High Throughput Screening with Multiphoton Excitation

Fluorescence detection is extensively used in high throughput screening. In HTS there is a continuous migration toward higher density plates and smaller sample volumes. In the present report we describe the advantages of two-photon or multiphoton excitation for HTS. Multiphoton excitation (MPE) is the simultaneous absorption of two long-wavelength photons to excite the lowest singlet state of the fluorophore. MPE is typically accomplished with short but high-intensity laser pulses, which allows simultaneous absorption of two or more photons. The intensity of the multiphoton-induced fluorescence is proportional to the square, cube, or higher power of the instantneous photon flux. Consequently, two-photon or multiphoton excitation only occurs at the focal point of the incident beam. This property of two-photon excitation allows the excited volume to be very small and to be localized in the center of each well in the HTS plate. We show that two-photon-induced fluorescence of fluorescein can be reliably measured in microwell plates. We also show the use of 6-carboxy fluorescein as a pH probe with two-photon excitation, and measure 4′-6-diamidino-2-phenylindole (DAPI) binding and two-photon-induced fluorescence. In further studies we measure the time-dependent intensity decays of DAPI bound to DNA and of calcium-dependent fluorophores. Finally, we demonstrate the possibility of three-photon excitation of several fluorophores, including indole, in the HTS plate. These results suggest that MPE can be used in high-density multiwell plates.

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Two-photon fluorescence excitation in detection of biomolecules

Biochemical Society Transactions ◽

10.1042/bst0280070 ◽

2000 ◽

Vol 28 (2) ◽

pp. 70-74 ◽

Cited By ~ 17

Author(s):

E. Soini ◽

N. J. Meltola ◽

A. E. Soini ◽

J. Soukka ◽

J. T. Soini ◽

...

Keyword(s):

Single Molecule ◽

High Throughput Screening ◽

Focal Point ◽

Single Photons ◽

Fluorescence Excitation ◽

Two Photon ◽

Photon Excitation ◽

Two Photon Fluorescence ◽

Photon Fluorescence ◽

Two Photon Excitation

Two-photon fluorescence excitation has been found to be a very powerful method for enhancing the sensitivity and resolution in far-field light microscopy. Two-photon fluorescence excitation also provides a substantially background-free detection on the single-molecule level. It allows direct monitoring of formation of labelled bio-molecule complexes in solution. Two-photon excitation is created when, by focusing an intensive light source, the density of photons per unit volume and per unit time becomes high enough for two photons to be absorbed into the same chromophore. In this case, the absorbed energy is the sum of the energies of the two photons. In two-photon excitation, dye molecules are excited only when both photons are absorbed simultaneously. The probability of absorption of two photons is equal to the product of probability distributions of absorption of the single photons. The emission of two photons is thus a quadratic process with respect to illumination intensity. Thus in two-photon excitation, only the fluorescence that is formed in the clearly restricted three-dimensional vicinity of the focal point is excited. We have developed an assay concept that is able to distinguish optically between the signal emitted from a microparticle in the focal point of the laser beam, and the signal emitted from the surrounding free labelled reagent. Moreover, the free labels outside the focal volume do not contribute any significant signal. This means that the assay is separation-free. The method based on two-photon fluorescence excitation makes possible fast single-step and separation-free immunoassays, for example, for whole blood samples. Since the method allows a separation-free assay in very small volumes, the method is very useful for high-throughput screening assays. Consequently we believe that two-photon fluorescence excitation will make a remarkable impact as a research tool and a routine method in many fields of analysis.

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Two-photon excitation microscopy in cellular biophysics

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100136684 ◽

1995 ◽

Vol 53 ◽

pp. 62-63

Author(s):

David W. Piston ◽

Brian D. Bennett ◽

Robert G. Summers

Keyword(s):

Confocal Microscopy ◽

Laser Beam ◽

Duty Cycle ◽

Excited Electronic State ◽

Input Power ◽

Two Photon ◽

Simultaneous Absorption ◽

Photon Excitation ◽

Very High ◽

Two Photon Excitation

Two-photon excitation microscopy (TPEM) provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging and photochemistry. Two-photon excitation arises from the simultaneous absorption of two photons in a single quantitized event whose probability is proportional to the square of the instantaneous intensity. For example, two red photons can cause the transition to an excited electronic state normally reached by absorption in the ultraviolet. In practice, two-photon excitation is made possible by the very high local instantaneous intensity provided by a combination of diffraction-limited focusing of a single laser beam in the microscope and the temporal concentration of 100 femtosecond pulses generated by a mode-locked laser. Resultant peak excitation intensities are 106 times greater than the CW intensities used in confocal microscopy, but the pulse duty cycle of 10-5 maintains the average input power on the order of 10 mW, only slightly greater than the power normally used in confocal microscopy.

