Апгрейд конфокального микроскопа


Конфокальный лазерный сканирующий микроскоп можно встретить почти в любой биохимической или клеточной лаборатории или ЦКП. Возможности такого микроскопа можно существенно расширить с использованием времяразрешенных методов.

Преимущества:

  • Lifetime FRET для количественных измерений эффективности FRET
  • Времяразрешенный имиджинг позволяет визуализировать такие параметры среды как концентрация ионов или pH
  • Время жизни флуоресценции не зависит от концентрации флуорофора
  • Разделение молекул с пересекающимися спектрами эмиссии по временам жизни
  • Сокращение числа необходимых детекторов — одного детектора достаточно для одновременного определения разных флуорофоров на основе их времени жизни, в том числе методом pattern matching
  • Отсечение упругого и рамановского рассеяния от флуоресценции, а также фонового сигнала по времени жизни
  • Время жизни как дополнительный параметр эксперимента повышает точность измерений

Апгрейд представляет собой набор дополнительных компонентов для микроскопа. Они просты в использовании, универсальны и не требуют большого числа настроек. Базовая установка включает три основных компонента: пикосекундный источник возбуждения, чувствительный к единичным фотонам детектор и устройство коррелированного по времени счета единичных фотонов (TCSPC).


Система возбуждения
  • Пикосекундные диодные лазеры с частотой повторов до 80 МГц внутри компактного блока лазеров.
  • Длина волны между 375 и 900 нм
  • Одно- или многоканальный лазерный драйвер
  • Опционально: внешний лазер (например сапфировый лазер)
  • Пикосекундное импульсное возбуждение на 560 нм с LDH-D-TA-560
Поддерживаемые LSM
  • Nikon: A1, C2+, C2, C1si
  • Olympus: FluoView FV3000, FVMPE-RS, FluoView FV1200 (MPE), FluoView FV1000 (MPE)
  • Scientifica:VivoScope, HyperScope
  • Zeiss: LSM 980, LSM 880, LSM 780, LSM 710
Обнаружение
  • До четырех параллельных каналов обнаружения
  • Descanned or non-descanned configuration
  • Подключение через оптические волокна к LSM
Детекторы
  • Hybrid-Photomultiplier Tubes
  • Single Photon Avalanche Diodes
  • Photomultiplier Tubes
Сбор данных
  • На основе метода подсчета единичных фотонов с временным коррелированием (TCSPC) в уникальном режиме с временной привязкой и временным разрешением (TTTR)
  • Одновременный сбор данных до четырех каналов
Программное обеспечение
  • SymPhoTime 64

(Eng)

rapidFLIM — Redefining standards for dynamic FLIM imaging

rapidFLIM measurements enable the imaging of dynamics in fluorescence lifetime. This new approach allows for fast FLIM acquisition with several frames per second as well as imaging of dynamic processes (e.g., protein interaction, chemical reaction, or ion flux), highly mobile species (e.g., mobility of cell organelles or particles, cell migration), and investigating FRET dynamics. More than 10 frames per second can be acquired, depending on sample brightness and image size. Unilamellar vesicles can be produced in a size regime ranging from giant (GUVs) to large (LUVs), and even to small (SUVs). The flexibility in membrane composition and possibility to introduce specifically labeled lipids increase the importance of unilamellar vesicles for studies in cell biology. Thus turning such vesicles a very powerful model to investigate e.g., membrane domain formation as well as lipid organization in membrane micro-domains. Until now, acquiring FLIM images took up to several minutes and due to the high mobility of GUV’s, imaging them accurately is difficult. Applying the rapidFLIM approach significantly decreases the acquisition time, allowing to record up to several frames per seconds. Thus even highly mobile GUVs can be precisely tracked. In this example, two fluorophore labeled lipids (C6-NBD-PC and N-Rhd-DOPE) were incorporated into GUVs. In non-phase separated GUVs, the lifetime of NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl) is strongly quenched (down to ~2 ns) due to FRET to the acceptor rhodamine. The video shown here contains 300 frames that were recorded with a frame rate of 5.6 fps.

