diamidino-2-phenylindole) to highlight the nuclear proteins in a cell along with fluorescently-labelled phalloidin to highlight the actin cytoskeleton.

The Jablonski Diagram of fluorescence

The Polish physicist Professor Aleksander Jablonski (1898-1980) was the first to describe the cycle between ground/excitation/emission in a three-level energy diagram which is known simply as a "Jablonski Diagram".  In 1930, he received his PhD from Warsaw University for his thesis entitled "On the influence of the change of the wavelength of excitation light on the fluorescence spectra". It was in 1933 that he published his paper ("Efficiency of Anti-Stokes Fluorescence in Dyes") in Nature which contained the Jablonski Diagram. This simple yet effective illustration of the behaviour of fluorophores highlights the excitation from ground state to excited state and back to ground with the emission of a photon of longer wavelength (Figure 3).

Although Figure 3 is a simplified Jablonski Diagram, it should be noted that fluorophores can exist in many different states of excitation and emission. Furthermore, fluorophores may return to ground via varying states of relaxation known as "triplet states" depending upon the electron spin of the molecules.

The Stokes Shift

Sir George Gabriel Stokes (1819-1903) was an Irish mathematician and physicist, and during his lifetime he made many discoveries and advances in the fields of optics and light. He was appointed the Lucasian Professor of Mathematics at Pembroke College, Cambridge in 1849 and remained in this position until his death in 1903. He wrote a beautifully descriptive paper which was published in 1852 and called "On the Change of Refrangibility of Light" [1]. The language used in this text is unlike that which we are used to in modern day scientific literature. Here are two brief extracts of the wonderful language which Stokes used;

"Towards the end of last autumn, when the lateness of the season afforded but few opportunities for observation, I learned from different sources that the kind of yellow glass which has already been mentioned as possessing in so high a degree the property of internal dispersion was coloured with oxide of uranium."

And:

"It was certainly a curious sight to see the tube instantaneously lighted up when plunged into the invisible rays: it was literally darkness visible. Altogether the phenomenon had something of an unearthly appearance."

The "Stokes Shift" was later named in his honour. When the excited electrons return to the ground state and emit a photon of light, the wavelength is always longer than that which was used to excite the fluorophore. This is due to the property of light whereby wavelength is inversely proportional to radiation energy. The Stokes Shift describes the difference (in nanometres) between the peak excitation and the peak emission wavelength of a fluorophore (Figure 4).

Distinguishing between the excitation and emission from a fluorophore is proportionally easier with an increase in the Stokes Shift. Fluorophores possess unique characteristics in terms of their Stokes Shift which is due to their electron configuration and molecular structure.

The discovery and use of Green Fluorescent Protein

It was Stokes who first used the term "fluorescence" to describe his observed phenomena, but the history stretches as far back as 1565. A Spanish physician and botanist called Nicolas Monardes described a strange blue colour of an infusion of wood from a Mexican tree, Lignum nephriticum. Despite these early observations, it was to be some 400 years later that a green fluorescent substance was reported in a living organism. In 1955 Davenport and Nicol published a paper [2] describing the photogenic tissue in the eosinophils of a subclass of jellyfish known as Hydromedusae. At the time, the authors of this work did not realise that the eosinophils contained a green fluorescent protein (GFP).

It wasn’t until 1962 that Osamu Shimomura (*1928) published a paper [3] in which the photogenic component was recognised as a protein. Working with his Professor (Frank Johnson) at Princeton University, they collected samples from the bioluminescent jellyfish Aequorea victoria. They had a unique way of isolating the fluorescent proteins from the jellyfish, namely squeezing the isolated bioluminescent tissue through a cotton bag to produce a solution which Shimomura called "squeezates".

In 1994, Martin Chalfie (*1947) Professor at Columbia University, compiled a paper demonstrating that the gene encoding GFP could be functionally expressed in prokaryotic and eukaryotic cells (namely Escherichia coli and the neurons of Caenorhabditis elegans) [4]. The paper stated that "because exogenous substrates and cofactors are not required for this fluorescence, GFP expression can be used to monitor gene expression and protein localization in living organisms." It was this publication which paved the way for the extensive use of GFP in biological research.

GFP expression in the nervous system.

