UV-VIS spectrophotometer JASCO V-670 This spectrometer is equipped with dual-grating, dual-detector design to fully cover the range to 2700 nm. The V-670 double-beam spectrophotometer utilizes a unique, single monochromator design covering a wavelength range from 190 to 2700 nm (3200 nm option). The monochromator features dual gratings (automatically exchanged): 1200 grooves/mm for the UV/VIS region; 300 grooves/mm for the NIR region. A PMT detector is provided for the UV/VIS region and a Peltier-cooled PbS detector is employed for the NIR region. Both gratings and detector are automatically exchanged within the user selectable 800 to 900 nm range
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Fluorescence lifetime spectrometer Edinburgh Instruments FL920 The FLS920-t is a modular, computer controlled fluorescence lifetime spectrometer. Based on an L – geometry hardware configuration, the FLS920-t utilizes the technique of Time Correlated Single Photon Counting (TCSPC) to measure time resolved luminescence spectra and luminescence lifetimes spanning the range from 100 picoseconds to 10 microseconds, with the accuracy and the resolution that only the technique of TCSPC can offer.
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Streak camera Streak camera is a device used to measure ultra-fast fluorescence. Several of the many advantages against other fluorescence measuring systems is that streak camera can register the entire dataset in a very short time and it has very high time resolution – up to 2 ps. The principle of operation of the camera is similar to analog oscilloscope. The main concept of the camera is to bend the trajectory of the electrons with a varying electric field, which increases over time. Thus, the last electrons which arrive at the camera get bent the most. Since only one coordinate of the camera is used for time resolution (electron trajectory deviation), the other coordinate can be used to get spectral information. This allows gathering all the spectra with extremely good time resolution in one sweep. Streak camera is extremely useful if the sample is degrading fast or if we need to measure only the start of fluorescence decay (50-200 ps). Our system uses 3W 1030nm "Pharos" as the main oscilator and "Hiro" harmonics genereator to produce laser pulses of ~80ps and 1030nm, 515nm, 343nm or 257nm. Frequency of the main oscilator (76MHz) can be reduced to 10kHz using Pockels cell.
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2D spectrometer The set-up is currently under construction. More info soon.
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Pump-probe Pump-probe technique is probably the most widely used variation of ultrafast spectroscopy technique. Its concept is very simple and uses two short laser pulses: an intense pulse, ‘the pump’, which induces a photoreaction in the studied system and a weaker pulse, ‘the probe’, which monitors the corresponding change in the absorption spectrum of the sample. The delay of the probe pulse can be varied with respect to that of the pump and thus, the corresponding absorption change in the samples can be recorded at different time instances after the arrival of the pump pulse. Absorption change dynamics shows various processes that happen during the relaxation of photoexcited molecules. The spectrometer is based on an amplified femtosecond laser Pharos 10-600-PP (Light Conversion Ltd.) generating 290 fs duration pulses at 1028 nm at a 200 kHz repetition rate. Optical parametric generator Orpheus PO15F2L (Light Conversion Ltd.) is used for tuning the wavelength of the pump pulse. We use a 2 mm thick sapphire or calcium fluoride plate in order to generate a white light continuum for probing differential absorption.
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CARS microscope CARS microscopy is label free technique which contrast mechanism based on intrinsic molecule vibrations enabling chemical selective imaging. CARS is orders of magnitude more sensitive than spontaneous Raman. Nonlinear nature of CARS signal provides 3D imaging possibility without using confocal microscopy configuration. The Anti-stokes signal is blue-shifted and therefore is free from one-photon fluorescence background. Our home-made CARS microscope setup includes a dual wavelength laser source and a scanning microscope. The laser consists of a picosecond frequency doubled Nd:YVO4 pump laser with the pulse repetition rate of 1 MHz equipped with a traveling wave optical parametric generator (OPG) which enables probing the 700 – 4500 cm-1 range of vibration frequencies. In our scheme for CARS implementation a signal radiation from the OPG (6 ps) is coupled with fundamental wavelength (1064 nm) and used as Pump and Stokes excitation beams, respectively.