Sample details:

  • GUVs with NBD and Rhodamin labeled lipids (no phase separation): DOPC + 0.5 mol % Palmitoyl-C6-NBD-PC + 0.5 mol % N-Rhd-DOPE
  • NBD fused to Palmitoyl-C6-NBD-PC (phosphatidyl choline)
  • Rhodamine fused to N-Rhd-DOPE (Di-oleyl-phosphatidyl-ethanolamin)

Set-up:

  • Excitation: 485 nm, 40 MHz
  • Long pass filter: 488 nm
  • 75 x 75 µm, 300 x 300 pixel, 1 µs/pixel
  • 300 frames with 5.6 fps

Lipid Order Determination Using Fluorescence Lifetime

FLIM measurements facilitate the differentiation between ordered and disordered membrane phases. The membrane dyes Laurdan and di-4-ANEPPDHQ can be used to image membrane order due to a spectral blue-shift in the fluorescence emission as well as a lifetime shift between the liquid-ordered and liquid-disordered phases. These images typically take the form of a normalized intensity ratio image known as a generalized polarization (GP) plot. Here, the known excited state photo physics is exploited via Time-Correlated Single-Photon Counting (TCSPC) to demonstrate GP contrast enhancement for these two probes. The image shows a GP plot of a Laurdan stained, fixed BAEC cell that combines lifetime and spectral changes. The plasma membrane at the cell surface shows higher order (red) compared to the inner cell compartments (blue).

Set-up:

Imaging specific newly synthesized proteins within cells by fluorescence resonance energy transfer

Shen L., Cai L., Liu J., Zhang S., Xu J.-J., Zhang X., Chen H.-Y. Chemical Science, Vol.008, p.748-754 (2017)

Reference to: LSM Upgrade Kit Related to: FLIM, FRET


Silver-coated nanoporous gold skeletons for fluorescence amplification

Lee M.-J., Yang W.-G., Kim J.H., Hwang K., Chae W.-S. Microporous and Mesoporous Materials, Vol.237, p.60-64 (2017)

Reference to: MicroTime 200, SymPhoTime Related to: FLIM


Intracellular fate of polymer therapeutics investigated by fluorescence lifetime imaging and fluorescence pattern analysis.

Panek J., Koziolova E., Stepanek P., Etrych T., Janouskova O. Physiological Research, Vol.065, p.217-224 (2016)

Reference to: LSM Upgrade Kit Related to: FLIM


A comparative study of the photophysics of phenyl, thienyl, and chalcogen substituted rhodamine dyes

Sabatini R.P., Mark M. F., Mark D.J., Kryman M.W., Hill J.E., Brennessel W.W., Detty M.R., Eisenberg R., McCamant D.W. Photochemical & Photobiological Sciences, Vol.015, p.1417-1432 (2016)

Reference to: PicoHarp 300, SymPhoTime


Mechanistic determinants of MBNL activity

Sznajder L.J., Michalak M., Taylor K., Cywoniuk P., Kabza M., Wojtkowiak-Szlachcic A., Matloka M., Konieczny P., Sobczak K. Nucleic Acids Research, Vol.044, p.10326-10342 (2016)

Reference to: LSM Upgrade Kit Related to: FLIM, FRET


Structure and dynamics of polyelectrolyte surfactant mixtures under conditions of surfactant excess

Hoffmann I., Simon M., Farago B., Schweins R., Falus P., Holderer O., Gradzielski M. The Journal of Chemical Physics, Vol.145, 124901 (2016)

Reference to: PicoHarp 300, LSM Upgrade Kit Related to: FCS


Investigating the DNA-binding interactions of small organic molecules utilizing ultrafast nonlinear spectroscopy

Doan P. Dissertation University of Michigan (2016)

Reference to: TimeHarp 100/200, LSM Upgrade Kit Related to: FLIM


Control of spontanous emission from quantum emitters using hyperbolic metamaterial substrates

Galfsky T. Dissertation City University of New York (2016)

Reference to: PicoHarp 300, SymPhoTime


Improving analytical methods for protein-protein interaction through implementation of chemically inducible dimerization

Andersen T.G., Nintemann S.J., Marek M., Halkier B.A., Schulz A., Burow M. Scientific Reports, Vol.006, 27766 (2016)

Reference to: MicroTime 200, LSM Upgrade Kit, SymPhoTime Related to: FLIM, FRET


Exploring the HYDRAtion method for loading siRNA on liposomes: the interplay between stability and biological activity in human undiluted ascites fluid

Dakwar G.R., Braeckmans K., Ceelen W., De Smedt S.C., Remaut K. Drug Delivery and Translational Research, Vol.007, p.241-251 (2016)

Reference to: LSM Upgrade Kit, SymPhoTime Related to: FCS