The instrument for quantitative cell biology at single-molecule detection.

Stimulated Emission Depletion (STED) is a powerful microscopy technique that allows for the observation of fluorescence structure with spatial resolution below the diffraction limit. The Alba-STED uses the pulsed excitation and pulsed depletion approach (pSTED) in combination with the digital frequency domain fluorescence lifetime imaging (FastFLIM) to record the time-resolved photons which allows for an increase in the image resolution and the separation of two labels with the same excitation wavelength.

Key Features of Alba-STED for FLIM/FFS:

  • pSTED (Pulsed excitation and pulsed STED)
  • FastFLIM for time-resolved pSTED acquisition
  • Improved image resolution using the phasor plot
  • Dual-label excitation
  • Fast image acquisition (dwell time: 0.2 µs)
  • High dynamic range (signal up to 60 million counts/s)

Measurements for Alba-STED for FLIM/FFS

FFS Measurements
  • Fluorescence Correlation Spectroscopy (single- and cross-correlation)
  • Photon Counting Histogram (PCH)
  • FFS measurements at target XYZ locations in an image
  • FLCS, Fluorescence Lifetime Correlation Spectroscopy
  • Scanning FCS
  • Number & Brightness (N&B)
  • Raster Image Correlation Spectroscopy (RICS)
Single-point Module Measurements
  • Intensity
  • Polarization
  • Kinetics
  • Lifetime
Imaging Module Measurements (Single plan and z-stack)
  • Intensity
  • Polarization
  • Ratiometric
  • FLIM
FLIM images (digital frequency-domain) (single plane and z-stack)
  • Acquired in digital frequency-domain (DFD). The routine acquires simultaneously a FLIM image and a steady-state image.
FLIM images time-domain (single plane and z-stack)
  • Acquired in time-correlated single photon counting (TCSPC)
  • STED
Single Molecule Module
  • Burst Analysis
  • FRET and Correlation Methods
  • PIE-FRET Methods

Specifications for Alba-STED for FLIM/FFS

Instrument Features
  • Individual pinholes on each acquisition channel
  • Computer-controlled selection of the pinhole variable aperture
  • Computer-controlled positioning of the pinhole in the imaging plane
  • Single-photon or multi-photon excitation
  • Up to four channel data acquisition
  • Auxiliary port for camera
Acquisition and Analysis Software
STED laser Pulsed, 775 nmAverage output power: 1 W
Pulsewidth: about 600 ps
Repetition rate: 0-100 MHz; or Ext. CLK
Beam quality: M2 < 1.1, TEM00
Amplitude noise: < 4.0% rms
Excitation laser Pulsed, 640 nmPulsewidth (at medium power): 40-90 ps
Repetition rate: 20, 50, 80 MHz; or Ext. CLK
Power (at 50 MHz): up to 5 mW
Laser Launcher
  • Models for 3-, 4-, 6-laser. Light is delivered to the microscope through a single-mode fiber optic.
  • 4 separate input signal
  • Dynamic range: up to 15 million counts/s per channel
  • Lifetime range: from picosecond to second
  • Inverted or Upright
  • Air objectives with 20X, 40X, 60X magnification and 1.5-8.1 working distances
  • Oil immersion objectives, 1.4 NA and 60X (standard); other aperture available
  • Water immersion objectives,1.2 NA 60X (standard), with coverslip correction (for 0.15-0.18 coverslip); other apertures available
  • Large distance movement (100x100x10 mm)
  • Stepper motor-controlled XYZ stage
  • Micro-distance movements
  • XYZ piezo-controlled stage, 100x100x50 µm with 5 nm step resolution.
Sample Holders
  • Microwell plates
  • Petri dishes
  • Coverslip
Light Detectors
  • GaAs PMT (Hamamatsu H7422P models)
  • Hybrid PMTs (Hamamatsu R10467U models)
  • SPADs
Image acquisition FLIM acquisition: 0.2 µs dwell time
OS Requirements
  • Windows 10, 64-bit
Power Requirements
  • Universal power input: 110-240 V, 50/60 Hz, 400 VAC
  • 885 mm (L) x 600 mm (W) x 330 mm (H)
  • 40 kg

Schematic Diagram of Alba-STED for FLIM/FFS

Measurement Examples from Alba-STED for FLIM/FFS

Confocal (green) vs. pSTED (red) images of 60-nm fluorescent beads, acquired by FastFLIM

Confocal (left) vs. pSTED (right) images of the actin labeled with the SiR dye in fixed glia cells, acquired by FastFLIM.

