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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 with Alba-STED for FLIM/FFS:

• 1p or 2p confocal images
• FLIM Fluorescence Lifetime Imaging (FastFLIM)
• PLIM, Phosphorescence Lifetime Imaging
• Polarization images
• Fluorescence Fluctuations Spectroscopy (FCS, FCCS, PCS, FLCS)
• RICS, N&B, Scanning FCS
• Burst Analysis
• FRET efficiency determination

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	VistaVision by ISS
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.
FastFLIM	4 separate input signal Dynamic range: up to 15 million counts/s per channel Lifetime range: from picosecond to second
Microscope	Inverted or Upright
Objectives	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
Stages	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
Dimensions	885 mm (L) x 600 mm (W) x 330 mm (H)
Weight	40 kg

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)
Superresolution	STED
Single Molecule Module	Burst Analysis FRET and Correlation Methods PIE-FRET Methods

Schematic Diagram of Alba-STED for FLIM/FFS

Measurement Examples from Alba-STED for FLIM/FFS

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

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

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

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.

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

Non Descanned Detection (NDD) Port

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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.

Laser Scanning Transmitted Detection Module with a Photon Counting PMT

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T-PMT is a transmitted detection module with a photon counting PMT that works with inverted microscopes.

The module is installed beside the microscope transmission illumination lamp and features a manual slider for selecting between the PMT and the lamp illumination.

Microscope Stages

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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.