Ultrafast Laser Spectroscopy

The Saskatchewan Structural Sciences Centre's Laser Technology Laboratory main purpose lies with the application of ultra-fast lasers. These short lasers pulses are a great aid in studying compounds with short fluorescent lifetime and in using non-linear optical phenomenon to optimize imaging of live cells. Disciplines from the life sciences, to chemistry, to electrical engineering make use of the facility for their research programs.

Fluorescence Up-Conversion

The SSSC fluorescence up-conversion system is used to measure the lifetime of short-lived fluorescent molecules in solution. The sample solution is exposed to an ultra-short laser pulse (400 nm) and the fluorescence emitted is monitored for intensity my mixing (up-conversion) it in a nonlinear crystal with another ultra-short laser pulse (800 nm), the probe. By delaying the time at which the probe pulse reaches the non-linear crystal, and mixes with the fluorescence, the intensity of the up-conversion can be monitored as a function of decay time. The up-conversion process is the sum frequency generated in the non-linear crystal by the combination of the fluorescence and the 800 nm probe light resulting in a wavelength shorter than that of the fluorescence. Lifetimes in the sub-nanosecond range can be measured using this technique.

Specifications

Excitation wavelength 400 nm
Measurements Decay lifetime
Rise-time
Cross-correlation FWHM 200 fs

Settings

This system uses the Verdi/Vitesse / RegA /OPA suite.

Time-Correlated Single Photon Counting

TCSPC is used to measure the fluorescence lifetime of compounds in specific environments. An ultra-fast laser pulse excites the sample and the light emitted from the sample is tagged for arrival time. A decay trace of the fluorescence as a function of time is used to determine the number of lifetime components present and their respective value. Lifetime measurements from 50 picoseconds to hundreds of nanoseconds can be measured.

Specifications

Excitation wavelength 240 to 330 nm
360 to 500 nm
9.5 kHz to 4.75 MHz (variable) or 76 MHz
Emission wavelength 400 – 910 nm (monochromator + MCP-PMT)
Time range 3.3 ns to 2μs
up to 4096 time channels
Cuvette Temperature control and magnetic stirring (FLASH 200, Quantum Northwest): -25 to +80 °C
Objective lens 63x/0.9NA, 2.2 mm working distance
Detection MCP-PMT, R3809U-51 (Hamamatsu)
FWHM 35 ps
160 to 190 nm
Acquisition Data acquisition board:
SPC-830 (Becker and Hickl)
SPC 630 (Becker and Hickl)
Advanced capabilities Detection below 400 nm (optical filter for wavelength selection) Detector: PMC-100-4 (Becker and Hickl) detector (185-820 nm and FWHM ~ 190 ps) Detection in ultra-violet and visible with CM110 monochromator (gratings AG1800-00450H or AG0600-00500) and PMC-100-4 detector Dual detectors: MCP-PMT and PMC-100-4 with CM110 (individual data acquisition boards)

Settings

Pulse repetition rate and width are adjusted to prevent sample bleaching.
Sample temperature control is available.
This system uses the Verdi / Mira / Pulse Picker / Harmonic Generator suite.

More Information

General statement for publications

The time correlated single photon counting data was acquired using a pulsed laser system as an excitation source and processed via the TCSPC data acquisition board SPC-630/SPC-830 (Becker and Hickl, Berlin, Germany). The laser was a Mira 900-D laser system (Coherent, Santa Clara, CA) in femto/picosecond mode, with a repetition rate modified to ______ Hz using a pulse picker PP9200, and a harmonic generator, HG 9300, provided the wavelength of ___ nm. A pick off optic was used to trigger the fast photodiode, a PHD-400 (Becker and Hickl, Berlin, Germany), to send a time decay synchronization pulse to the SPC-630/SPC-830. The fluorescence emission was collected at Magic angle (54.7°degrees) relative to excitation beam polarization. The emission light intensity was controlled using absorbance neutral density filters to maintain a photon counting ratio of ____% relative to the excitation beam repetition rate. The emission wavelength was selected using a monochromator (CM112, Spectral Products, Putnam, CT) outfitted with a AG0600-00500/AG1200-00750 grating and ____ mm slits. The resulting light was detected using a MCPPMT R3809U-51 (Hamamatsu, Japan), its signal was amplified using a HFAC-26 (Becker and Hickl, Berlin, Germany), and then sent to the CFD input of the SPC-630/SPC-830 which was set for a total acquisition time of ____ seconds/minutes, overflow correction on/off/stop, and ___ time channels.