Increasing the resolution of pSTED images using the Phasor Plot

Confocal images of 60nm fluorescence beads (left); pSTED images (middle); sharpening the pSTED image using a binary filter based on the phasor plots (right).

Separation of the locations of two labels using FastFLIM and the Phasor Plot

Dual labels can be separated using pSTED and FastFLIM. Atto 647N and Atto 655 were used as labels; they both are excited by the 640 nm laser. The two dyes are first separated using the phasor plots, and then assigned with two different false colors (Atto 647N - yellow, Atto 655 - purple) to produce the processed and merged pSTED image of the two labels.

General Theory

A Novel Pulsed STED Microscopy Method Using FastFLIM and the Phasor Plots
Sun, Y.,Tortarolo G., Teng, K.-W., Ishitsuka, Y., Coskun, U.C., Liao, S.-C., Diaspro, A., Vicidomini, G., Selvin, P.R., Barbieri, B.
Proc. SPIE 10069, Multiphoton Microscopy in the Biomedical Sciences XVII, 100691C (February 21, 2017)

Options and Accessories available for Alba-STED for FLIM/FFS

Non Descanned Detection (NDD) Port

Detection through the Non Descanned Detection Port (NDD) is used in conjunction with multiphoton excitation; the fluorescence photons generated in the excitation spot of the laser are scattered back and collected right after the objective (without passing through the optics in the scanning path).

The Figure displays the NDD port on the Nikon Model Ti microscope coupled to the Alba. A raiser is introduced on the Nikon microscope above the epifluorescence port for connecting the NDD port and adding the filters-cartridge where the dichroic filters for the NDD detection are inserted. The detectors are mounted on an orthogonal mount complete of dichroic and filter holders. The NDD port uses either GaAs PMTs or hybrid detectors. The output of the detectors is diverted either to the data acquisition unit.

Microscope Stages

XYZ-stepper motor controlled stage for Microwell plates (8-, 96- and 384-wellplate)

The XYZ stage provides high resolution, highly repeatable, and fast controls for the X, Y, and Z position of the microscope stage; it utilizes crossed-roller slides, a high-precision lead screw, and zero-backlash miniature geared DC servomotors for smooth and accurate motion. Controlled through the USB port, it is the ideal stage when measuring samples in a microwell plate.

VistaVision includes protocols for the automatic measurement at single points (FFS, lifetime, polarization); the user can select the sequential measurements on all the wells; alternatively, a set of wells can be selected for the measurements.

XYZ Piezo-controlled Stage

The XYZ PZT is an actuated linear nanopositioning stage of exceptional resolution and stability. Manufactured by Mad City Labs, it is packaged for ISS to be utilized in the Alba under the control of VistaVision.

The 66 x 66 mm aperture in the stage center is ideal for applications involving transmitted beams, multiple probes or inverted optics. With its large distance of travel and high stability, the PZT is ideal for the most challenging microscopy and positioning applications. The PZT comes complete with position sensitive detectors for closed loop operation.

The stage is manufactured from a high performance Al alloy. actuators are preloaded within the PZT and supply the driving force for stage movement. The flexure hinges, which form the guidance mechanism, are cut into the stage using electric discharge machining (EDM). EDM is also used to form integrated amplifiers that increase the range of motion of the PZT actuators for the X, Y and Z axis. The PZT actuators are oriented perpendicular to the stage motion direction and within these amplifiers.