Active Illumination

Active Illumination (AI) describes a rapidly evolving range of optical techniques with an increasing impact on scientific enquiry and experimentation. All AI techniques target optical radiation at user-defined region(s) of the specimen and use optical imaging to observe, and often to measure, the impact of the “perturbation”.

Model Description
Mosaic
Simultaneously illuminate multiple regions of interest.
MicroPoint
Diffraction limited illumination from a pulsed dye-laser.
FRAPPA
Unique design for galvo-scanned photobleaching and activation.

Overview

Active Illumination, or AI, describes a rapidly evolving range of optical techniques with an increasing impact on scientific enquiry and experimentation. AI has developed over the last two decades alongside the revolution of fluorescent proteins in biology (ref 7, 8), the instrumental and technological developments of confocal laser scanning microscopy (CLSM), solid state light sources (lasers and LEDs), fast galvo and optical MEMS technology, and of course the ubiquitous personal computer. We include among these techniques:

  • Photoactivation and switching
  • Constrained or adaptive illumination
  • FRAP
  • Ablation, cutting and marking
  • Uncaging of caged compounds
  • Optogenetics e.g. channelrhodopsin2

Mosaic

Mosaic & Mosaic Duet

Simultaneous illumination of multiple regions of interest in real time and with zero delta acquisition time
Mosaic is a patented instrument platform built around MEMS Digital Mirror Devices (DMD). DMDs were developed at Texas Instruments in 1987 and are now in widespread use in digital projectors and other display devices. The DMD comprises an array of individually addressable micro-mirrors that can be switched “on and off” (tilted) with MEMS “hinge” elements. DMD arrays contain hundreds of thousands to millions of micro-mirrors.

  • Unlimited flexibility in shape, size, complexity of illumination mask
  • Simultaneous illumination of multiple regions of interest
  • Precise illumination of areas of interest that protects target specimen and fluorophore
  • Zero delta acquisition time for true digital excitation
  • Complementary illumination option enables on and off control

Mosaic exploits DMD in a proprietary programmable platform, integrated with scientific light sources including lasers, LEDs and arc lamps, and operates from 360 to 800 nm. It is offered with a range of high performance microscope adapter optics and can be integrated with CLSM, spinning disk and wide field imaging modalities.

High speed frame switching (60 Hz) makes Mosaic suitable for many dynamic applications including bleaching, uncaging, photoswitching, optogenetics and constrained illumination. Variable intensity distributions can be achieved by rapid gating of mirror patterns.

Mosaic has a unique capability to illuminate in parallel an arbitrary number of complex regions (sometimes called “zero delta t”) that sets it apart from galvo-based devices and makes it especially attractive for uncaging, photoswitching and light activation. It is a unique tool for the study of optically stimulated intra and inter-cellular activity in neuroscience and physiology, as well as for function-structure studies with photoswitching fluorescent proteins. Mosaic uses dichroic coupling to the microscope light path and is therefore capable of simultaneous stimulation and imaging.

Mosaic is a patented instrument platform built around MEMS Digital Mirror Devices (DMD). DMDs were developed at Texas Instruments in 1987 and are now in widespread use in digital projectors and other display devices.

Specifications Summary
Transmission 360 nm to 800 nm
Intensity stability Absolute
Extinction ratio > 1000:1
Minimum resolvable spot Diffraction limited with 100x objective
Optical pixel rise / fall time < 1 µs
Minimum optical pulse width 60 ms external trigger 100 msec internal trigger
Maximum frame repetition rate 600 frames / sec
Certification CDRH IIIb (if fitted with a laser source)
Features & Benefits
Unlimited flexibility in shape, size, complexity of illumination mask
Simultaneous illumination of multiple regions of interest
Precise illumination of areas of interest that protects target specimen and fluorophore
Zero delta acquisition time for true digital excitation
Complementary illumination option enables on and off control in optogenetics studies
Longest lifetime and lowest maintenance with rugged semiconductor device

MicroPoint

MicroPoint

Simultaneous and precise illumination and ablation

MicroPoint provides a flexible and field-proven tool for photo-stimulation. Supplied with a patented compact, pulsed nitrogen pumped tunable dye laser it is capable of ablation, bleaching and uncaging over a wavelength range of 365 to 656 nm. Broad wavelength range and energy control allow MicroPoint to be optimized for a wide range of scenarios. More than 20 wavelengths can be utilized with available dye resonator cells, while appropriate dichroic filter sets enable simultaneous imaging and photo-stimulation of the specimen. MicroPoint is supplied with a UV-Vis imaging quality Epi illumination adapter for both current and previous generation microscopes from Leica, Nikon, Olympus and Zeiss.

