Time Resolved Fluorescence Emission – Fluorometer


Principle of Operation

Decay Measurement Time Correlated Single Photon Counting (TCSPC)

A Pulsed laser source (DeltaDiode) sends Start signals to the timing electronics. This pulsed laser is focused onto the sample and the fluorophores within the sample fluoresces in all directions. To reduce stray light, the emission optics are mounted at right angles to the excitation source. The emission monochromator (TDM) wavelength is set as desired. When a fluorescence photon reaches the detector (PPD) a Stop is detected and the timing electronics (DeltaHub) are reset. The time between the start and stop is measured and computed in a histogram. If no photon is detected during the specified time range, the electronics are reset.

The ND filters are in place to reduce the Stop/Start rate to < 2 %. At levels >2 % the fluorescence decay may be distorted to a shorter decay time due to pile up effects. That is if 2 photons reach the detector before the electronics reset only the 1st one is measured as the 2nd one cannot be measured as the electronics are resetting. Think of throwing tennis balls to your dog. If you throw one ball your dog can run and catch it (this is the time you are recording), then it will take some time for him to return it to you (call this time deadtime). If you throw a second tennis ball during the time your dog takes to run back to you, your dog will ignore it and only return the second tennis ball.

The longpass or bandpass filter is present to stop 2nd order emission.

trfluorometer

The instrument response is measured using fast Rayleigh scattering. A scattering silica colloid such as dilute Ludox SM-AS is used to maximise Rayleigh scattering. To maximise Rayleigh scattering both polariser’s are at the vertical orientation and the emission monochromator is set to the excitation wavelength of the Pulsed Laser and any long pass filters are removed. ND filters are present to ensure that the STOP/START rate is less than 2 %. Usually a peak preset of 10000 counts is used.

For the sample the excitation polariser is at the vertical orientation and the emission polariser is set to the magic angle. ND filters are present to ensure that the STOP/START rate is less than 2 %. Usually a peak preset of 10000 counts is used.

We then carry out reconvolution analysis of the prompt and decay to extract the decay time(s) and pre-exponential factors.

Fluorescence decays are usually computed in log format on the intensity axis and linear format on the time axis. The log of a mono-exponential is a straight line.

Time Resolved Emission Spectra

The time-resolved emission spectra consists of a series of fluorescence decays with respect to the emission wavelength. Instead of a peak preset all decays are measured for a specified dwell time. This creates a measurement that has time on one axis, emission wavelength on the other and intensity on the third axis. This dataset is measured alongside the instrumental response measured to a peak preset of 10000 counts as before.

 

TRES

From this 3D dataset the data may be sliced. We may look at early time slices to view fast effects such as Raman scattering. Then we may look at medium time slices where the fast scattering processes will be substantially diminished meaning we can look at fluorescence processes in more detail without distortion from scattering. At long time ranges fluorescence processes will likewise diminish and only the longer lived phosphorescence processes will remain.

From this 3D dataset we can also carry out reconvolution analysis to extract the decay time(s) and compute spectra for each component using their pre-exponential factors.

Fluorescence decays are usually computed in log format on the intensity axis and linear format on the time axis. The log of a mono-exponential is a straight line. Steady-state spectra are usually shown in linear format however.

Fluorescence Anisotropy

The first concept to understand for anisotropy measurements is the concept of Brownian motion. Although water at room temperature contained in a glass to the eye may look very still, on the molecular level each water molecule has kinetic energy and thus there are a continuous number of collisions between water molecules. A nanoparticle (yellow dot in the figure) suspended in solution will undergo a random walk due to the summation of these underlying collisions.

Image Reference: https://en.wikipedia.org/wiki/Brownian_motion

The rotational correlation time that is the time it takes for the molecule to rotate 1 radian is dependant on the viscosity, temperature, Boltzmann constant and volume of the nanoparticle.

When nanoparticles suspended in solution are excited with a polarised pulse, molecules whose transition moment is parallel are preferentially excited. In solution at room temperature these nanoparticles experience continuous bombardment by water molecules known as Brownian motion.

For a monoexponential decay when measured with the polariser configuration at VM, no polarisation bias is observed. A mono-exponential lifetime standard will therefore have a single decay time τ and an associated positive pre-exponential factor.

The fluorescence decay when measured with polarisers in the VV configuration will have 2 decay times. The first will have a positive pre-exponential factor and the decay time will match τ the decay time measured in the unpolarised decay. The second decay time will also have a positive pre-exponential factor and its decay time will be associated with the speed of Brownian motion. Essentially additional fluorescence is lost as vertically polarised fluorophores rotate and are no longer picked up by the detector behind the vertically polarised polariser.