A year later, Professor Roger Tsien (1952-2016) from the University of Califronia, San Diego (who had been researching mutants of the wild-type GFP) published his work in the scientific correspondence in Nature [5]. This discovery of a single-point mutation (S65T) was chosen by Tsien and colleagues as it had the longest excitation and emission wavelengths (490/510 nm) which rendered it more photostable and resulted in the well-known GFP excitation/emission peaks.

In 2008, Shimomura, Tsien and Chalfie were jointly awarded the Nobel Prize in Chemistry for their roles and discovery of GFP. When Shimomura gave his Nobel lecture he said that “When I found the chromophore of GFP in 1979, I thought I had done all I could do with GFP, and decided to terminate my work on GFP in order to concentrate my efforts in the study of bioluminescence”. He went on to admit that “GFP was a beautiful protein but it remained useless for the next 30 years after the discovery.”

Since Tsien’s discoveries, his lab have engineered many variants of GFP which cover much of the visible spectrum and have indeed revolutionised the field of optical microscopy and imaging. Thanks to such genetic engineering, GFP is available in colours ranging from the blue part of the spectrum (EBFP; 380/460 nm) through to the yellow part of the spectrum (YFP; 514/527 nm).

Reporters and probes

As well as the field of imaging, fluorophores can be used as "reporters" to study gene expression in cells and organisms. The enzyme luciferase, as well as the gene encoding GFP, are amongst those which are commonly used to examine whether specific genes are expressed by cells. Organisms or cells are transfected with genetically modified viral DNA or plasmids which fluoresce when the target gene is expressed. The organelles or sites of interest within the cells can then be visualised and examined in the transfected cells.

Antibodies which are labelled (or "tagged") with fluorophores are commonly referred to as "fluorescent probes". Before the widespread use of GFP and the other fluorophores which followed its discovery, two of the most commonly used fluorophores to tag antibodies were FITC (fluorescein isothiocyanate) and TRITC (tetramethyl rhodamine isothiocyanate). Although FITC and TRITC are still used, there have been major advances in the field and the choice of fluorophores and probes is extensive to say the least.

Certainly one of the most extensive ranges of fluorophores used in laboratories today is the "Alexa Fluor" dyes. The current range of Alexa Fluor dyes available spans the visible spectrum with excitation wavelengths ranging from 346 to 784 nm.

Quantum Dots

Quantum Dots (or "Qdots") were first discovered in a glass matrix in 1981 by the Russian scientist Alexey Ekimov whilst working at the Vavilov State Optical Institute in St. Petersburg. However, the first colloidal solutions of Qdots were discovered by Louis Brus who was working on semiconductors at the AT&T Bell Laboratories in New Jersey. He is now the Professor of Chemistry at Columbia University. In two papers published in 1983 and 1984, Brus described Qdots as "small semiconductor crystallites".

Quantum dots behave in a peculiar manner- although they contain between as little as 100 to 100,000 atoms, they exhibit properties as though they were comprised of a single atom. However, they do conform to the properties of a fluorophore whereby they can absorb light energy, become excited and release photons of light when returning to ground state. But the wavelength of light which they emit is dependent on the size of the Qdot- the smaller the Qdot, the shorter the emission wavelength. This property is due to smaller Qdots possessing a larger "minimum band gap". This is the amount of energy which is required to excite an electron to a higher energy state. As smaller Qdots require more energy for excitation, they subsequently emit lower wavelengths of light (wavelength is inversely proportional to excitation energy).

It was almost 20 years later, in 2002, when Qdots became commercially available to life science researchers from the Quantum Dot Corporation, California.

Quantum dots are very bright and stable tools for use in fluorescence applications. Studies have shown that Qdots are several orders of magnitude brighter than conventional fluorophores, although there exists some variation in exactly how much brighter they are compared to organic dyes [6]. In terms of photostability, Qdots have been reported to be up to 100-times more stable than conventional fluorophores and in one in vivo imaging study, they remained fluorescent for up to four months [7].

In conventional fluorescent probes, one or more fluorophores can be attached to a single antibody of interest. However, due to the very large surface area of Qdots, many molecules and antibodies can be attached to a single dot which can have many advantages including fluorescent signal amplification. The use of Qdots also offers an advantage in multiplex assays in which a variety of wavelengths can be simultaneously imaged. In such assays, the only variable is the size (and hence emission wavelength) of Qdot which is selected by the researcher. Excitation of a wide range of Qdots can be performed simultaneously with white light which negates the use of multiple lasers and many adjustments to form the final image.