Recommended Reading

  • Phillips, David, and Desmond V. O’Connor. Time-Correlated Single Photon Counting. London: Academic Press, 1984.
  • Becker, Wolfgang. Advanced Time-Correlated Single Photon Counting Techniques. Berlin: Springer, 2005.

Fluorescence Lifetime Imaging Microscopy (FLIM)

FLIM is the measurement of the lifetime of a fluorophore in an excited electronic state as a function of position on an image. This gives a picture of environment of the fluorophore. The lifetime of a fluorophore can change with chemical composition (pH, calcium concentration) or because of quenching (FRET, oxygen). At the SSSC, FLIM is based on the time domain acquisition of information in conjunction with the laser scanning confocal microscope. The pulsed laser system can used in pico- or femtosecond mode with a variety of repetition rates, and it can be used in the visible or infrared regions.

Specifications

Excitation wavelength Two-phonon mode: 700 to 1000 nm
Visible: 400 to 490 nm
Objective lens 63x/0.9NA, 2.2 mm working distance
Detection Descanned (2 detectors with emission filter wheel) for visible light excitation
Non-descanned
PMC-100-4 (Becker and Hickl) FWHM 190 ps
Acquisition Data acquisition board SPC-830 (Becker and Hickl)
Advanced capabilities 63x/0.9NA, 2.2 mm working distance
Detection MCP-PMT, R3809U-51 (Hamamatsu)
FWHM 35 ps
160 to 190 nm
Acquisition Data acquisition board:
SPC-830 (Becker and Hickl)
SPC 630 (Becker and Hickl)
Advanced capabilities Objective inverter (LSM Technologies) for upright microscopy applications FCS2 Temperature control chamber (contact Dr. Jim Xiang for use of the FCS2) Detector: Cooled MCP-PMT R3809U-51 (Hamamatsu) with FWHM ~ 35 picosecond

Settings

This system uses the Verdi / Mira / Pulse Picker / Harmonic Generator suite.

More Information

General statement for publications

You may use the following as a guideline for publication details. It is your responsibility to meet publisher requirements prior to submitting. One-photon excitation and descanned detector (RFL2 filter wheel): The FLIM time domain data was acquired using a laser scanning confocal microscope, a pulsed laser system set at ____ nm, and a SPC-830 (Becker and Hickl, Berlin, Germany) FLIM data acquisition board. The LSCM was a LSM410 (Zeiss, Thornwood, NY) upgraded by LSM Technologies (Etters, PA), the bandpass/longpass emission filter in the confocal path was a ___-___ nm (part # and manufacturer) and the pinhole was set to _____ airy units. The light path was redirected to a single photon counting photomultiplier tube, a PMC-100-4 (Becker and Hickl, Berlin, Germany), whose signal became the CFD input to the SPC-830. The laser was a Mira 900-D laser system (Coherent, Santa Clara, CA) in femto/picosecond mode, with a repetition rate modified to ______ Hz using a pulse picker PP9200, and a harmonic generator, HG 9300, changed the wavelength to ___ nm. A pick off optic was used to trigger the fast photodiode, a PHD-400 (Becker and Hickl, Berlin, Germany), to send a time decay synchronization pulse to the SPC-830. The SPC-830 was set for a total acquisition time of ____ seconds/minutes, ___ time channels, and receiving scanning synchronization information from the LSM410 for image construction of ___ pixels by ___ pixels. The scan rate of the LSM410 was carefully chosen for timing of excitation of each pixel based on the repetition rate of the laser.