The fluorescence decay when measured with polarisers in the VH configuration will also have 2 decay times. The first will have a positive pre-exponential factor and the decay time will match τ the decay time measured in the unpolarised decay. The second decay time will have a negative pre-exponential factor and its decay time will be associated with the speed of Brownian motion. Essentially additional fluorescence is obtained as vertically polarised fluorophores rotate and begin to be picked up by the detector behind the horizontically polarised polariser.

The fluorescence anisotropy decay combines both decays at VV and VH. The decays HH and HV are measured for a grating factor, that is to compensate for the bias of HV over HH. Note the grating factor is wavelength dependent. From the anisotropy decay you can extract the rotational correlation time. As larger objects take longer to rotate you can use this to infer the size of a nanoparticle.

Anisotropy

 

DeltaFlex Hybrid

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This FluoroCube and Hub A has been upgraded to incorporate the state of the art electronics of the DeltaHub and DeltaDiode offered by HORIBA Scientific IBH (Glasgow). The old Hub A is still used to control the optics but the timing electronics of the DeltaHub and the Deltadiode light sources essentially make it a DeltaFlex. This is a high throughput system.

Capabilities: Fluorescence Lifetime, Fluorescence Anisotropy, Steady State, Time Resolved Emission Spectra, Sequential TCSPC

Hub and TAC Specifications: DeltaHub <10 ns Deadtime (best on the market).

Detector Specifications: TBX-850c 250-850 nm.

Light Sources: DeltaDiodes, NanoLEDs and SpectraLEDs see TCSPC Shared Components for inventory

Monochromators: Excitation and Emission 5000M

PC: Dell Optiplex 790, Windows 7 64 bit DataStation 2.7 and Decay Analysis Software 6.8.2

Principle of operation (click to maximise)

Weblinks: Tutorial Videos, Technical Notes, DeltaSeries Brochere, DeltaDiode Brochere

FluoroCube

Capabilities: Fluorescence Lifetime, Fluorescence Anisotropy, Steady State, Time Resolved Emission Spectra

Hub and TAC Specifications: FluoroHub A ~ 5 µs deadtime.

Detector Specifications: TBX-650 180-650 nm.

Light Sources: NanoLEDs and SpectraLEDs see TCSPC Shared Components for inventory

Monochromators: Excitation and Emission 5000M

PC: Personal Custom Build incorporating a ASRock H81M Motherboard into a Cooler Master N200 case with a Corsair CX430M power supply. A PCIe-x1 to 2 serial card was also required. An Intel Pentium G3220 Processor, 4 GB RAM and 1 TB HDD were also added. The MCA3, NI6602 cards are used as timing electronics. Software required is: Windows 7 32 Bit Datastation 2.6.7 and Decay Analysis Software 6.8.2.

Weblinks: Tutorial Videos, Technical Notes.

TemPro Hybrid

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This is a FluoroCube without monochromators making it essentially a TemPro. It is attached to a Coherent Chameleon and APE Pulse picker.

Capabilities: 1 and 2 Photon Fluorescence Lifetime, 1 and 2 photon Fluorescence Anisotropy

Hub and TAC Specifications: FluoroHub A ~5 µs deadtime

Detector Specifications: TBX-850 250-850 nm.

Light Sources: Light Sources: NanoLEDs and SpectraLEDs see TCSPC Shared Components for inventory and Coherent Chameleon (2 photon)

Emission selected via long pass filters.

PC: Personal Custom Build incorporating a ASRock H81M Motherboard into a Cooler Master N200 case with a Corsair CX430M power supply. A PCIe-x1 to 2 serial card was also required. An Intel Pentium G3220 Processor, 4 GB RAM and 1 TB HDD were also added. The MCA3, NI6602 cards are used as timing electronics. Software required is: Windows 7 32 Bit Datastation 2.6.7 and Decay Analysis Software 6.8.2.

Weblinks: Tutorial Videos, Technical Notes.

Light Sources

DeltaDiode (FWHM ~200 ps) Wavelengths 378, 482, 503 and 670 nm. (250 kHz-100 MHz)

NanoLED Lasers (FWHM <300 ps) Wavelengths 373, 374, 405, 435, 437, 469, 474, 638, 649 and 665(1 MHz).

NanoLED Diodes (FWHM ~1.4 ns) Wavelengths 265, 279, 295, 296, 296, 339. 370, 458, 490, 562, 590 and 605 nm (1 MHz).

SpectraLED Wavelength 377, 471 nm

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Filters

Neutral Density Filters, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 1.0, 1.3, 2.0, 3.0

Longpass Filters Wavelengths: 180, 250, 305, 335, 345, 360, 365, 370, 375, 385, 400, 405, 410, 410, 410, 430, 440, 475, 485, 495, 500, 505, 530, 560, 570, 590, 605, 610, 610, 630, 645, 665, 670, 670, 695, 710, 785, 785, 835 and 845 nm

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Add spectra of the filters….

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