References

1. On the change of refrangibility of light; G. G. Stokes, M. A., F. R. S. Phil. Trans. R. Soc. Lond. 1852 142, 463-562, published 1 January 1852; http://rstl.royalsocietypublishing.org/content/142/463.full.pdf+html

2. Luminescence in Hydromedusae D. Davenport; J. A. C. Nicol, Published 29 November 1955. DOI: 10.1098/rspb.1955.0066; http://rspb.royalsocietypublishing.org/content/144/916/399

3. Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol. 1962 Jun;59:223-39.; http://www.ncbi.nlm.nih.gov/pubmed/13911999?dopt=Abstract

4. Green fluorescent protein as a marker for gene expression; Science  11 Feb 1994: Vol. 263, Issue 5148, pp. 802-805 DOI: 10.1126/science.8303295; http://www.sciencemag.org/content/263/5148/802?ijkey=e60d69b507876bb025397e40e06389c288d2e92e&keytype2=tf_ipsecsha

5. Improved Green Fluorescence; Nature Volume 373, 23. February 1995; http://www.tsienlab.ucsd.edu/Publications/Heim%201995%20Nature%20-%20Improved%20GFP.PDF

6. QDs versus Alexa: reality of promising tools for immunocytochemistry; Journal of Nanobiotechnology; http://www.jnanobiotechnology.com/content/7/1/4

7. Noninvasive Imaging of Quantum Dots in Mice; Bioconjugate Chem., 2004, 15 (1), pp 79–86 DOI: 10.1021/bc034153y; Publication Date (Web): December 30, 2003; http://pubs.acs.org/doi/abs/10.1021/bc034153y

Article

Fluorescent Proteins - From the Beginnings to the Nobel Prize

Fluorescent proteins are the fundament of recent fluorescence microscopy and its modern applications. Their discovery and consequent development was one of the most exciting innovations for life sciences in the last century and the starting point of the deciphering of numberless natural phenomena. This article is dedicated to the people who were involved in the fate of fluorescent proteins by the input of their scientific engagement. It should give insights into the long road of one of the most beautiful biochemical tools from its beginnings to the Nobel Prize.

Authors

- Christoph Greb , Dr.

Topics & Tags

- Fluorescence Microscopy

- Fluorescence

- GFP

- History

Early fluorescence observations

The interest of man in fluorescent proteins can be traced back to the first century A.D. when the roman natural philosopher Pliny the Elder [1] (Gaius Plinius Secundus, 23 A.D. – 79 A.D.) described a glowing jellyfish (Pulmo marinus) in the Mediterranean Sea. In his eyes these animals emitted light of such intensity that it could almost be used as a torch (Bohn, 1855). Beside this jellyfish there is an abundance of other organisms attracting our attention because they glow in the dark. In fact they don’t do this to catch our admiration but, amongst other theories, to communicate with their conspecifics (fireflies exert a pull on their mates e.g.), attract prey (frogfish e.g.) or to frighten predators (bobtail squid e.g.). Some of these fascinating creatures are living in the world of darkness – such as the deep sea – where light is a raw property. Whereas only the upper 200 m of the oceans are penetrated by sunlight, underneath that border the only existing illumination is produced by organisms which are living there.

Using chemiluminescence and fluorescence to emit light, evolution developed one of the most impressive and powerful tools of recent biochemistry and cell biology which is based on the scientific curiosity of several persons. Their efforts and discoveries should be mentioned in the following.

With the upcoming interest in natural sciences after the Middle Ages, people like the Spanish physician and botanist Nicolás Monardes described a gleaming wood extract made of Lignum nephriticum in 1565. Later on this light could be ascribed to an oxidation product of a flavonoid which is found in this plant and showed that glowing was not only restricted to animals. More than three centuries later (1858) George Gabriel Stokes, an Irish mathematician and physicist, formed a name for that kind of natural occurring phenomena: Fluorescence. He thought up this term after the blue light emitting mineral fluorite and broadened the observation of radiating objects from animals over plants to stones.