Two-photon excitation and non-descanned detector: The FLIM time domain data was acquired using a laser scanning confocal microscope, a pulsed laser system set at ____ nm, and a SPC-830 (Becker and Hickl, Berlin, Germany) FLIM data acquisition board. The LSCM was a LSM410 (Zeiss, Thornwood, NY) upgraded by LSM Technologies (Etters, PA) for two-photon excitation and a non-descanned emission path with a 700 nm short pass filter (E700SP-2P, Chroma, Rockingham, VT). The single photon counting photomultiplier tube detector was a PMC-100-4 (Becker and Hickl, Berlin, Germany) and its signal became the CFD input to the SPC-830. The laser was a Mira 900-D laser system (Coherent, Santa Clara, CA) in femto/picosecond mode and set to ___ nm, with a repetition rate modified to ______ Hz using a pulse picker PP9200. A pick off optic was used to trigger the fast photodiode, a PHD-400 (Becker and Hickl, Berlin, Germany), to send a time decay synchronization pulse to the SPC-830. The SPC- 830 was set for a total acquisition time of ____ seconds/minutes, ___ time channels, and receiving scanning synchronization information from the LSM410 for image construction of ___ pixels by ___ pixels. The scan rate of the LSM410 was carefully chosen for timing of excitation of each pixel based on the repetition rate of the laser.

Recommended Reading

  • Becker, Wolfgang. Advanced Time-Correlated Single Photon Counting Techniques. Berlin: Springer, 2005.

Laser Scanning Confocal Microscope

The LSCM is primarily used for imaging fluorescent samples. Organelles and proteins in cells or tissues are imaged using native fluorescence or by adding fluorescent tags. The LSCM is based on point scanning approach to image formation whereby the information for each pixel is obtained sequentially instead of the broad area excitation approach used for wide-field imaging. A laser is used for selective excitation of a fluorophore and an optical bandpass filter limits the light reaching the detector. The LSCM is useful for three-dimensional reconstruction of data. Spatial resolution ~ 300 nm is achievable with objectives of numerical aperture of 1.3.

Specifications

Excitation wavelength multi-line argon ion laser (457, 488, 514 nm)
three HeNe lasers (543, 594, 633 nm)
Filters Bandpass
Longpass
Objective lens 10x/0.3NA
20x/0.75NA
40x/1.3NA
63x/0.9NA (dipping objective)
100x/1.3NA
Simultaneous excitation 2 channels
488 nm and 594 nm (recommended)
Acquisition Time sequence
Differential interference contrast Using 10x and 100x objective lenses
Advances capabilities Objective inverter (LSM Technologies) for upright microscopy applications Widefield imaging, CCD camera as detector (broadband excitation and emission) ~405nm excitation (achieved using the ultra-fast laser system and SHG) FCS2 Temperature control chamber (contact Dr. Jim Xiang for use of the FCS2) Two-Photon Excitation Microscopy Fluorescence Lifetime Imaging Microscopy

Settings

User may excite fluorescence of elements within the sample, or add fluorescent tags. Excitation energy is selected by use of one, or two lasers simultaneously. Sequential colour-imaging, and z-sectioning procedures may be used to acquire images. Widefield images may be collected for comparison.
This system uses the Verdi / Mira / Pulse Picker / Harmonic Generator suite.

More Information

General statement for publications

You may use the following as a guideline for publication details. It is your responsibility to meet publisher requirements prior to submitting. The images were acquired using a LSM410 (Zeiss, Thornwood, NY) upgraded by LSM Technologies (Etters, PA). An excitation wavelength of ___ nm from a multiline argon ion laser / HeNe laser was used and a bandpass/longpass emission filter (part # and manufacturer) were used for imaging. The total image acquisition time was ___ seconds and the pinhole was set to _____ airy units. The step size for three-dimension reconstruction was ____ micrometers and a total of ___ images were acquired.

Recommended Reading

  • Conn, P. Michael, ed. Confocal Microscopy. San Diego: Academic Press, 1999.
  • Diaspro, Alberto, ed. Confocal and Two-Photon Microscopy, Foundations, Applications, and Advances. New York: Wiley-Liss, 2002.
  • Paddock, Stephen ed. Confocal Microscopy (Methods in Molecular Biology Volume 122). Totowa, NJ: Humana Press, 1998.
  • Pawley, James B., ed. Handbook of Biological Confocal Microscopy, 2nd edition. New York: Plenum Press, 1995.
  • Periasamy, Ammasi and Richard N. Day, eds. Molecular Imaging: FRET Microscopy and Spectroscopy. New York: Oxford UP, 2005.
  • Yuste, Rafael, et al., eds. Imaging Neurons: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Lab Press, 1999.

