Ultra-short-pulse solid-state laser sources have improved contrast within fluorescence imaging and also opened new
windows of investigation in biological imaging applications. Additionally, the pulsed illumination enables harmonic
scattering microscopy which yields intrinsic structure, symmetry and contrast from viable embryos, cells and tissues.
Numerous human diseases are being investigated by the combination of (more) intact dynamic tissue imaging of cellular
function with gene-targeted specificity and electrophysiology context. The major limitation to more widespread use of
multi-photon microscopy has been the complete system cost and added complexity above and beyond commercial
camera and confocal systems. The current status of all-solid-state ultrafast lasers as excitation sources will be reviewed
since these lasers offer tremendous potential for affordable, reliable, "turnkey" multiphoton imaging systems. This
effort highlights the single box laser systems currently commercially available, with defined suggestions for the ranges
for individual laser parameters as derived from a biological and fluorophore limited perspective. The standard two-photon
dose is defined by 800nm, 10mW, 200fs, and 80Mhz - at the sample plane for tissue culture cells, i.e. after the
full scanning microscope system. Selected application-derived excitation wavelengths are well represented by 700nm,
780nm, ~830nm, ~960nm, 1050nm, and 1250nm. Many of the one-box lasers have fixed or very limited excitation
wavelengths available, so the lasers will be lumped near 780nm, 800nm, 900nm, 1050nm, and 1250nm. The following
laser parameter ranges are discussed: average power from 200mW to 2W, pulse duration from 70fs to 700fs, pulse
repetition rate from 20MHz to 200MHz, with the laser output linearly polarized with an extinction ratio at least 100:1.
Computer-controlled, repeatable ultra-short pulse lasers and a broad wavelength range autocorrelator have opened up practical spectral investigations of the group delay dispersion in laser scanning microscope systems. The laser output pulse duration was measured via intensity-based (two-photon absorption) autocorrelation and the laser
spectral bandwidth was measured via spinning spectrometer. The separate measurements provided the ~0.39 timebandwidth product for two different Coherent ultra-short pulse lasers. The laser-scanning system pulse durations were measured at the sample plane with high numerical aperture objective lenses. The pulse broadening of ultra-short laser pulses through a laser-scanning multi-photon system has been characterised experimentally from 720nm to 950nm. The pulse spreading of individual laser-scanning system components was estimated from Gaussian pulse dispersion with data acquired using a standard, characterized objective lens and an external GaAs:P diode signal. The objective lens was found to produce the most pulse broadening, and most apochromatic objective lenses showed two-fold increases in the magnitude of dispersion values. All spectra of measured components followed the same shape and trend, as expected for normal dispersion. During this study, the average power incident on the Carpe autocorrelator was kept below ~50mW (130fs and 800nm), but this limitation has been removed in current versions of the autocorrelator. Dielectric broadband reflector mirrors introduce significant dispersion, which changes sign with wavelength; this can reduce or increase the system pulse spreading, but will certainly make component spectral measurements challenging.
We report on the application of a novel blue laser diode source to confocal microscopy. The source has the potential to be a replacement for argon lasers in a range of fluorescence based imaging systems. It has been demonstrated that with the use of a minimal number of optical components, high quality confocal images can be obtained from laser diodes operating around 406nm. Improvements in image quality through the use of anamorphic prisms to modify the beam profile have been investigated. Living mammalian cells stained with a range of biologically significant compounds have been imaged with high resolution. The stains excited range from fluorescein based compounds to green fluorescent protein. Through the use of the absorption wings a wide range of shorter wavelength fluorophores have been excites, including those more normally excited using UV laser systems. It is expected that this will lead to reduced photo-toxicity within the sample and conventional rather than UV transmitting objective lenses can be used.
The development of laser scanning fluorescence microscopy will be outlined. The focus will be technical instrumentation applied to solve biological problems through dynamic, high- resolution imaging. Laser scanning confocal microscopy will be presented first, followed by two-photon excitation fluorescence microscopy. Ideal imaging modes for two-photon imaging will be highlighted: deep tissue imaging and live cell imaging. Contributions from selected pioneers over the last decade of multi-photon imaging field will be highlighted in specific biological applications areas where two-photon imaging has already been established as the best (or only) option: intact tissues, developing embryos, and whole animal studies. The specific, unifying thread will focus in the quest for the observation of microtubule dynamics during the first few, asymmetric cell divisions in Caenorhabditis elegans embryos.