The breakthrough for GFP

One of the first researchers in modern times, who started the systematical exploration of fluorescent proteins, was the Japanese biochemist Osamu Shimomura. He shared the interest of Pliny the Elder in fluorescent jellyfish and established Aequorea victoria as his "object of desire" in the early 1960’s. Shimomura was fascinated by A. victoria because it is able to emit green light by means of fluorescence. Its habitat is in the Pacific Ocean were it feeds on shellfish and other jellyfish. The light is produced in the so called luminescent organs arranged in a ring on the underside of the organism. Shimomura, who did studies about luciferin of some seed shrimp during his graduation already, dissected these organs and squeezed them through a sieve with the result of a faint luminescent slurry. By doing so with thousands of jellyfish Shimomura’s effort was rewarded by the extraction and purification of the chemiluminescent protein aequorin [2]. Later on it was discovered that aequorin was responsible for the emission of blue light which was due to its role as a luciferase in the oxidation of coelenterazine in a calcium-dependent reaction. Luciferases are enzymes which are oxidizing their substrates (Luciferines) in a chemical reaction, associated with the emission of light.

Fig. 1, 2: Jellyfish Aequorea Victoria. Source: Ssblakely, via Wikimedia Commons

Nevertheless there was this puzzling question why there was blue light emitted in the extraction while the intact organism glowed in green? It took Shimomura and colleagues several years to solve this mystery. But finally their studies revealed that the blue light coming from aequorin, was only the source of energy for another gleaming protein – the jellyfish green fluorescent protein (GFP). They realized that the light emitted by aequorin was absorbed by GFP which used this energy to produce a green fluorescence.

GFP)

The next path-breaking step in the triumphal procession of GFP had to wait for the invention of cloning techniques in the 1970s. It was the work of the American microbiologist Douglas Prasher who cloned the complete GFP gene for the first time in 1992 [3]. Before he prepared an A. victoria cDNA library together with his colleague Milton Cormier at the University of Georgia, knowing already the prospective of GFP. Unfortunately Prashers funding by the American Cancer Society ran out before he could start to express the recombinant GFP in bacteria.

Initial doubts that GFP would not work outside jellyfishes were erased by Martin Chalfie, an American biologist who could benefit from Prasher’s preliminary work. By introducing the GFP gene into E. coli and the nematode C. elegans he could show that there were no other jellyfish specific proteins or factors necessary to produce the green glow [4].

GFP in live cell imaging

With that experience the door was opened for the green fluorescent protein to start its astonishing career in life sciences. GFP became the key to observe structures in living cells and organisms without the necessity of synthetic labels or fluorescent antibodies. In the case of immunofluorescence staining, the access of antibodies to accordant target structures inside the cell has to be guaranteed. This is achieved by permeabilizing the cells with detergents (e.g.), which inevitably induces cell death. Furthermore most antibodies have a preference for denatured antigens. Therefore immunofluorescence techniques use agents like para formaldehyde to denature cellular protein target structures. Altogether the use of GFP could overcome these non physiological conditions and paved the way for life cell imaging.

Another person who was realizing and developing the great potential of GFP was Roger Tsien. Coming from the Calcium regulation field, the American cell biologist was getting interested in tracking macromolecular interactions inside living cells. After studying and working in Harvard, Cambridge and Berkeley he finally settled down at the University of California, San Diego. As a Professor of Pharmacology, Chemistry and Biochemistry he improved the efficiency of GFP by taking advantage of a very common principle in evolution: Mutation. In 1994, he and his group established a single point mutant (S65T) of GFP with a much better intensity and photo stability than the wild type version. Besides a brighter glow the S65T mutant had another striking technical benefit. Whereas the wild type GFP had two excitation maxima at 395 nm and 475 nm, the mutated version had only one at 484 nm. By keeping its emission wavelength at 509 nm, the spectral characteristics of the "new" GFP fitted almost into classical FITC fluorescence properties (FITCex: 496 nm, FITCem: 520–530 nm). Because of its enhancements this GFP variant was called “enhanced” GFP or EGFP.

By doing structural research on GFP Tsien and coworkers developed further fluorescent derivates. With the GFP structure in their hands they established a variant (T203Y) which was shining in bright yellow and was therefore given the name "yellow fluorescent protein" or YFP. Cyan (CFP) and blue (BFP) forms followed [5].