Two-photon excitation microscopy

Two-photon excitation microscopy is best at reducing out-of-focus excitation, reducing the risk of bleaching a volume of the sample before the imaging process is completed. The two-photon process requires high photon density, which is achievable at the focus point of the objective with ultra-short light pulses. TPEM may reduce photo-toxicity and help to improve live cell imaging. The longer wavelength range required for the technique can make it possible to image thicker samples compared to excitation in the ultraviolet and visible ranges.

Specifications

Light Source Ultra-fast Mira laser in fs mode
Excitation wavelength 700 to 1000 nm
Pulse repetition rate 76 MHz tunable to 4.75 MHz
Objective lens 63x/0.9NA, 2.2 mm working distance
Detection Descanned (2 detectors with emission filter wheel)
Non-descanned

Settings


This system uses the Verdi / Mira / Pulse Picker / Harmonic Generator suite.
Pulse width and repetition rate are selected to avoid bleaching the sample.

More Information

General statement for publications

You may use the following as a guideline for publication details. It is your responsibility to meet publisher requirements prior to submitting. The images were acquired using a LSM410 (Zeiss, Thornwood, NY) upgraded by LSM Technologies (Etters, PA) for two-photon excitation. The excitation wavelength was ___ nm from a femtosecond pulsed Mira 900-D laser system (Coherent, Santa Clara, CA) and a ____ nm bandpass/longpass emission filter (part # and manufacturer) were used for imaging. The image was acquired using a descanned/non-descanned light path. The total image acquisition time was ___ seconds and the pinhole was set to _______ airy units. The step size for three-dimension reconstruction was ____ micrometers and a total of ___ images were acquired. Note: Emission filter in non-descanned path: E700SP-2P, Chroma, Rockingham, VT

Recommended Reading

  • Diaspro, Alberto, ed. Confocal and Two-Photon Microscopy, Foundations, Applications, and Advances. New York: Wiley-Liss, 2002.

Verdi/Vitesse / RegA / OPA

This system is used as a light source for the Up-Conversion Fluorescence Lifetime instrument.

Specifications

Excitation wavelength 800 nm
OPA 480-700 nm and 1100-1600 nm
Repetition rate 800 Hz to 300 kHz
Measurements Decay lifetime
Rise-time
Cross-correlation FWHM 200 fs
Optical imaging
Simulated radiation testing

Settings

Repetition rate and pulse width are tunable for time-correlated measurements, and control damage to the sample.

More Information

General statements for publications

You may use the following as a guideline for publication details. It is your responsibility to meet publisher requirements prior to submitting. Vitesse / RegA / OPA The laser system (Coherent, Santa Clara, CA) was a Vitesse Duo pumped RegA 9000 producing 800 nm light at a rate of ____ kHz for an average power of ____ mW. The RegA pumped the OPA9400 to produce ____ nm (____ mW power).

Verdi / Mira / Pulse Picker / Harmonic Generator

This system is used as a light source for the TCSPC and LSCM. It is used for two-photon excitation imaging and fluorescence lifetime measurements (FLIM and TCSPC).

Specifications

Wavelength 700 to 1000 nm
2nd harmonic: 360-500 nm
3rd harmonic: 240-330 nm
Repetition rate BLAH300
76 MHz tunable to 4.75 MHz
Measurements Fluorescence Lifetime Imaging Microscopy (FLIM),
Laser Scanning Confocal Microscope (LSCM),
Time-Correlated Single Photon Counting (TCSPC),
Two-photon Excitation Microscopy (TPEM)

Settings

Repetition rate and pulse width are tunable for time correlated measurements, and control damage to the sample. Accessories for experiments include: LSM 410 confocal microscope, SPC830 time counting board, FLIM

More Information

General statements for publications

You may use the following as a guideline for publication details. It is your responsibility to meet publisher requirements prior to submitting. Verdi / Mira / Pulse Picker / Harmonic Generator The laser system (Coherent, Santa Clara, CA) was a Verdi V-10 pumped Mira 900-D tunable Ti:Sapphire laser set in the pico/femtosecond mode at ___ nm. The repetition rate of the laser beam was set to ___ Hz using a pulse picker, PP9200. The wavelength was modified to ___ nm using a harmonic genrator, HG9300. The average output power was ____ mW.