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Single molecule microscope with spectrometer Our single-molecule fluorescence microscope - spectrometer allows us to acquire signals (intensity over time or fluorescence emission spectrum) of individual fluorescent molecules either immobilised on the surface or freely diffusing in solution. The setup can be operated in two excitation-detection configurations: By exciting a wide sample field in total internal reflection (TIR) mode and detecting fluorescence signal from a number of individual molecules in parallel. Fluorescence image is split into two spectral components. The temporal resolution is limited, however, to approximately a video rate. The main advantage of this detection mode – the possibility of observation of a large number of molecules in parallel. It also allows us to monitor single-molecule FRET signals [1]. By exciting individual molecules in confocal mode and monitoring fluorescence from separate molecules one at a time. In this mode, it is possible to acquire full fluorescence spectra and achieve better temporal resolution. Temporal resolution for spectral acquisition is a few milliseconds, whereas if opting for the detection of only two spectral components, temporal resolution is up to a few tens of nanoseconds. Moreover, in the confocal mode, it is possible to collect fluorescence bursts from molecules diffusing freely in solution. This way vast data statistics are collected relatively fast. By analysing such data with correlation analysis, it is possible to determine, for example, the efficiency of the excitation energy transfer inside the molecule of interest. Recently our setup was upgraded with an alternated laser excitation (ALEX) [2] and a beam rotation in the back focal plane of the objective, which allows us to achieve more even wide field illumination.
References: 1. D. Rutkauskas, M. Petkelytė, P. Naujalis, G. Sasnauskas, G. Tamulaitis, M. Zaremba, V. Šikšnys. Restriction enzyme Ecl18kI-induced DNA looping dynamics by single-molecule FRET. Journal of Physical Chemistry B 118, (2014), 8575-8582. 2. Kapanidis AN, Lee NK, Laurence T, Doose S, Margeat E, Weiss S. Fluorescence-aided molecule sorting: analysis of structure and interactions by alternating-laser excitation of single molecules. Proc Natl Acad Sci. 2004;101: 8936-41.
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Glove Box
This glovebox (Plas-Labs basic glove box) is engineered to fit general laboratory isolation applications. It can be easily modified for specific uses. Unit is very handy for isolating sensitive research studies from a hostile exterior environment. This unit is compact, portable, lightweight and self-contained. Main features:
- Clear one piece acrylic top,
- Double layered, closed cellular neoprene gaskets for airtight seal between top and base,
- Adjustable SS clamps compensate for wear
- 8 in. O.D. ports with ambidextrous Hypalon® glove with SS O-rings
- Transparent transfer chamber (12 L × 11 in. I.D.) with adjustable vacuum gauge
- Four purge valves, two on transfer chamber, two on main chamber
Multiple electrical outlet strip, CE UL and CSA approved
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TEA_MT - Intensity versus time trace of fluorescent spots extraction and analysis software Author: Marijonas Tutkus
General info
- The analysis package is written in “Igor Pro” language.
- This software can only be distributed upon direct request to the author!
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- The software was written so that all of the daily single-molecule data analysis tasks are accessible at one place:
- fluorescent spot detection,
- co-localization of the fluorescent spots from 2 channels,
- intensity versus time traces extraction for selected type of fluorescent spots,
- filtering of traces
- normalization of traces to an excitation field
- intensity change point detection in the traces,
- characterization of detected states,
- manual selection of extracted traces,
- 2D histogram plotting of the analysis results.
- It can handle most of the single-molecule data formats.
- The software has internal memory for entered parameters, the size of windows and their positions.
- Each part of the program gives output as tables of igor binary format. There is an additional function in the main panel to convert them into delimited text format.
- Software development was under MacOS, therefore, it may happen that Windows users find some small problems.
- I am making updates of software constantly, therefore, feedback is highly appreciated:
marijonas.tutkus@ftmc.lt
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