GFP, yellow:YFP, red: DsRed

GFP Mice

Fluorescent Proteins in Anthozoa

The colour palette of fluorescent proteins was extended into the red range of the spectrum when the biochemist Sergey A. Lukyanov, who is a corresponding member of the Russian Academy of Sciences since 2003, discovered red fluorescing proteins in corals [7]. After buying some of these Cnidaria in a moscow pet shop Lukyanov studied the fluorescent behavior of these primitive animals destined for russian marine aquariums. Amongst others he identified a red fluorescing protein coming from Discosoma – a Corallimorpharia – which he called DsRed. Besides DsRed, Lukyanov and coworkers identified a whole palette of other glowing proteins in Anthozoa, which were optimized for biochemical use time after time.

The Nobel Prize

All the effort researches put into the discovery, understanding and enhancement of fluorescing proteins lead to a huge range of applications in life sciences. GFP and its variants opened the door for scientists to watch f.e. metastasis or angiogenesis in living organisms. Moreover the use of fluorescent multicolored neurons (Brainbow) will help to understand complex neuronal networks in the brain. With the possibility to decorate parasites like the Malaria pathogen Plasmodium falciparum with fluorescent proteins it is realizable to watch their fate in host cells. The list of opportunities could be continued almost endless and does not only include clinical relevant projects but also other basic science ventures.

All together the use of GFP and its mutants changed our view on life and its pathological modifications in a dramatic way, that three people who were involved in the discovery and development of GFP were rewarded by giving the Nobel Prize in Chemistry 2008 to them. Osamu Shimomura, Martin Chalfie and Roger Y. Tsien were decorated with the highest honors in science for their work on "the discovery and development of the green fluorescent protein, GFP".

www.nobelprize.org/nobel_prizes/chemistry/laureates/2008/

In the eyes of the Nobel Committee for Chemistry (2008), represented by Professor Måns Ehrenberg "… The discovery and development of the green fluorescent protein (GFP) have radically changed the scientific agenda. Improved variants of GFP and GFP-like proteins in synergy with high-resolution microscopes, computational techniques and powerful theoretical approaches are currently fuelling a scientific revolution focused on quantitative analyses of complex biological systems. The gradual appearance of a world of hitherto unseen structures and dynamic principles is now impacting virtually all aspects of biological, medical and pharmaceutical research …"

This quotation of the Presentation Speech for the 2008 Nobel Prize in Chemistry represents once more the high impact of fluorescent proteins on current life sciences and also arouses our curiosity on upcoming utilizations in the future. Recent developments, like highlighter proteins (photoactivateable, photoswitchable or photoconvertable FPs) and the establishment of new optical facilities like super-resolution microscopy make clear, that evolution of fluorescence is still in progress and far away from the end.

References

1. Pliny JB and Riley HT: The natural history of Pliny, Book XXXII. Remedies derived from aquatic animals. Chapter 52 – Other aquatic productions. Adarca or Calamochnos: three remedies. Reeds: eight remedies. The ink of the sæpia. Gaius Plinius Secundus (Pliny the Elder). AD77. Bohn HG, London (1855).

2. Shimomura O, Johnson FH and Saiga Y: J Cell Comp Physiol 59 (1962) 223–239.

3. Prasher DC, Eckenrode VK, Ward WW, Prendergast FG and Cormier MJ: Gene 111 (1992) 229–233.

4. Chalfie M, Tu Y, Euskirchen G, Ward WW and Prasher DC: Science 263 (1994) 802–805.

5. Heim R, Prasher DC, Tsien RY: Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc Natl Acad Sci USA 91 (1994) 12501–04.

6. Ormo M, Cubitt AB, Kallio K, Gross LA, Tsien RY, Remington SJ: Crystal structure of the Aequorea victoria green fluorescent protein. Science 273 (1996) 1392–95.

7. Matz MV, Fradkov AF, Labas YA, Savitisky AP, Zaraisky AG, Markelov ML, Lukyanov SA: Fluorescent proteins from nonbioluminescent Anthozoa species. Nature Biotech 17 (1999) 969–73.

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Modern Fluorescent Proteins and their Biological Applications

Fluorescence in Microscopy