A most-important-variables analysis of practical, successful multiphoton excitation fluorescence microscopy is presented. The key strength of multiphoton imaging -- localization of the excitation volume -- helps to decouple the excitation from the emission; the emission no longer needs to be imaged. The presentation starts with maximizing the detected signal and proceeds to the laser source considerations. The main goal is to match the instrumentation to the biological problem. The main aspects covered are... Emission collection: potential signal; Focal plane mismatch: chromatic aberration; Light detected: actual signal; Live cell imaging: preservation of biological function; Deep imaging: preservation of image contrast; Signal production: potential limitations.
Most multiphoton imaging has been undertaken using tuneable femtosecond Ti:Sapphire lasers which are large, expensive and require a level of laser expertise to operate. Although new commercial, computer controlled systems are becoming available they are still complex instruments. We report on the development of a range of novel laser sources for multiphoton microscopy based upon optically pumped semiconductor materials.
Fluorescence microscopy is an invaluable technique for investigating structural and biochemical changes in cells and tissues. While it is preferable to study these changes in living specimens, such studies are often compromised by the destructive properties of light which can cause cellular damage either directly (photoablation) or indirectly by generating toxic by-products (phototoxicity). To minimize these problems, new methods of illuminating cells are being developed. In particular, ultrafast infrared lasers have been employed to excite fluorophores at one-half and one- third the wavelength of the laser by a process called multiphoton excitation. This process limits excitation to a small volume of indicator which, together with fast scanning of the sample, may reduce photodamage. One source of photodamage is light-induced stimulation of H2O2 in cells. In this report, we tested whether scanning with an ultrafast Ti:sapphire laser could stimulate H2O2 production in cultured human and monkey cells measured with the fluorescent indicator dichlorodihydrofluorescein. We demonstrate that illumination at 800 - 900 nm induced H2O2 production in cells when laser power was increased above 10 mW (at the specimen plane). The frequency of scanning (duty cycle) also influenced H2O2 production indicating that a trade-off between power and exposure time may be an appropriate way to control this type of toxicity. Alternatively, high power and increased exposure time could provide an effective means for controlling H2O2 production and subsequent damage to cellular structures.
The use of fluorescent probes is a powerful technique for the study of living specimens. Unfortunately, living tissues are vulnerable to photodamage from the excitation illumination and they make poor optical specimens due to their light-scattering nature. Multiphoton (two or more photon) excitation imaging offers significant advantages compared to laser-scanning confocal fluorescence microscopy for fluorescence microscopy of live specimens: considerable reduction in total sample fluorophore excitation and hence less photodamage, increased depth penetration due to increased tolerance for scattering, and increased detection sensitivity as more signal photons can be used for imaging. These advantages become more significant if 3D or 4D (multifocal plane, time-lapse) imaging is undertaken. In addition, multiphoton excitation imaging allows UV excited probes such as DAPI or INDO I or endogenous fluorophores such as NAD(P)H and serotonin to be imaged without UV excitation. We, and others, have been evaluating the potential of multi-photon excitation imaging for biological microscopy and have found all of the aforementioned advantages particularly significant for laser-scanning fluorescence imaging of developing embryos; a summary of currently pursued developmental biology applications will be presented. The current status of all-solid-state ultrafast lasers as excitation sources will also be reviewed since these lasers offer tremendous potential for affordable, reliable, 'turnkey' multiphoton imaging systems. The combination of demonstrated applications, simple ultrafast laser sources, and affordable commercial systems may promote a revolution in the study of embryogenesis with the light microscope.
Fluorescence microscopy is a ubiquitous and powerful tool for the biologist mainly due to the availability of a wealth of highly specific fluorescent probes. Multiphoton (two or more photon) excitation fluorescence microscopy is an optical sectioning technique that offers significant advantages over other optical sectioning techniques in terms of improved viability of living material and the ability to penetrate deeper into specimens. The use of a longer excitation wavelength (typically twice that of the excitation peak of the fluorophore) increases the penetration of the excitation into the sample, yet essentially eliminates single-photon excitation in the bulk of the sample. In order to attain the high peak-power densities necessary for the production of multiphoton events while keeping mean power levels below damaging levels, ultrashort-pulsed excitation sources are used. Some sources, such as mode-locked, Ti:sapphire lasers, can produce pulses less than 100 fs. Pulses this short need to be pre-chirped in order to compensate for the group velocity dispersion of the microscope optics so that the pulse width is maintained at the sample. Without such pre-compensation we show that the average power required to produce a fixed level of two-photon excited signal, using typical microscope optics, is fairly constant from 60fs to 250fs. We argue that the choice of pulse width is an important consideration for a biological imaging system since varying the source pulse width may be used to change the relative amounts of two- and three- photon excitation. With a pre-chirped (compensated) system, if the pulse length is quadrupled then twice the power will be required to attain the previous level of two-photon excited fluorescence, but only half the three-photon excitation (or absorption) will be produced. Pulse widths may be varied on compensated systems by adjusting the pre-compensation. This may be used to favor three-photon excitation of UV-excited fluorophores, or, on the other hand, it may be desirable to reduce levels of three-photon excitation during two-photon imaging of live samples using 700 nm - 800 nm radiation as deleterious excitation of endogenous fluorophores or absorption by (and therefore damage to) proteins and nucleic acids could occur. Variable pulse widths may therefore prove to be an important parameter for live cell studies. Alternatively, for a given range of applications, a simpler and cheaper fixed-pulse length source with the desired characteristics may be chosen.