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Two-photon excitation fluorescence microscopy in living systems

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100146618 ◽

1993 ◽

Vol 51 ◽

pp. 154-155 ◽

Cited By ~ 1

Author(s):

David W. Piston

Keyword(s):

Confocal Microscopy ◽

Fluorescence Microscopy ◽

Singlet State ◽

Excited Electronic State ◽

Input Power ◽

Two Photon ◽

Simultaneous Absorption ◽

Photon Excitation ◽

Very High ◽

Two Photon Excitation

Two-photon excitation fluorescence microscopy provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging. Two-photon excitation arises from the simultaneous absorption of two photons in a single quantitized event whose probability is proportional to the square of the instantaneous intensity. For example, two red photons can cause the transition to an excited electronic state normally reached by absorption in the ultraviolet. In our fluorescence experiments, the final excited state is the same singlet state that is populated during a conventional fluorescence experiment. Thus, the fluorophore exhibits the same emission properties (e.g. wavelength shifts, environmental sensitivity) used in typical biological microscopy studies. In practice, two-photon excitation is made possible by the very high local instantaneous intensity provided by a combination of diffraction-limited focusing of a single laser beam in the microscope and the temporal concentration of 100 femtosecond pulses generated by a mode-locked laser. Resultant peak excitation intensities are 106 times greater than the CW intensities used in confocal microscopy, but the pulse duty cycle of 10−5 maintains the average input power on the order of 10 mW, only slightly greater than the power normally used in confocal microscopy.

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Photochemistry with high power ultraviolet lasers

Canadian Journal of Chemistry ◽

10.1139/v83-180 ◽

1983 ◽

Vol 61 (5) ◽

pp. 1023-1026 ◽

Cited By ~ 10

Author(s):

R. J. Donovan ◽

C. Fotakis ◽

A. Hopkirk ◽

C. B. McKendrick ◽

A. Torre

Keyword(s):

Energy Distribution ◽

High Power ◽

Single Photon ◽

Laser Induced Fluorescence ◽

Multiphoton Excitation ◽

Dissociation Process ◽

Two Photon ◽

Photon Excitation ◽

Ultraviolet Lasers ◽

Two Photon Excitation

Rotationally resolved photofragment fluorescence from OH(A2Σ+) following the coherent two-photon excitation of H2O with a KrF laser (248 nm), is reported and the dynamics of the dissociation process are discussed. Fluorescence from CN(B2Σ+) following two-photon excitation of ICN is also described. In both cases the energy distribution in the photofragments is shown to differ significantly from that observed with single-photon excitation at closely similar energies.Two further examples of multiphoton excitation, involving CS2 and SO2, are briefly discussed. In these cases absorption of a further photon, by fragments produced in the primary step, gives rise to strong laser-induced fluorescence.

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Two-photon controlled sol–gel condensation for the microfabrication of silica based microstructures. The role of photoacids and photobases

RSC Advances ◽

10.1039/c7ra08608c ◽

2017 ◽

Vol 7 (74) ◽

pp. 46615-46620 ◽

Cited By ~ 4

Author(s):

J. Kustra ◽

E. Martin ◽

D. Chateau ◽

F. Lerouge ◽

C. Monnereau ◽

...

Keyword(s):

Laser Beam ◽

Focal Point ◽

Sol Gel ◽

Ph Changes ◽

Two Photon ◽

Photon Excitation ◽

Sol Gel Process ◽

And Control ◽

Two Photon Excitation

Two-photon excitation of photobases is used to induce pH changes and control the condensation step of the sol–gel process at the focal point of a laser beam in a confocal configuration.