MicroPoint systems consist of a wavelength tuneable pulsed laser, coupling optics, beam steering optics, a microscope adapter, a selection of beam splitters and interference filters, and a motorized or manually driven optical attenuator to adjust spot size and power. There are three MicroPoint versions available:

MicroPoint Manual

Angular and spatial alignment of the illumination at the sample target is manually controlled via a 2-axis joystick. This manual version can be upgraded in-situ to provide galvo or bluetooth control.
MicroPoint Galvo
Galvanometer based beam steering is provided through PC control, enables precise and repeatable laser ablation and/or illumination in synchronization with other experiment parameters.
MicroPoint Bluetooth
In laser marking or circuit isolation applications a computer is often unnecessary, the advanced and automated features of the MicroPoint can be controlled through the handheld PDA interface.

Specifications Summary
Transmission 365 nm to 656 nm
Spectral bandwidth 4 nm FWHM
Transmission variation 0.1%-100%
Resolvable spot size Near diffraction limited
Average power 750 μW, 15 Hz / 50 μJ
Peak power 12 kW
Pulse width 3 to 5 nsec
Pulse repetition rate 0 to 15Hz
Certification CDRH IIIb
Features & Benefits
Simultaneous laser delivery, microscope viewing and image acquisition
Low maintenance with fiber optic delivery that maintains alignment when system is moved
Quick set-up with manual beam positioning or automatic pattern generation
User control of ablation and illumination plane provided by z-axis telescope
Precise control of energy provided by motorized variable attenuator slide

FRAPPA

FRAPPA

Share all laser lines for photo stimulation and imaging

FRAPPA is a galvo scanning instrument, named by conjoining acronyms for fluorescence recovery after photobleaching (FRAP) and photoactivation (PA). FRAPPA has a unique switching design that allows it to be configured in the imaging path. In bypass mode it acts as a relay optic, projecting an image to the detector, while in scanning mode it acts as a laser scanner, targeting user-defined regions of the specimen. This “in-line” configuration allows it to utilize the same wavelength for imaging and photo-stimulation.

Andor’s FRAPPA uses a dual galvanometer scan head to provide a computer-steered laser beam delivery system. It can be configured in line with a CSU and/or camera and operates in two modes CSU By-Pass Mode and Frap Mode.

  • Bypass” mode provides 1:1 relay imaging for “in-line” configuration
  • “FRAPPA” mode performs laser scanning via imaging C-port
  • Diffraction limited spot size ~0.6 μm @488 nm FWHM
  • “In-line” operation enables use of all laser lines for FRAPPA actions
  • Mode switching in < 10 ms

The cost of “in-line” operation is sequential execution of imaging and photostimulation, but the switching speed is optimized at ~10 ms to minimize its impact. FRAPPA utilizes galvo technology, has a single laser input and is designed to deliver a diffraction limited spot from CW and pulsed lasers in the wavelength range 400-800 nm.

These features make it an attractive option for photobleaching, switching and activation as well as DNA damage studies.

Specifications Summary
Transmission 400 to 800 nm
Resolvable spot size Near diffraction limited
Laser inpute Single mode fiber FC
Beam power Up to 2 Watts optical
Intensity control AOTF 0.1 to 100%
Laser compatibility Pulsed or CW
Pixel dwell time (minimum) 20 μs
Certification CDRH IIIb
Features & Benefits
“Bypass” mode provides 1:1 relay imaging for “in-line” configuration
“FRAPPA” mode performs laser scanning via imaging C-port
Diffraction limited spot size ~0.6 μm @488 nm FWHM
“In-line” operation enables use of all laser lines for FRAPPA actions
Mode switching in < 10 ms
Integrated control with iQ2 software provides “point-and-shoot” and protocol modes
Arbitrary multi-region scanning of points, rectangles and polygons
Share imaging lasers with Andor’s unique multi-port adapter
Active blanking output for ALC integration
Product Components
FRAPPA is available in a single model for “in-line” or dichroic configuration. With C-mount input and output, FRAPPA is easy to configure with the “in-line” setup, FRAPPA uses the bypass mode for imaging and the scanning mode for photo-stimulation. Its output can be coupled directly to a camera or a confocal scanner e.g. CSUX.
FRAPPA can be used for simultaneous photo-stimulation and imaging when configured on a separate microscope C-port and used with a dichroic mirror. FRAPPA is compatible with visible to near IR lasers operating in CW or pulsed mode (400 – 800 nm) for photostimulation.
Left: In bypass mode the FRAPPA acts as a 1:1 relay imaging system. As shown by the orange beam path the X galvo mirror is positioned at the instrument pupil and completes a 4f imaging path..
Right: In FRAPPA mode, the laser beam is injected from the laser input port and is steered to the Y galvo. The X galvo is positioned to scan the laser beam into the microscope to execute the defined photo-stimulation.

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