VisiTech HAWK - Combined Multi-Point Imaging and Single Point Illumination

Location: HWB room 3/51
Contact: Dr Kees Straatman


Download:
Startup guide.
Bleaching guide.
VoxCell software user guide
.

The latest purchase of AIF is a VisiTech HAWK system; an Infinity 3 confocal microscope (see: http://www.visitech.co.uk/vt-infinity3.html) with bleaching/photo activation laser included. This system was funded by the University Equipment Fund and will boost our live cell imaging capability as well as imaging of larger tissue sections like drosophila brain (see figure 1).

Drosophila brain scan on VisiTech Infinity3 confocal laser microscope

Figure 1; An extended depth of field projection from the right hemisphere of a 3 days old male Drosophila showing Pigment Dispersing Factor (PDF) expression in Lateral Ventral neurons imaged on the VisiTech Infinity3 system using a 20x objective. The image has 77 z-sections and was taken in 84 seconds (Dr Giorgio Fedele). 

The system is build on an inverted NikonTi-E microscope stand. It allows for fast confocal imaging with speeds up to 1000 fps. However, in most cases the number of photons released by the sample will be the limiting factor for acquisition speed.

Super resolution reconstruction

The VisiTech system can also be used to acquire images for super resolution reconstruction using Super-Resolution Radial Fluctuations calculations. This is possible for both fixed and live samples using any of the colours you would normally use for this system. Please see this page.

Specifications

The system uses a Hamamatsu C11440 -22CU Flash 4.0 V2 sCMOS camera for image acquisition. The system still has to scan the sample and uses a fixed scan rate of 360 Hz and therefore the actual exposure time at the sample has to be divided by 2.8. However, there is a little more to this than just the scan rate. The system also has to speed up and slow down the scanning 2D plate and the laser power is adjusted accordingly so that the total illumination at each point at the sample plane remains constant. The pinhole sizes and positions do not affect this value, each position on the sample is exposed equally.

Objectives

Default objectives on the system:
10x/0.3 Pan Fluor DIC L/N1; WD = 16 mm; pixel size: 0.6472 x 0.6472 µm/pixel
20x/0.75 Plan Apo ƛ; WD = 1.0 mm; pixel size: 0.3240 x 0.3240 µm/pixel
100x/1.45 Plan Apo ƛ; WD = 0.13 mm; pixel size: 0.0647 x 0.0647 µm/pixel
Other objectives can be added on request.

Currently extra on the system:
60x/1.4 Plan Apo VC; WD = 0.13 mm; pixel size: 0.1082 x 0.1082 µm/pixel

Lasers

The system is equipped with four laser lines:
405 nm - Solid state laser diode (SSLD)
488 nm - SSLD
561 nm - Optically pumped semiconductor (OPSL)
642 nm - SSLD
Laser lines are available for acquisition and photoactivation/bleaching experiments (using hawk unit; http://www.visitech.co.uk/vt-hawk.html).

Filters

The specification for the quad dichroic mirror from Envin Scientific in the system is below:
VisiTech dichroic

The system has 3 options for emission filters.

1. The system has a Chroma ZET405/488/561/640m quad band emission filter. This will allow fast imaging of all channels available as no emission filters have to be changed. However there is a change of bleed through from one channel in the other channel. See specs below:

quad emission

2. The system has a Cairn OptoSpin IV filter wheel with emission filters. Using the individual emission filters will give the lowest change of bleed through. Available filters:
440/42
525/50
605/70
700/75

3. The system has also a Cairn Optosplit II image splitter which allows simultaneous imaging of GFP and RFP and split this signal into two images on the camera chip. It uses the dichroic T565 LPXR-UF2 and emission filter combination ET525/50 and  ET605/70, see figure below:

Splitter figure

VisiTech Analysis Action Bar

The VisiTech acquisition software VoxCell saves the data in the *.ome.tiff file format. This format is developed by the Open Microscopy Environment consortium to standardize microscope formats. To open ome.tiff files you can use ImageJ with the Bio-Formats plugin installed or you can use Fiji (Fiji Is Just ImageJ) which comes with Bio-Formats included. To make analysis of the images easier I have created the VisiTech Analysis Action Bar.  (More soon) 

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