Multi-photon (two or more photon) excitation imaging offers three significant advantages compared to laser-scanning confocal fluorescence microscopy for 3-D and 4-D fluorescence microscopy: considerable reduction in total sample excitation, increased depth penetration, and increased detection sensitivity. All-solid-state ultra-fast lasers offer tremendous potential for affordable, reliable, 'turn-key' multi-photon excitation sources. We have been developing a multi-photon system that utilizes an all-solid- state Nd:YLF excitation source. We have been evaluating the potential of this source for biological microscopy and have been optimizing system parameters for this application area. We have found that the 1047 nm radiation from these lasers can excite by two-photon fluorescence many commonly used fluorophores that are normally excited from blue to yellow light. In addition, we have found that this wavelength readily excites several normally UV excited fluorophores by the mechanism of three-photon excitation. The Nd:YLF laser has proven reliable in operation with nearly 6000 hours logged without significant loss of power. However, the original system produced rather long pulses for multi-photon excitation (300 fs) and a beam shape that was not ideal. We have recently commissioned the development of an improved pulse compressor from the manufacturers that gives narrower pulses (120 fs), improved beam shape, and a smaller insertion loss. This optimized excitation system has 6 times more potential two-photon excited fluorescence and 22 times more potential three-photon excited fluorescence than the prototype system. In addition, by optimizing coatings in the excitation and signal paths, we have improved the descanned detection sensitivity by 20% for two-photon excited fluorescence and 315% for three-photon excited fluorescence. The excitation optical transfer efficiency (1047 nm) of our imaging system is currently 60% to the back aperture of the objective. The emission optical transfer efficiency (670 nm) is currently 47% for descanned detection and 83% for non- descanned detection; both from the objective back aperture. Surprisingly, we find there is a signal-to-background increase of a factor of 17 between descanned and non- descanned modes of detection using a Nile Red solution sample.
Two-photon excitation imaging is a recently described optical sectioning technique where fluorophore excitation is confined to--and therefore defines--the optical section being observed. This characteristic offers a significant advantage over laser-scanning confocal microscopy; the volume of fluorophore excited in the minimum necessary for imaging, thereby minimizing the destructive effects of fluorophore excitation in living tissues. In addition, a confocal pinhole is not required for optical scattering--thus further reducing the excitation needed for efficient photon collection. We have set up a two-photon excitation imaging system which uses an all-solid-state, short-pulse, long-wavelength laser as an excitation source. The source is a diode-pumped, mode-locked Nd:YLF laser operating in the infrared (1047nm). This laser is small, has modest power requirements, and has proven reliable and stable in operation. The short laser pulses from the laser are affected by the system optical path; this has been investigated with second harmonic generation derived from a nonlinear crystal. The system has been specifically designed for the study of live biological specimens. Two cell types especially sensitive to high-energy illumination, the developing Caenorhabditis elegans embryo and the crawling sperm of the nematode, Ascaris, were used to demonstrate the dramatic increase in viability when fluorescence is generated by two-photon excitation. The system has the capability of switching between two-photon and confocal imaging modes to facilitate direct comparison of theory of these two optical sectioning techniques on the same specimen. A heavily stained zebra fish embryo was used to demonstrate the increase in sectioning depth when fluorescence is generated by infrared two-photon excitation. Two-photon excitation with the 1047nm laser produces bright images with a variety of red emitting fluorophores, and some green emitting fluorophores, commonly used in biological research. Fortuitously, we have found that at least four blue emitting fluorophores normally excited by UV light are excited by the pulsed 1047nm laser, by what we believe to be three-photon excitation. Multi-photon excitation is demonstrated by a double labelled C. elegans embryo.