Leica DMi8 for FRAP
Yes, the Leica WF FRAP is added to the Leica DMi8 via one Infinity Port. This leaves the second Infinity Port free for the addition of fluorescent light sources and does not affect the overall operation of the microscope.
The Leica WF FRAP is a laser safety class 1 device.
A 450 nm diode laser is integrated into the Leica WF FRAP module.
Often, recovery times are very fast, so using a camera that can capture events at a high speed will ensure reliable results. A high-speed triggered camera, such as the Leica DFC7000 T or the Andor Zyla, is needed for the Leica WF FRAP. The Leica DFC7000 T is especially interesting for users who perform simultaneous multiple fluorescence imaging.
LAS X version 1.5 or higher can be configured with the Leica WF FRAP wizard.
Yes, using an incubation chamber to regulate temperature and humidity will help maintain healthy cells throughout your experiment.
The Leica WF FRAP can be added to the Leica DMi8 via the Infinity Port by Leica Microsystems’ technicians. FRAP experiments are performed on living cells, and it’s important to set up an environment that is cell-friendly for your experiments.
More information about configuring your Leica DMi8 for live cell imaging
The Leica WF FRAP module within LAS X software guides users through their FRAP experiment. Step-by-step guides make setting up, running and analyzing the experiment straightforward.
The Leica WF FRAP is used exclusively with the widefield microscope, the Leica DMi8. Combined with a high speed camera, you can minimize the imaging exposure time as well as laser pulse duration. This means that the amount of light that your cells are exposed to can be reduced even further than in scanning based approaches.
When cells are exposed to light, such as from a laser or other fluorescent light source, they can be damaged over time. Therefore, reducing the exposure to light as much as possible is an important factor when doing live cell imaging.
The Leica WF FRAP has 12 different shapes that can be chosen for an experiment. When a photobleaching experiment is performed, these masks define the shape of the region of interest that will be bleached. Users can choose the size and shape that works best for their experiments.
Using the Leica WF FRAP and software wizard in LAS X you select a defined region of interest (ROI) in your cells that matches the 12 bleaching masks available.
Living cells that express fluorescently labelled molecules such as GFP can be photobleached by selecting a region of interest and then analyzing that region over time. Generally there are three phases that need to be analyzed; pre-bleach, bleach, and post-bleach time points are needed.
FRAP experiments are used to better understand how molecules are organized in a cell. By studying the effect of photobleaching and the timing of recovery afterwards, one can discern freely diffuse molecules from those localized to membranes, organelles, or other cellular components.
For more information, see the Science Lab article: Fluorescence Recovery after Photobleaching (FRAP) and its Offspring
Fluorescence Recovery After Photobleaching is a technique that uses a laser pulse to bleach the fluorescent molecules in a defined region of a cell, and then monitor how quickly and to what degree the fluorescent molecules return to that area.