Microtime 200 rapidFLIM STED

Microtime 200 STED

Fluorescence Lifetime Imaging (FLIM) adds another dimension to conventional intensity based imaging methods. FLIM is not affected by fluctuations in the fluorescence intensity and permits to discrimination between fluorophores with similar emission spectra (and from auto-fluorescence. It can be used to probe local environmental conditions (e.g., pH value), to determine ion concentrations, to study intracellular signal transduction or to distinguish between different tissue components.

Microscope body: Inverted
Motroised Stage: No
Temp Control: Stage top
CO2: Stage Top
Software: SymPho time

 

Common Applications

rapidFLIM

rapidFLIM measurements enable the imaging of dynamic processes via fluorescence lifetime imaging (FLIM). This new approach allows for fast FLIM acquisition up to several frames per second for 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.

STED

STED microscopy uses the principle of stimulated emission depletion. After exciting fluorophores in the laser focus, a second, donut-shaped focus of a laser with longer wavelength is used to actively de-excite the molecules in the periphery via stimulated emission. In the MicroTime 200 STED, the donut is created using a so-called EASYDonut phase plate. It is inserted into the beam path and changes the STED laser focus to a donut-shape, while leaving the excitation laser unaffected. This simple implementation makes spatial alignment of the two laser beams, which emerge from the same optical fiber, unnecessary and yields a spatial resolution of less than 50 nm. Due to the time-resolved data acquisition principle of the MicroTime 200 STED, it is additionally possible to apply time-gates to all measured data. This gated STED (or gSTED) approach leads to an enhanced resolution in images and a reduced observation volume for FCS.

FCS

Fluorescence Correlating Spectroscopy allows for determining diffusion coefficients and concentrations of fluorescently labelled molecules at nanomolar concentrations, both in vitro and in live cells. It is mostly useful for indirect studies of molecular activity in plasma membrane, in cytosol and in nucleus via following relative changes in diffusive behavior and/or concentration. This technique can also determine the mobility of molecules (e.g. movement, aggregation, association, dissociation, and conformational changes), help studying the intracellular dynamics (e.g. diffusion, active transport) and determine the stoichiometry of complexes

FRET

FRET (Lasers Förster Resonance Energy Transfer or Fluorescence Resonance Energy Transfer) is a technique using two carefully selected fluorophores that when in close enough proximity (10–100 Å), and suitable orientation, energy is transferred nonradiatively from the excited donor fluorophore to another acceptor fluorophore. It is largely used to investigate inter- and intramolecular distances, between one or more molecules. It can also monitor conformational changes of molecules and environmental parameters (e.g., pH) via FRET sensors.

Objectives

Magnification

Numerical Aperture

Corrections

Immersion

20x

0.4

UPLFLN

air

60x

0.64

UPLSAPO

Water

100x

1.4

UPLSAPO

Oil

Light Sources

Pulsed diode lasers with repetition rates up to 80 MHz:

443 nm 479 nm 510nm 638 nm

 

For STED

  • 595 and 640 excitation, 765 nm (STED depletion laser)

Scanning

Galvo scanner FLIMbee:

  • Image size ranging from 10 × 10 to 2048 × 2048 pixel
  • Maximum field-of-view: 250 × 250 µm (60x objective)
  • Up to 2.6 kHz line frequency (bi-directional scanning), 5.2 FPS @ 512 × 512 pixel
  • Optional z-axis control, e.g., for z-stacks (piezo-based, up to 100 µm)
  • Pixel dwell times from 0.5 µs up to 1 s

Detectors

rapidFLIM: 2 channel detection with 2x PMA Hybrid detector

FLIM: 4 Channel detection with 2x PMA Hybrid detectors and 2x SPAD detectors

STED: 2 channel detection with  red-sensitive SPAD detection