Andor iQ USER GUIDE

Add to my manuals
578 Pages

advertisement

Andor iQ USER GUIDE | Manualzz

Andor iQ USER GUIDE

3.3 Differential Spinning Disk (DSD) Calibration Wizard

The Andor Revolution DSD is a spinning disk structured illumination microscopy scanner or SD-SIM. Because it uses a structured illumination technique, it requires computer processing of two image halves to create a single confocal or epifluorescence result. The image processing required is simple enough, either weighted addition or subtraction, but in order to obtain high quality results the images must be very closely aligned e.g. 0.1 pixels. The DSD Calibration Wizard is described in section 3.3.2.

3.3.1 DSD and SD-SIM Principle of operation

The DSD is the first confocal instrument to successfully deliver high-performance confocal imaging using a cost-effective white-light source rather than lasers. The differential spinning disk (DSD) scanner can provide multifluorophore imaging from a single light source at a cost substantially lower than laser scanning instruments.

However, unlike previous white-light systems, DSD performance is comparable to that of point scanning systems, but with higher speed and lower photobleaching.

DSD is competitively priced and uses a patented optical detection principle known as

“aperture correlation” to reject out-of-focus light. Key benefits of the DSD scanner include frame rates up to 10 times those of most point scanning systems; optical sectioning with objectives ranging from 10× to 100×; operation with many fluorophores by selection of appropriate filters; and, as will be discussed, acquisition of confocal and epifluorescence images simultaneously.

In a DSD system, the spinning element comprises a single synthetic quartz disk supporting a thin layer of aluminum in which the structured illumination pattern (SIP) is created by photolithography. The aluminum SIP has a 1:1 mark to space ratio (half metal and half space), which means that approximately half of the light falling upon it is reflected (R) and half transmitted.

The disk is located at an image plane of the microscope optical system, so that an image of the SIP is projected into the specimen, and about half of the illumination

112

© Andor Technology

Andor iQ USER GUIDE intensity arrives at the specimen while the remainder is reflected back into the illumination pathway, where it is baffled to minimize background. In the detection pathway, the resulting fluorescence signal comprising what is in focus (confocal, or

C) and out of focus (wide field, or W) is imaged back onto the disk, where we make

use of its transmissive and reflective properties.

The fluorescent light transmitted by the DSD disk comprises the C signal plus about half of the W signal, while the light reflected from the disk comprises about half of the

W minus the C signal. In confocal terminology, the SIP is located in a conjugate

image plane and hence acts as both the confocal source and detection apertures.

However, as it is not a pinhole, we must undertake some further image processing to separate confocal and wide-field signals. The optical path is illustrated in Figure above - green is the excitation path and yellow and orange the fluorescence transmitted and reflected paths respectively.

From the description above, we can see that the transmitted and reflected signals can be approximated as follows:

T = 0.5 W + C; R = 0.5 W

C, (Eq. 1) and simple algebra shows us that

113

© Andor Technology

Andor iQ USER GUIDE

2C = T

R; W = T + R.

(Eq. 2)

As Equation 2 highlights, we need to collect both transmitted and reflected light signals (images) to compute the confocal signal. Furthermore, we can easily compute the wide-field (conventional epi-fluorescence) signal as well as the confocal. The figure below shows how the T and R images are projected onto the

CCD detector - transmitted image, T on the left and reflected image, R on the right.

The simple mathematics of Equations 1 and 2 hide one complexity in the principle of the DSD; i.e., the T and R images must be very well-registered for the calculations to provide high-quality images. Any misalignment between the two will result in registration noise, so an essential feature of image processing for the DSD is a highperformance, real-time registration algorithm. The DSD Calibration wizard delivers the parameters needed for the algorithm to be successful.

3.3.2 DSD Calibration Wizard

The DSD has an internal calibration mask which can be switched into the imaging path and used as a reference pattern to align the two image halves projected onto the image sensor. The calibration mask pattern has a unique high contrast pattern to ease image matching and the T and R images of the pattern follow the same optical paths as the T and R images from a real specimen. Consequently the pattern

114

© Andor Technology

Andor iQ USER GUIDE images are distorted in exactly the same manner. The distortion is wavelength dependent and so the registration algorithm is applied at all filter positions and parameters stored so that they can be applied during image capture based on the filter combination in use.

The DSD calibration wizard also measures the background signal (or image) for each wavelength, with the light source in the correct state and filter position. So the wizard corrects for optical distortion and background to provide the best quality result with the widest dynamic range.

The Wizard executes automatically from a single button click on the

Camera user interface as shown left.

Simply click on the “Calibrate

Alignment” button highlighted in red.

Note you can select and deselect the

Background Correction option by clicking on the blue highlighted checkbox. The only reason to deselect is for reasons of speed as the process adds about 90 seconds to the

Alignment step of about 2 minutes.

When the wizard is running it shows you a progress dialogs as shown below for

Registration and Background.

115

© Andor Technology

Andor iQ USER GUIDE

The DSD calibration Wizard should be run each day before starting work - the system will remind you if you do not do this. The wizard should also be run whenever a filter turret is changed. A filter turret change Wizard is also available from the

Wizard menu or the DSD device control tab as shown below. Note that Andor DSD turrets (shown below) can be switched with the DSD attached to the microscope.

116

© Andor Technology

Andor iQ USER GUIDE

Once the system has been calibrated it is very easy to use.

The Camera dialog houses a new tab called DSD Processing as shown here. This should be enabled and then the user can select the Imaging Mode as

Confocal, Wide field or both and this will determine which images iQ captures during a Protocol. The

Wide field mode can be very helpful in focusing the specimen in the first instance.

A slider is provided to set the Confocal Black level. We recommend that the black level is set by imaging a region of the specimen where there is little no real signal or where the specimen is out of focus and ensure that the resulting confocal grey levels are in the range of a few tens.

117

© Andor Technology

Andor iQ USER GUIDE

The following figures show wide field and confocal images of the same field in a three-color tissue preparation. The high levels of detail, lack of flare and dark background is apparent in the confocal image.

118

© Andor Technology

advertisement

Was this manual useful for you? Yes No
Thank you for your participation!

* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project

Related manuals

Download PDF

advertisement

Table of contents