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Multi-Photon Excitation Microscopy: An Old Idea in Quantum Theory Applied to Modern Scientific Problems

Microscopy and Microanalysis ◽

10.1017/s1431927600038393 ◽

2000 ◽

Vol 6 (S2) ◽

pp. 1180-1181

Author(s):

David W. Piston

Keyword(s):

Physical Principle ◽

Excitation Intensity ◽

Appreciable Amount ◽

Photon Density ◽

Two Photon ◽

Simultaneous Absorption ◽

Photon Excitation ◽

Dramatic Difference ◽

Basic Physical Principle ◽

Two Photon Excitation

Multi-photon excitation microscopy provides attractive advantages over confocal microscopy for three-dimensionalry resolved fluorescence imaging and photochemistry. The most commonly used type of multi-photon excitation is two-photon excitation where simultaneous absorption of two photons leads to a single quantitized event. The powerful advantages of using two-photon excitation microscopy arise from the basic physical principle that the absorption depends on the square of the excitation intensity. In practice, two-photon excitation is generated by focusing a single pulsed laser through the microscope. As the laser beam is focused, the photons become more crowded, but the only place at which they are crowded enough to generate an appreciable amount of two-photon excitation is at the focus. Above and below the focus, the photon density is not high enough for two of them to interact with a single fluorophore at the same time. This dramatic difference between confocal and two-photon excitation microscopy is shown in Fig. 1.

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High throughput instrument to screen fluorescent proteins under two-photon excitation

10.1101/2020.09.04.283572 ◽

2020 ◽

Author(s):

Rosana S. Molina ◽

Jonathan King ◽

Jacob Franklin ◽

Nathan Clack ◽

Christopher McRaven ◽

...

Keyword(s):

High Throughput ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Agar Plate ◽

Focal Volume ◽

E Coli ◽

Two Photon Microscopy ◽

Two Photon ◽

Photon Excitation ◽

Two Photon Excitation

AbstractTwo-photon microscopy together with fluorescent proteins and fluorescent protein-based biosensors are commonly used tools in neuroscience. To enhance their experimental scope, it is important to optimize fluorescent proteins for two-photon excitation. Directed evolution of fluorescent proteins under one-photon excitation is common, but many one-photon properties do not correlate with two-photon properties. A simple system for expressing fluorescent protein mutants is E. coli colonies on an agar plate. The small focal volume of two-photon excitation makes creating a high throughput screen in this system a challenge for a conventional point-scanning approach. We present an instrument and accompanying software that solves this challenge by selectively scanning each colony based on a colony map captured under one-photon excitation. This instrument, called the GIZMO, can measure the two-photon excited fluorescence of 10,000 E. coli colonies in 7 hours. We show that the GIZMO can be used to evolve a fluorescent protein under two-photon excitation.

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High throughput instrument to screen fluorescent proteins under two-photon excitation

Biomedical Optics Express ◽

10.1364/boe.409353 ◽

2020 ◽

Vol 11 (12) ◽

pp. 7192

Author(s):

Rosana S. Molina ◽

Jonathan King ◽

Jacob Franklin ◽

Nathan Clack ◽

Christopher McRaven ◽

...

Keyword(s):

High Throughput ◽

Fluorescent Proteins ◽

Two Photon ◽

Photon Excitation ◽

Two Photon Excitation

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Imaging of Cellular Dynamics by Two-Photon Excitation Microscopy

Microscopy and Microanalysis ◽

10.1017/s1431927695110259 ◽

1995 ◽

Vol 1 (1) ◽

pp. 25-34 ◽

Cited By ~ 2

Author(s):

David W. Piston ◽

Brian D. Bennett ◽

Guangtao Ying

Keyword(s):

Uv Light ◽

Excited Electronic State ◽

Red Light ◽

Three Dimensional ◽

Focal Volume ◽

Optical Sectioning ◽

Two Photon ◽

Simultaneous Absorption ◽

Photon Excitation ◽

Two Photon Excitation

Two-photon excitation fluorescence microscopy provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging. Two-photon excitation arises from the simultaneous absorption of two photons in a single quantitized event whose probability is proportional to the square of the instantaneous intensity. For example, two red photons (∼700 nm) can cause the transition to an excited electronic state normally reached by absorption in the ultraviolet (∼350 nm). In the fluorescence experiments described here, the final excited state is the same singlet state that is populated during a conventional fluorescence experiment. Thus, the fluorophore exhibits the same emission properties (e.g., wavelength shifts, environmental sensitivity) used in typical biological microscopy studies. Three properties of two-photon excitation give this method its advantage over conventional optical sectioning microscopies: (1) the excitation is limited to the focal volume, thus providing inherent three-dimensional resolution and minimizing photobleaching and photodamage; (2) the two-photon technique allows imaging of UV fluorophores with only conventional visible light optics; (3) red light is far less damaging to most living cells and tissues than UV light and permits deeper sectioning, because both absorbance and scattering are reduced. Many cell biological applications of two-photon excitation microscopy have been successfully realized, demonstrating the wide ranging power of this technique.

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